Split (1)

train · 133k rows

text stringlengths 0 868k
Lignocaine (n - diethylaminoacetyl-2,6-xylidide) was first synthesized by lofgen a swedish chemist in 1943 and was first introduced into clinical use in 1948 . Lignocaine hydrochloride (c14h22n2o.hcl) is most soluble in water and so this is most commonly used injectable solution as local anesthetic agents . The efficacy profile of lidocaine as a local anesthetic is characterized by a rapid onset of action and intermediate duration of efficacy . Lidocaine or lignocaine along with adrenaline has the advantage of a rapid onset of action . Epinephrine (adrenaline) vasoconstricts arteries, reducing bleeding and also delays the resorption of lidocaine, almost doubling the duration of anesthesia . For surface anesthesia, several available formulations can be used, e.g. For endoscopies, before intubations, etc . Topical lidocaine has been shown in some patients to relieve the pain of postherpetic neuralgia (a complication of shingles), though not enough study evidence exists to recommend it as a first - line treatment . Although not completely curing the disorder, it has been shown to reduce the effects by around two - thirds . Lidocaine is also the most important class-1b antiarrhythmic drug; it is used intravenously for the treatment of ventricular arrhythmias (for acute myocardial infarction, digoxin poisoning, cardioversion, or cardiac catheterization) if amiodarone is not available or contraindicated . Lidocaine should be given for this indication after defibrillation, cardiopulmonary resuscitation, and vasopressors have been initiated . A routine prophylactic administration is no longer recommended for acute cardiac infarction; the overall benefit of this measure is not convincing . The onset of action of lidocaine is about 4590 s and its duration is 1020 min . It is about 95% metabolized (dealkylated) in the liver mainly by cyp3a4 to the pharmacologically active metabolites monoethylglycinexylidide (megx) and then subsequently to the inactive glycine xylidide . Megx has a longer half - life than lidocaine but also is a less potent sodium channel blocker . The elimination half - life of lidocaine is biphasic and around 90120 min in most patients . This may be prolonged in patients with hepatic impairment (average 343 min) or congestive heart failure (average 136 min). Lidocaine is excreted in the urine (90% as metabolites and 10% as unchanged drug). Bupivacaine (1-butyl-2, 6- pipecoloxylidide) was first synthesized in 1957 by ekenstam, a scandinavian chemist, and was first introduced into clinical use in 1963 . It is the longer side chain with four methylene groups on the piperidine ring that is responsible for the different properties of bupivacaine when compared to lignocaine . It has a molecular weight of 288.4 and an empirical formula of c18h28n2o and exists as white crystalline base with a melting point of 251258c . The base is not soluble in water, but the acid salt, bupivacaine hydrochloride (c18h28n2o.hcl) is slightly soluble . The rate of systemic absorption of bupivacaine and other local anesthetics is dependent upon the dose and concentration of drug administered, the route of administration, the vascularity of the administration site, and the presence or absence of epinephrine in the preparation . Duration of action (route and dose - dependent): 29 h. half - life in neonates is 8.1 h and in adults is 2.7 h. time to peak plasma concentration (for peripheral, epidural or caudal block) is 3045 min . The rate of systemic absorption of local anesthetics is dependent upon the total dose and concentration of drug administered, the route of administration, the vascularity of the administration site, and the presence or absence of epinephrine in the anesthetic solution . A dilute concentration of epinephrine (1:200,000 or 5 mcg / ml) usually reduces the rate of absorption and peak plasma concentration of bupivacaine, permitting the use of moderately larger total doses and sometimes prolonging the duration of action . The onset of action with bupivacaine is rapid and anesthesia is long lasting . The duration of anesthesia is significantly longer with bupivacaine than with any other commonly used local anesthetic . It has also been noted that there is a period of analgesia that persists after the return of sensation, during which time the need for strong analgesics is reduced . Local anesthetics are bound to plasma proteins in varying degrees . In general, the lower the plasma concentration of drug the higher the percentage of drug bound to plasma proteins . The rate and degree of diffusion are governed by (1) the degree of plasma protein binding, (2) the degree of ionization, and (3) the degree of lipid solubility . Fetal / maternal ratios of local anesthetics appear to be inversely related to the degree of plasma protein binding because only the free, unbound drug is available for placental transfer . Bupivacaine with a high protein binding capacity (95%) has a low fetal / maternal ratio (0.20.4). Depending upon the route of administration, local anesthetics are distributed to some extent to all body tissues, with high concentrations found in highly perfused organs such as the liver, lungs, heart, and brain . Pharmacokinetic studies on the plasma profile of bupivacaine after direct intravenous injection suggest a three - compartment open model . The second compartment represents the equilibration of the drug throughout the highly perfused organs such as the brain, myocardium, lungs, kidneys, and liver . The third compartment represents an equilibration of the drug with poorly perfused tissues, such as muscle and fat . The elimination of drug from tissue distribution depends largely upon the ability of binding sites in the circulation to carry it to the liver where it is metabolized . After injection of bupivacaine hydrochloride for caudal, epidural, or peripheral nerve block in man, peak levels of bupivacaine in the blood are reached in 3045 min, followed by a decline to insignificant levels during the next 36 h. various pharmacokinetic parameters of the local anesthetics can be significantly altered by the presence of hepatic or renal disease, addition of epinephrine, factors affecting urinary ph, renal blood flow, the route of drug administration, and the age of the patient . The half - life of bupivacaine in adults is 2.7 h and in neonates 8.1 h. in clinical studies, elderly patients reached the maximal spread of analgesia and maximal motor blockade more rapidly than younger patients . Amide - type local anesthetics such as bupivacaine are metabolized primarily in the liver via conjugation with glucuronic acid . Patients with hepatic disease, especially those with severe hepatic disease, may be more susceptible to the potential toxicities of the amide - type local anesthetics . When administered in recommended doses and concentrations, bupivacaine hydrochloride does not ordinarily produce irritation or tissue damage and does not cause methemoglobinemia . Local anesthetics block the generation and the conduction of nerve impulses, presumably by increasing the threshold for electrical excitation in the nerve, by slowing the propagation of the nerve impulse, and by reducing the rate of rise of the action potential . In general, the progression of anesthesia is related to the diameter, myelination, and conduction velocity of affected nerve fibers . Clinically, the order of loss of nerve function is as follows: (1) pain, (2) temperature, (3) touch, (4) proprioception, and (5) skeletal muscle tone . Systemic absorption of local anesthetics produces effects on the cardiovascular and central nervous systems (cns). At blood concentrations achieved with normal therapeutic doses, changes in cardiac conduction, however, toxic blood concentrations depress cardiac conduction and excitability, which may lead to atrioventricular block, ventricular arrhythmias, and cardiac arrest, sometimes resulting in fatalities . In addition, myocardial contractility is depressed, and peripheral vasodilation occurs, leading to decreased cardiac output and arterial blood pressure . Recent clinical reports and animal research suggest that these cardiovascular changes are more likely to occur after unintended intravascular injection of bupivacaine apparent central stimulation is manifested as restlessness, tremors and shivering progressing to convulsions, followed by depression and coma progressing ultimately to respiratory arrest . However, the local anesthetics have a primary depressant effect on the medulla and on higher centers in most of the studies 0.5% bupivacaine with adrenaline 1:200,000 and 2% lignocaine with adrenaline 1:200,000 were used as local anesthetic agents for third molar surgery as they are roughly equipotent . Studies have proved that bupivacaine is superior to lidocaine plus diflunisal in controlling postoperative pain after lower third molar surgery . Surprisingly there have been records of patients with no or mild pain at 8 h after the bupivacaine procedure was almost twice as many as after the lidocaine procedure, but is explained by the reason for preference being shorter duration of the anesthetic agent . In an australian study, most of the patients preferred the long - acting anesthetic agent bupivacaine . Many authors suggest that it seems reasonable that the combination of a long - acting local anesthetic with a weak analgesic is more efficient in relieving pain than the combination of a rather short - acting local anesthetic and a weak analgesic . Bupivacaine is considered to have a therapeutic ratio of 2:0 while lignocaine in combination with adrenaline has a therapeutic ratio of 2:3 . However, it has shown that the injection route alters the relative toxicity of local anesthetics . In a study by de jong and bonin in 1980 they found out that the bupivacaine has a greater therapeutic ratio than lignocaine when used for surgical removal of impacted third molars . Studies have proved that long - acting bupivacaine can be safely administered for surgical removal of lower third molar and it does have a long period of nalgesia postoperatively compared to lidocaine, but the cardio depressant property of bupivacaine should be kept in mind and should be administered judicially . Even studies have also shown that bupivacaine combined with methylprednisolone reduced the postoperative pain and swelling compared with the use of lidocaine and placebo, lidocaine and methylprednisolone, or bupivacaine and placebo . Right now clinical trials are going on bupivacaine versus lidocaine anesthesia under university of british columbia 2014 with the idea that the longer duration of anesthesia offered by bupivacaine when administered preoperatively in elective outpatient hand surgeries, will offer more effective postoperative pain control compared to using lidocaine only . It has been found that both bupivacaine and lignocaine have their merits and demerits but beyond any doubt it has been proven by the clinical trials that bupivacaine provides better and prolonged analgesia and anesthesia post operatively during minor surgical procedures done at chair side along with surgical removal of impacted third molars . Hence, bupivacaine can be regularly used as the anaesthetic solution along with adrenaline 1:200,000 for surgical removal of impacted third molars provided care being taken regarding the dosage and the cardiodepressant property of bupivacaine . Right now, further studies are going on.
Coccidia are a diverse group of protozoan parasites within the phylum apicomplexa and include pathogenic species of significance to animal and human health . Atoxoplasma), primarily parasitize a single host throughout their life cycle and include important avian pathogens (mcdougald, 1998, barta et al ., 2005, berto et al ., 2011). Heteroxenous coccidian protozoa including toxoplasma gondii, neospora caninum and sarcocystis neurona infect multiple hosts throughout their life cycle and their transmission is facilitated by predator prey relationships (sibley, 2003). In the intermediate host (often herbivorous or omnivorous), infectious stages are formed within tissue cysts that can be ingested by the definitive host (often a carnivorous predator). The definitive host is so called because the parasite life cycle is perpetuated through sexual multiplication in these animals . Definitive hosts shed environmentally - resistant oocysts, (or sporocysts from sporulated oocysts in the case of s. neurona), which are infective after sporulation for intermediate hosts that ingest fecally - contaminated food or water (sibley, 2003). Although the only known definitive hosts of t. gondii, n. caninum and s. neurona are terrestrial animals, specifically felids, canids and opossums (didelphis spp . ), respectively, there is evidence that marine mammals can also become infected with these and other related coccidia (dubey et al ., 2000,, 2001a, dubey et al ., 2001b, tenter et al ., 2000, dubey et al ., 2003,, 2011, gibson et al ., 2011, carlson - bremer et al ., the most likely modes of transmission of these pathogens to aquatic animals are via ingestion of water - borne oocysts or sporocysts originating in land - based surface runoff, or infected prey (miller et al ., 2002, conrad et al ., 2005, massie et al ., 2010, gibson et al ., 2011, one of the most serious consequences of s. neurona and t. gondii infection in marine mammals is fatal protozoal encephalitis which has been described most commonly in cetaceans, including southern sea otters (enhydra lutris nereis), california sea lions (zalophus californianus) and pacific harbor seals (phoca vitulana richardsi) (thomas and cole, 1996, lapointe et al ., 1998, cole et al ., 2000, miller et al, 2001a, dubey et al ., 2003, kreuder et al ., 2003, miller et al ., 2004, miller et al ., 2009, miller et al ., 2010, gibson et al ., 2011). More recently, severe myositis in a hospitalized california sea lion was recognized as a new clinical syndrome associated with s. neurona infection (carlson - bremer et al ., 2012b). Pathogenic infections with other, less well characterized, tissue - encysting coccidia have also been documented in marine mammals in association with protozoal lymphadenitis, hepatitis, myocarditis, encephalitis and non - suppurative necrotizing meningoencephalitis (dubey et al ., 2003, lapointe et al ., 2003, california sea lions inhabit waters of the pacific coast of north america between southwestern canada and baja california (lowry et al ., 1992). As long - lived coastal residents with large fat stores and piscivorous prey preferences that are shared with humans, sea lions have the potential to act as sentinel species, or indicators of aquatic ecosystem and human health (bossart, 2011). During postmortem examination of rescued california sea lions that died at the marine mammal center (tmmc, sausalito, california), sexual and asexual stages of three novel coccidia with genetic similarity to neospora spp . Were identified in sea lion enterocytes, and these organisms were putatively named coccidia a, b and c (colegrove et al ., 2011, carlson - bremer et al ., 2012a). Subsequent fecal analysis of stranded and rehabilitated sea lions sampled between 2007 and 2009 confirmed that sea lions shed coccidia a and b, particularly as yearlings, and that shedding of coccidia a could be detected in feces for up to 22 days (carlson - bremer et al ., 2012a). Evidence to date suggests that california sea lions act as both definitive and intermediate hosts of novel protozoa in the absence of clinical signs or pathologic evidence of disseminated infection . Yet, the identification of severe protozoal hepatitis, myocarditis and encephalitis in a neonatal harbor seal infected with coccidia c is of particular concern because it indicates that sea lions may be capable of shedding coccidian protozoa that are pathogenic to harbor seals and perhaps other aquatic wildlife (colegrove et al ., 2011). A better understanding of the biology, epidemiology, and pathogenesis of tissue - encysting coccidian organisms that parasitize marine mammals is needed to properly assess the risks and burden of protozoal disease in aquatic ecosystems such as the pacific coast of north america . As part of ongoing health surveillance in aquatic species of the pacific ocean, and accompanying studies of pathogen pollution in near - coastal california waters, we examined the diversity of coccidian parasites shed by hospitalized and free - ranging sea lions at coastal haul - out locations in central california . In addition, we characterized the phenotypes of sea lion - derived coccidian protozoa in both a mouse model of pathogenesis and in mammalian cell culture . Staff at tmmc collected fecal samples from individual california sea lions (csl) stranded along the central and northern california coast between september 2010 and may 2012 as previously described (carlson - bremer et al ., 2012a). Samples were typically collected within the first week of hospital admission, although some animals were sampled up to 3 weeks later . Animals and corresponding fecal samples when coccidial oocysts were identified at tmmc, one or more fecal samples from an individual animal were shipped on cold packs to the university of california davis for oocyst harvesting and genetic characterization . Age classifications for sea lions (adult, subadult, juvenile, yearling, and pup) were estimated as previously described (carlson - bremer et al ., 2012a). Between may 2011 and october 2012, sea lion fecal samples from free - ranging animals were also collected from coastal haul out sites at ao nuevo state park (37.108, 122.337), white rock (35.532, 121.088), and seal rock at point lobos (36.516, 122.336), placed into individual 50 ml conical vials and kept at 4 c during transport to uc davis for oocyst detection, harvesting and genetic characterization . Coccidial oocysts were harvested from fecal samples by a double centrifugation flotation method as described by dabritz and co - authors (dabritz et al ., 2007) with some modifications . A detailed protocol for oocyst harvesting from sea lions is available in the supplemental information associated with this article (see supplementary data). When quantities were sufficient, a portion of freshly - harvested oocysts were not frozen, but were reserved and sporulated for bioassay in mice and inoculation into cell cultures . Newly - harvested oocysts were washed two times in sterile pbs, and the final pellet resuspended in 5 ml pbs, to which was added clorox bleach at a final concentration of 10% . Following 20 min incubation with occasional gentle mixing, the sample was washed 3 times in 50 ml molecular biology grade water, with each centrifugation at 1000 g for 10 min . After final centrifugation, supernatant was carefully removed and the pellet mixed with 35 ml pbs (higher volume for samples with higher oocyst concentration). Approximately 1 ml of oocyst suspension in pbs was added to 5 ml filtered seawater (0.22 m, acrodisc syringe filters, pall corporation) with 250 l amphotericin b (250 g / ml) in a t-25 tissue culture flask with ventilated lid . Each flask was set on a gentle rocker at rt and visually inspected every 2 days by light microscopy for evidence of sporulation . Sporulated oocysts were washed two times in 40 ml sterile pbs, counted, and prepared for oral inoculation into mice . For excystation and preparation for cell culture, sporulated oocysts were suspended in 0.5 ml warm (37 c) dulbecco's minimum essential media (dmem, gibco), supplemented with 10% (v / v) heat - inactivated fetal bovine serum, 2 mm l - glutamine, 100 u / ml penicillin, 100 g / ml streptomycin, 10 mm hepes and 55 m 2-mercaptethanol and transferred to 2 ml screw cap tubes containing 350 mg of 200400 mm sterile acid - washed glass beads (thermofisher scientific). Oocyst disruption and sporozoite release was accomplished through vortexing in the presence of glass beads for 2030 s. to confirm the presence of free sporozoites, a 5 l aliquot of the suspension was examined following each 10 s of vortexing . When sporozoites were visible, the sample was immediately transferred to monolayer cultures of embryonic monkey kidney (ma104) cells (biowhittaker, walkersville, md) and incubated for 2 h at 37 c in 5% co2, after which the inoculum was removed and replaced with fresh 37 c prepared dmem . Cells were checked 3 times weekly for parasite growth alongside uninoculated ma104 cells . When zoites became visible in cell culture, supernatant was collected and pelleted for dna extraction, cryopreservation, and antigen slide preparation for serological testing . During microscopic analysis of oocysts isolated from sea lion feces, multiple photomicrographs were taken at 4001000 magnification using a camera (spot rt3 scientific digital ccd camera, spot imaging solutions, sterling heights, mi) mounted on a zeiss axioskop epifluorescent microscope equipped with a uv emission filter set (emitter 460/50 nm band pass filter; chroma #11000 v3). Micrographs were used to measure the long axis and short axis lengths of oocysts (in unsporulated and sporulated forms) and sporocysts (in sporulated forms) using spot advanced software (spot imaging solutions, sterling heights, mi, usa). The mean and standard deviation of 1020 sporulated and unsporulated oocysts were calculated separately for each coccidian species . In oocyst - inoculated cell cultures, approximately 10 zoites per culture were measured at 200 magnification from photomicrographs using spot software . All animal experiments were conducted with the approval and oversight of the institutional animal care and use committee at the university of california, davis, which is accredited by the association for assessment and accreditation of laboratory animal care, international (iacuc #17501). Mouse strains were chosen based on known susceptibility to t. gondii (rytel and jones, 1966, villegas et al ., 2002, gavrilescu and denkers, 2003). Prior to inoculation, 68 week old female mice were screened by the indirect fluorescent antibody test (ifat, see methods below) using a 1:40 serum dilution to ensure seronegativity to t. gondii, n. caninum and s. neurona . Approximately 1000 sporulated oocysts were inoculated into two mice of each genetic background: b6.129s7-ifng / j (c57bl/6 background), cba / caj (the jackson laboratory, bar harbor, me) and swiss webster (cfw) (charles river laboratories, wilmington, ma). Of the two mice in each genetic background group, one was orally inoculated by gastric lavage and the other was subcutaneously inoculated in the interscapular region as previously described (fritz et al ., 2012). Control mice of each genetic background were given inocula free of oocysts consisting of clinicare canine / feline liquid diet (abbott health care) for gastric lavage controls or pbs for subcutaneous controls . Mice were checked once per week for clinical signs of disease and were bled at 3 and 6 weeks post - infection for ifat serology . Mice were humanely euthanized by co2 asphyxiation for necropsy at 5070 days post - inoculation, at which time additional blood was collected from the heart cavity for ifat serology . During necropsy, portions of the tongue, heart, and brain were collected, divided into three pieces, and frozen at 80 c until dna extraction . A portion of the mouse brains was homogenized, trypsinized and placed over monolayer cultures of ma104 cells for coccidial parasite detection as previously described (miller et al ., 2001b). Remaining mouse tissues were placed in 10% formalin and submitted to the california animal health and food safety laboratory (cahfs, davis, ca) for histological and immunohistochemical analysis . The following mouse tissues were fixed in 10% neutral buffered formalin and examined histologically: liver, kidney, heart, lung, spleen, subcutaneous fat, skeletal muscle, pancreas, salivary gland, uterus, lymph node, thymus, multiple sections of gastrointestinal tract and brain, gonad, trachea, adrenal gland . Formalin - fixed tissues were trimmed, routinely processed for paraffin embedding, sectioned at 5 m, and stained with hematoxylin and eosin . Immunohistochemical staining for the presence of t. gondii, s. neurona and n. caninum antigen was performed as previously described (miller et al ., 2001b) on selected sections of formalin - fixed, paraffin - embedded tissues . Antigen slides for ifat analysis were prepared with parasites harvested from the supernatants of ma104 cultures containing visible zoites using previously described methods (miller et al ., 2001b). Mouse blood was collected by venipuncture, cardiac puncture or retro - orbital bleed, allowed to clot, and centrifuged at 1000 g for 10 min . Sea lion serum was similarly separated after collection via venipuncture of the caudal gluteal vein during hospitalization at tmmc and was shipped to uc davis for ifa testing . Serum was serially diluted in pbs from 1:40 to 1:81,920 and screened for immunoreactivity to t. gondii, s. neurona and n. caninum antigen using a fluorescein isothiocyanate (fitc)-conjugated rabbit anti - canine igg (jackson immunoresearch laboratories, west grove, pa). Mouse sera were screened at a 1:40 dilution for immunoreactivity to antigens of the same three parasites using fitc - conjugated goat anti - mouse igg (jackson immunoresearch laboratories). Test controls consisted of previously analyzed animal sera with titers ranging from 1:12801:10,240 (positive controls) or titers <1:40 (negative controls). Sea lion and mouse serum specimens that showed no reactivity at 1:40 dilution were considered test - negative . For the purpose of this study, sea lion sera that were reactive to t. gondii antigen at 1:640 dilution were considered test - positive to identify sea lions likely infected with t. gondii . Sea lion sera that were reactive to s. neurona antigen at 1:320 were considered test - positive as has been used previously to identify s. neurona infection in sympatric species (kreuder et al ., 2005, prior to dna extraction, frozen, pelleted oocysts that had been harvested from sea lion feces were subjected to a single freeze dna was extracted from oocysts and mouse tissues using the qiagen dneasy blood and tissue kit using manufacturer's recommendation for animal tissues . Two out of three mouse tissue sections were analyzed per tissue type per mouse, with preference for mice that demonstrated histological lesions, leading to a total of 164 individual samples, including controls, which were extracted and analyzed by pcr . Negative controls (water) were included in the extraction process to monitor for environmental contamination . For molecular characterization of coccidian dna in oocysts, mouse tissues and zoite cultures, nested pcr was performed using pan - coccidian primers that span the internal transcribed spacer 1 (its-1) region, and s. neurona / s . Falcatula - specific primers (its1500) that amplify 500 bp of the its1 region in those organisms (miller et al ., 2009, gibson et al ., 2011). For characterization of s. neurona - like dna, pcr protocols were employed to amplify microsatellites sn7 (nested pcr) and sn9 (hemi - nested pcr), (asmundsson and rosenthal, 2006, rejmanek et al ., 2010) and surface antigens snsag3, snsag4 (nested pcr) (rejmanek et al ., 2010) and either snsag1, snsag5, or snsag6 which amplify using snsag1 - 5 - 6 primers (wendte et al ., 2010). Additional primers were used to amplify a secondary region of s. neurona its-1, approximately 150 bp upstream of the area amplified by its1500 primers: snits1-f, ccgggatgatgtcgtcaag and snits1-r, acagatgatgtgccccgc (carlson - bremer, 2011). All pcr reactions were performed using qiagen hotstar taq plus (qiagen, valencia, ca) according to manufacturer's recommendations, utilizing 5 l of template dna . The following reaction conditions were used for all primer sets: initial denaturation at 95 c for 5 min, followed by 35 cycles at 94 c for 40 s, 58 c for 40 s (external primers or non - nested pcr) or 59 c for 40 s (internal primers for nested / hemi - nested pcr), and 72 c for 90 s, followed by a 10 min extension at 72 c . In the case of nested or hemi - nested pcr protocols, 1 l of first round pcr product was added to the second round master mix . Positive controls for pcr included either hammondia heydorni dna isolated from a domestic dog, neospora hughesi isolated from a horse, and/or s. neurona strain snucd-1 (rejmanek et al . Pcr products were purified using the qiaquick pcr purification kit (qiagen) and were sequenced by the ucdna sequencing facility (davis, ca). Forward and reverse sequences were assembled into contigs using geneious pro v. 5.3.4 (drummond et al ., 2009). Consensus sequences were aligned to reference sequences obtained from ncbi (http://www.ncbi.nlm.nih.gov/nuccore) using muscle or mafft (katoh et al ., 2002, edgar, 2004) with default parameters, followed by manual editing . Posterior sets of phylogenetic trees were generated using mrbayes (huelsenbeck and ronquist, 2001) implemented in geneious pro v. 5.3.4 and run for 1,000,000 generations using the gtr + g nucleotide substitution model . Trees were sampled every 1000 generations and 25% of trees were discarded from the initial burn - in period . Pairwise genetic distance values were determined through uncorrected - p distance matrices generated in paup * (sinaeur associates inc .) Using prepared alignments of 420 bp for its-1 . Individual sequences were analyzed using blast (basic local alignment search tool) available on the ncbi website http://blast.ncbi.nlm.nih.gov/blast.cgi . Kp999999 and kp990539 for t. gondii m4 and ao11, respectively), and snsag3 (kr011915 for sarcocystis sp . Groups were compared for significant differences using the two - tailed two - sample test of proportions (stata / ic v. 11.0, statacorp lp, college station, tx). Fecal samples collected from 139 sea lions stranded in nine counties located on the california coast between 2010 and 2012 were screened upon hospitalization for the presence of coccidian oocysts . The majority of animals stranded in monterey (n = 48), san luis obispo (n = 36) and santa cruz (n = 32) counties, followed by san mateo (n = 7), san francisco (n = 5), sonoma (n = 4), marin (n = 4), mendocino (n = 2) and humboldt (n = 1). Coccidian oocysts were identified in the feces of 16 of the 139 (11.5%) sea lions sampled the majority of which were from monterey (8.3%, 4/48) and san luis obispo (13.9%, 5/36) counties . Only one of the 32 stranded animals (3.1%) from santa cruz county was shedding oocysts . Table 1 describes the clinical status and results of serological testing and coccidian oocyst dna analysis for the 16 sea lions that had oocysts detected in their feces upon presentation at tmmc . Oocysts observed in the feces of 16 stranded sea lions (table 1) were 810 m in diameter . Sporocysts were not observed by light microscopy following fecal flotation in any of the sea lions sampled . A similar proportion of males (9/89) and females (7/51) shed oocysts (p = 0.518). Yearlings most commonly shed oocysts (8/33, 24.2%) followed by subadults (2/16, 12.5%), pups (2/19, 10.5%), juveniles (3/50, 6.0%) and adults (1/21, 4.8%). The proportion of yearlings shedding oocysts (8/33) was significantly greater than the proportion of all other age classes shedding oocysts (8/106; p = 0.009). 1 shows the close relationship of coccidian dna sequences amplified from fecal samples of the stranded hospitalized sea lions in this study (e.g. Csl ncmmc1047 humboldt and csl9878 monterey) to n. caninum and dna sequences amplified from marine mammal fecal samples and tissues in previous studies (colegrove et al ., 2011, gibson et al ., 2011, carlson - bremer et al ., 2011), coccidia a (csl ncmmc1047) and b (csl9878) its-1 sequences shared 79.6% and 78.8% pairwise nucleotide similarity, respectively, to n. caninum and 74.1% and 71.8% similarity, respectively, to t. gondii . Based on sequence analysis of the its-1 locus from the 16 hospitalized sea lions that were shedding coccidia during this study, 6 (37.5%) animals were confirmed to be shedding coccidia a, 6 (37.5%) were shedding coccidia b, and 2 (12.5%) were shedding both a and b simultaneously, indicating dual infections (table 1). Sera from 5 (31.3%) of these sea lions had antibodies to t. gondii antigens detectable by ifat, but only one of these animals (csl9830) had an antibody titer above 1:640 (table 1). One animal from monterey (csl9878) was seroreactive to s. neurona antigen at a 1:2560 dilution . No animals tested during this time period were seroreactive to n. caninum despite the apparent genetic relatedness of coccidia a and b to neospora spp . At the its-1 locus (fig . 1) (colegrove et al ., 2011, carlson - bremer et al ., unsporulated oocysts had a round to ovoid shape and autofluoresced under uv illumination (fig . 2) as observed for t. gondii and other coccidian oocysts (lindquist et al ., 2003). Unsporulated coccidia a oocysts averaged 9.9 0.59 m in length and 9.5 0.69 m in width (n = 20 oocysts from csl ncmmc 1047, measured at 200 magnification). Unsporulated coccidia b oocysts averaged 8.9 0.31 m in length and 8.5 0.51 m in width (n = 20 oocysts from csl9878, measured at 1000 magnification) (fig . 2). Coccidian oocysts a and b were previously identified in the feces of stranded california sea lions surveyed at tmmc between 2007 and 2009 (carlson - bremer et al ., 2012a). Here, we summarized shedding prevalence for all sea lions sampled between 2007 and 2012 (table 2). Over the six year period, 558 sea lions were sampled, 31 (5.6%) of which were shedding coccidia a or b, in similar frequency . Hospitalized stranded sea lions were typically diagnosed with malnutrition and trauma with no apparent clinical effects attributed to protozoal infection (table 1). Between august 2011 and september 2012, california sea lion fecal samples were collected from coastal haul - out sites located at ao nuevo state reserve (n = 48), white rock (n = 94) and point lobos (n = 70) and the samples were screened for oocysts . The prevalence of oocysts identified by microscopy during isolation was 8.3% (4/48) at the ao nuevo haul - out site, 3.2% (3/94) at white rock, and 8.6% (6/70) at point lobos (table 3). Sporocysts were not observed by light microscopy following fecal flotation in any of the free - ranging sea lion samples . Dna amplification and sequence alignment revealed that both coccidia a and b were present in fecal samples collected at ao nuevo and point lobos, while only coccidia a was identified in samples collected at white rock (table 3). Fecal sample 11 collected at ao nuevo state park (ao11) represented a novel sequence type with 9798% pairwise similarity at the its-1 locus to previously published coccidian dna isolates in guadalupe fur seal tissues (see neospora sp . The csl ao11 isolate had 92% pairwise similarity to coccidia a and b and shared even less similarity (87.7%) to coccidia c at the its-1 locus . This sequence as well as representative sequences of coccidia a and b dna isolated from field - collected specimens (csl ao53 and csl ao50, respectively) are shown in fig . 1, and the geographic distribution of coccidian oocyst species shed by free - ranging animals is shown in fig . When sufficient quantities were available, oocysts harvested from fecal samples were sporulated and inoculated into mice to investigate coccidian parasite infectivity and pathogenesis . Sporulated coccidia a oocysts were, on average, 9.8 0.83 m in length and 8.6 0.68 m in width, with sporocysts measuring 5.3 0.69 m long and 4.0 0.53 m wide (n = 20 oocysts from csl ncmmc1047, measured at 4001000 magnification) (fig . Sporulated coccidia b oocysts were 9.9 0.88 m in length and 9.0 1.2 m in width, with sporocysts measuring 6.0 0.4 m long and 4.6 0.5 m wide (n = 10 oocysts from csl9878, measured at 4001000 magnification) (fig . Sufficient quantities of oocysts from animals that were shedding either coccidia a or b (or both) including csl9830, csl9878, csl ncmmc1047, csl10089, csl10092, and csl10100, were available for mouse bioassay . All mice were seronegative for t. gondii, s. neurona and n. caninum prior to inoculation and at 3 weeks and 6 weeks post - inoculation, as well as at the time of necropsy (5070 days post - inoculation). No clinical signs were observed in coccidia a- and b- inoculated or mock - inoculated control mice throughout the observation period . Mouse tissues were generally unremarkable histologically, although one or more of the following histological changes were observed in approximately 40% of mice: slight nephritis, slight pneumonia, inflammation in the lung, spleen or lymph node, and steatitis . Mouse brain, heart and tongue tissues tested negative for the presence of coccidia a or b dna using pan - coccidian primers that span the its-1 region . In an effort to propagate and isolate coccidian protozoa identified by light microscopy in sea lion feces, sporulated oocysts from csl10089 (coccidia a), csl10092 (coccidia a & b), csl10100 (coccidia a & b), and csl10266 (coccidia a) were prepared and inoculated into flasks containing ma104 cells . All cultures were fed and examined for the presence of live coccidian zoites three times per week . After 14 days, 21 days and 35 days of observation, cell cultures inoculated with sporulated oocysts from csl10089, csl10100, and csl10092, respectively, showed propagating organisms with morphologic similarity to sarcocystis spp . Extracellular zoites were 811 m in length and 12 m in width when measured with spot advanced software at 200 magnification . Schizonts were seen periodically as the culture matured and the number of free zoites increased; suggesting that these were merozoites produced by schizogony . Free zoites had active, circumaxial motility characteristic of s. neurona merozoites observed in vitro (miller et al ., 2001a). Similar organisms did not grow in cultures inoculated with sporulated oocysts from a fourth sea lion, csl10266 or in un - inoculated cultures of ma104 cells (table 4). By ifat, antigen slides prepared from zoite cultures of all three sea lions, csl10089, csl10092 and csl10100, tested positive using serum from a horse with equine protozoal myeloencephalitis (fig . The horse's s. neurona infection was confirmed by ifat and western blot on serum (1:320 titer to laboratory strain snucd-1) and cerebral spinal fluid, and by immunohistochemistry used to detect s. neurona merozoites in the lumbar spine . In addition, all three zoite isolates from sea lion oocyst cultures reacted to sera from hospitalized sea lion csl9878 (table 4) which was itself seroreactive to merozoites of snucd-1 (1:2560) (table 1 and fig . Sea lion culture antigen was ifat - negative (<1:40) to goat serum containing t. gondii - reactive antibodies, bovine fetal serum containing n. caninum - reactive antibodies, and, as shown in fig . 5e, to horse serum that was seronegative to s. neurona and n. hughesi . As soon as zoites were observed, supernatant from the ma104 cell cultures inoculated with sporulated, excysted oocysts from the fecal samples of csl10089, csl10100 and csl10092 were collected for molecular characterization approximately once per week for 1114 weeks . Pan - coccidian primers that span the its-1 region produced a 1000 bp band when dna was amplified from zoites taken from cultures from the 3 sea lions throughout their collection period . This is the expected band size for amplification of s. neurona dna using pan - coccidian its-1 primers (gibson et al ., 2011). In addition, a second band 400 bp in size was amplified simultaneously in culture supernatant of csl10100 and csl10092 using the pan - coccidian primers, but only in the first week of sample collection following zoite observation (table 4). Four hundred basepairs is the expected band size for amplification of coccidia a, b or c using pan - coccidian its-1 primers (colegrove et al ., 2011, gibson et al ., 2011). In culture csl10266, in which no zoites (only oocysts) were observed, only the 400 bp band was amplified by pcr during the first two weeks of incubation . Subsequent supernatant samples of culture csl10266 and all uninoculated ma104 control cultures were pcr - negative (table 4). Sequence analysis confirmed that the 400 bp amplicon dna generated in early sampling of cultures csl10100, csl10092 and csl10266 was identical to dna amplified from frozen, pelleted oocysts originally harvested from the same animal; namely, coccidia a alone (in the case of csl10092 and csl10266) or a dual infection with coccidia a and b (in the case of csl10100) (table 4). By blast analysis, good quality partial sequences (700 bp; abbreviated due to the presence of a homopolymer region) generated from the 1000 bp amplicon in csl10089, csl10092 and csl10100 zoite cultures were 99% similar to s. neurona dna isolated from horses (af204230 & af081944) and from a skunk (ay082648) (see supplementary data). Dinucleotide mixtures in the its-1 sequences indicated the presence of at least two s. neurona - like genotypes in the sea lion cultures . These polymorphic regions shared nucleotide similarity to horse strains snucd-1 (af081944) and sn - mu1 (af204230), sea otter strains so3639 (dq084486) and so5259, and a skunk isolate (ay082648) (see supplementary data). To further characterize the s. neurona - like organisms cultured from sea lion feces, we amplified seven additional genetic loci in dna extracted from zoites in the sea lion oocyst culture supernatant . Table 4 displays the pcr and sequencing results for these additional loci . Using its1500 primers, with known specificity for s. neurona or sarcocystis falcatula (gibson et al ., 2011), a 500 bp product was consistently amplified in all culture supernatant samples from csl10089, csl10092, csl10100 in which zoites had been observed . By blast analysis, good quality partial its1500 amplicon sequences (300 bp, abbreviated due to the presence of a homopolymer region) were 98% identical to s. neurona isolated in southern sea otters including isolates so3528, so3539, so3485, and so3501 (genbank dq084485 dq084488). Microsatellite primers amplified 150 bp products containing di - nucleotide repeats ca17 (sn7) and gt18 (sn9) found in s. neurona strain sn - mu1 and other s. neurona strains isolated from opossums, sea otters, and cats (rejmanek et al ., 2010). Merozoite surface antigen sag1 gene sequences of csl10089, csl10092 and csl10100, amplified by snsag 1 - 5 - 6 primers, were 100% identical across 959 bp to s. neurona strain snucd-1 (af401682) (ellison et al ., 2002) and 99% similar across 968 bp to the representative sequence of the snsag1 allele amplified in samples from terrestrial and marine mammals (gq851951) (wendte et al ., 2010). Sea lion cultures csl10089, csl10092 and csl10100 were identical to each other across a 948 bp sequence of the merozoite surface antigen gene snsag4 and 100% identical to sequences of s. neurona isolated from a sea otter (so3106, gq851957) and an opossum (op134, gq386979). A unique sequence in sea lion s. neurona - like isolates was identified in the merozoite surface antigen 3 (snsag3) locus in which csl10089 was confirmed by bi - directional sequencing to contain an at insertion at positions 519520 (table 5). The same insert was observed in single direction sequencing for csl10092 but could not be confirmed in csl10100 due to poor sequence quality . The same insert was not identified in snsag3 sequences available in genbank originating in a variety of host species including sea otters, opossums and horses (table 5). Lack of the position 519520 insert in laboratory strain snucd-1, derived from a horse in california (table 5), ruled out the possibility that s. neurona growth in sea lion oocyst cultures was due to laboratory contamination . At other polymorphic snsag3 nucleotide positions, including 239 and 1059, sea lion s. neurona - like isolates were similar to published opossum and sea otter isolates (table 5). In light of the unexpected findings revealed during cell culture analysis of fecal samples from stranded hospitalized sea lions, we screened all dna extracted from frozen, pelleted oocysts of both hospitalized and field - collected fecal samples using s. neurona / s . Falcatula - specific its1500 primers . While none of the additional frozen, pelleted oocyst samples collected from hospitalized animals were pcr - positive for s. neurona - like dna (including those remaining for csl10089, csl10092 and csl10100), two fecal samples collected at point lobos (fig . 3, samples pl11 and pl27) produced 500 bp products in the its1500 pcr reaction (table 3). Additional primers (sn - its) targeting the its-1 region of s. neurona produced amplicons with sequences identical to hospitalized sea lion zoites collected in the present study as well as s. neurona isolated in other animals (see supplementary data). All other attempts to amplify s. neurona in samples pl11 and pl27 using alternative primers failed with the exception of microsatellite sn7 which, in sample pl27, produced a sequence containing the di - nucleotide repeat ca17 found in s. neurona and zoites isolated in csl10089, csl10092 and csl10100 (table 4). The investigation of infectious diseases in coastal aquatic wildlife allows us to probe the relationships between terrestrial - origin pathogen distribution, patterns of overland water runoff, and the risk of pathogen pollution in the coastal and ocean environment . The present research was undertaken to investigate the role of california sea lions in the dispersal and life cycle of terrestrial and aquatic coccidian parasites that pose a risk to marine mammal health, and to genetically and phenotypically characterize protozoa shed by sea lions . Building on data acquired from previous protozoal pathogen surveillance efforts in california sea lions from the coastal waters of california (carlson - bremer et al ., 2012a), we confirmed that oocysts (810 m in size) that are morphologically similar to, but genetically distinct from, t. gondii and n. caninum are frequently shed by stranded sea lions that are hospitalized and by free - ranging populations . Genetic variability in the partial its-1 region of these coccidia a and b isolates was not observed within this study or in previous reports (colegrove et al ., 2011, carlson - bremer et al ., consistent with other studies of stranded sea lions, shedding of coccidia a and b occurred primarily in yearlings, and those shedding oocysts had stranded due to causes other than protozoal infection . While our data further establish that sea lions are naturally asymptomatic definitive hosts of coccidia a and b, the pathogenic potential of these protozoa in other aquatic species requires further investigation, particularly considering the known virulence of coccidia c and other novel tissue - cyst forming coccidia in harbor seals and guadalupe fur seals (colegrove et al . Phylogenetic analyses of the its-1 locus performed here and elsewhere (gibson et al ., 2011, carlson - bremer et al ., 2012a) demonstrate that coccidia a, b, c, csl ao11, and isolates from a harbor seal and guadalupe fur seals share a common ancestor with n. caninum . Morphologically, however, coccidia a and b measured in this study were smaller than n. caninum oocysts and had <80% pairwise similarity to n. caninum at the its-1 locus . While unsporulated coccidia a and b oocysts shed by sea lions are 810 m in diameter, unsporulated n. caninum oocysts shed by dogs are typically 1011 m in diameter (mcallister et al ., 1998). Sporulated n. caninum oocysts are reportedly an average of 11.7 11.3 m (lindsay et al ., 1999) while sporulated forms of coccidia a and b are an average of 9.8 8.6 m, and 9.9 9.0 m, respectively . By comparison, unsporulated t. gondii oocysts are 10 12 m in diameter and sporulated forms are 11 13 m in diameter (dubey et al ., 1998). More detailed genetic and morphologic studies (e.g. Whole genome sequencing, electron microscopy, and histological examination of life stages in situ) are necessary to clarify the taxonomy of novel coccidia identified in aquatic animals, and will likely reveal that these organisms belong to either new neospora species or a new genus within the family sarcocystidae . At the phenotypic level, we observed that the sporulated oocysts of coccidia a and b were not cultivable in the african green monkey kidney cell line, ma104, commonly used to propagate other tissue - cyst - forming coccidia including t. gondii, n. caninum and s. neurona (miller et al ., 2001a, miller et al ., 2004, holmberg et al ., 2006). While coccidia a dna was detectable by pcr in a culture prepared from oocyst isolate csl10266 for up to two weeks after inoculation, no protozoal organisms were ever observed . In ma104 cultures of oocyst isolates csl10092 and csl10100, coccidia a and b dna as well as s. neurona - like dna were amplified for up to 56 weeks post - inoculation; after which time only s. neurona - like dna and live s. neurona - like zoites were detected for the duration of observation . While it is possible in the latter cases that coccidia a and b did not replicate in vitro due to competition from the sarcosystsis sp . During co - culture, we hypothesize, rather, that the ma104 cell line may not be susceptible to infection with coccidia a and b, or the culture conditions were not optimal for these parasites . Other cell culture systems should be investigated for their propagation, particularly in the interest of developing a serological assay for the purpose of screening marine mammals for infections with these and other potentially pathogenic protozoa . Coccidia a and b did not cause disease in mice with known susceptibility to t. gondii including b6.129s7-ifng / j, cba / caj and swiss webster (cfw) (rytel and jones, 1966, villegas et al ., 2002, gavrilescu and denkers, 2003). Surprisingly, coccidia a and b were not pathogenic to cba / caj mice despite previous studies showing that cba / ca mice can be used as a model of n. caninum pathogenesis (mcguire et al . These findings, taken together with the lack of growth in ma104 cells or reactivity of serum from coccidia a- and b - infected sea lions with n. caninum, t. gondii or s. neurona antigen by ifat, is further evidence of the taxonomic divergence of the sea lion parasites from known tissue - cyst forming coccidia with terrestrial life cycles . A significant outcome of this study was the discovery of s. neurona or s. neurona - like zoites growing in cell cultures inoculated with fecal material (including sporulated, excysted oocysts of coccidia a and b) prepared from three stranded hospitalized sea lions sampled between 2010 and 2012 . The cultivation of live protozoal zoites with strong genetic similarity to s. neurona across multiple genes during parasite isolation from sea lion feces, particularly in the absence of microscopic evidence of sarcocystis sp . A possible explanation for these findings is that samples taken for microscopic observation during fecal flotation only represent a small portion of the overall feces sampled and the overall pool of oocysts being shed . If the original pool of isolated protozoa from feces consisted of a mixture of variable numbers of coccidian oocysts (coccidia a and/or b) and sporocysts (s. neurona - like), it is possible that the subdivision of the oocyst pool for flotation and microscopy, pelleting / freezing, sporulation for mouse bioassay, and sporulation / excystation for cell culture, could explain discrepancies in the ability to detect different parasites, depending on the sensitivity of each assay . We hypothesize that a small number of s. neurona - like sporocysts existed in the pool of protozoa prepared for cell culture, but were not present in the subsamples designated for microscopy, pelleting and freezing or mouse bioassay . In addition, in vitro cultivation may have facilitated the expansion of small numbers of s. neurona sporozoites that were able to develop into schizonts that produced merozoites . The isolation of s. neurona - like dna from feces of two free - ranging sea lions at point lobos indicates that the organism may occur naturally in sea lions, as opposed to occurring via nosocomial exposure in the hospitalized setting . Exposure of sea lions to s. neurona, or an s. neurona - like organism, most likely occurs in coastal waters contaminated with opossum feces transported from land - to - sea via freshwater run - off (carlson - bremer et al ., 2012b, shapiro et al ., 2012) or may be concentrated in sea lion prey, as has been shown with t. gondii shed by terrestrial felids (miller et al ., 2002, johnson et al ., 2009). Although the extent of environmental contamination with s. neurona sporocyts has not been documented, infections in southern sea otters and a number of other marine mammals of the pacific northwest have been reported (miller et al ., 2001b, miller et al ., 2009, miller et al ., 2010, all mouse tissues were pcr - negative for s. neurona using its1500 primers, indicating that the sea lion s. neurona - like sporocysts were either not present in the inoculum, were present but below the threshold of infection, or the mice utilized were not susceptible . Because of the similarity of coccidia a and b to t. gondii, mice in this study were selected for their susceptibility to t. gondii, not s. neurona . Shed by sea lions would benefit from the utilization of interferon gamma gene knock - out mice (e.g. C57bl/6-black) with well - characterized susceptibility to s. neurona (dubey et al . Sarcocystis neurona is the etiologic agent of equine protozoal myeloencephalitis a serious and progressively debilitating neurological disease in horses and is one of several coccidian parasites known to cause protozoal encephalitis in pacific harbor seals, southern sea otters, and other marine mammals (lapointe et al ., 1998, rosonke et al ., 1999, lindsay et al ., 2000, lindsay et al ., 2001, miller et al ., 2001a, lapointe et al ., 2003, thomas et al ., 2007, gibson et al ., 2011, dubey et al ., 2015 although not frequently reported in the literature, when s. neurona infections do occur in california sea lions, they are either clinically irrelevant (gibson et al ., 2011) or associated with myositis (carlson - bremer et al ., 2012b). Tissue cysts caused by other sarcocystis spp ., including sarcocystis canis - like organisms, have been detected in sea lion muscle (mense et al ., 1992, miller, 2008) and liver tissue (mense et al ., 1992, the three animals that shed s. neurona - like protozoa were admitted to tmmc for malnutrition or trauma, and had no overt signs of protozoal infection . Lack of serum titers to s. neurona in these animals indicated that they had not yet mounted a detectable antibody response, or that they never had systemic infections . An elevated s. neurona titer (1:2560) was detected in another hospitalized sea lion, csl9878, but this animal also had no signs of disease and was not shedding detectable levels of s. neurona - like sporocysts or dna at the time of sampling for this study . Overall, findings from this study suggest that california sea lions are likely acting as mechanical vectors of s. neurona or an s. neurona - like organism, similar to the way canines can mechanically transmit t. gondii (lindsay et al ., 1997). Using a mouse bioassay to detect the presence of infectious t. gondii oocysts in canine tissues and feces, lindsay et al . Sporulated t. gondii oocysts can traverse the canine intestinal tract and be excreted in an infectious state . Several experimental outcomes indicate that an s. neurona - like organism is excreted by sea lions in low concentrations, which is expected during mechanical transmission: (1) s. neurona - like sporocysts were not observed by light microscopy following fecal flotation (2) s. neurona - like dna was not amplified from concentrated oocysts (pellets) isolated from hospitalized sea lion fecal samples, (3) s. neurona - like dna was only amplified by pcr on high copy number genes in concentrated oocysts isolated from feces of free - ranging animals, and (4) s. neurona - like merozoites were only observed after long - term cultivation in ma104 cells . The genetic similarity of sea lion s. neurona - like isolates to previously characterized protozoa in aquatic and terrestrial animals across much of the genome is not surprising considering the extensive overlap in genetic identity in s. neurona isolates from the tissues of opossums, sea otters and horses that was previously demonstrated (rejmanek et al ., 2010, the key snsag3 polymorphism that we identified in the zoites from sea lion fecal samples may represent a novel s. neurona genotype which, in the future, may facilitate identification of the origin of infection . This is particularly important as we seek to understand the flow of terrestrial pathogens into oceans and risk factors for s. neurona infections in other aquatic species . Continued investigations into the pathogenesis of s. neurona in sea lions through necropsy and systematic surveillance for s. neurona exposure, shedding, and disease will help clarify the importance of this host species in parasite transmission within the pacific coastal ecosystem . In conclusion, continued surveillance for tissue - cyst forming coccidia in marine mammals should remain a priority for programs that monitor the population health of aquatic wildlife . Efforts to explore the distribution, pathogenesis and genetic diversity of sarcocystidae in land and sea isolates promise to improve our understanding of the interconnectedness between terrestrial and aquatic microbial diseases, and to direct interventions that will benefit our oceans and their inhabitants.
Bacterial infections as well as the emergence and spread of antibiotic resistance in human pathogens are serious public health problems in hospital and community settings across the globe . New strategies to prevent and treat bacterial infections are needed, including methods to overcome antibacterial resistance that results from the outer membrane permeability barrier in gram - negative organisms and targeting approaches that afford species- or pathogen - specific therapeutics . Almost all bacterial species have a metabolic iron requirement and therefore employ various strategies to acquire this metal ion when colonizing . Siderophores are high - affinity fe(iii) chelators that are produced by bacteria under conditions of iron limitation, such as those encountered in the vertebrate host, to scavenge this metal ion from the environment . Siderophore producers also express dedicated ferric siderophore import machinery and employ various mechanisms to release siderophore - bound iron following cellular uptake (e.g., reductive and/or hydrolytic release mediated by reductases and/or esterases, respectively). Numerous studies support the importance of siderophore - based iron acquisition during bacterial infections . Thus, the potential of using siderophores, or targeting siderophore biosynthetic and transport machineries, in therapeutic development continues to attract significant interest . Of particular relevance to the advances described herein are prior investigations pertaining to the development of siderophore this strategy has received particular attention for the delivery of antibiotics into gram - negative bacteria because these organisms are inherently less sensitive to many antibiotics used in the clinic as a result of the outer membrane permeability barrier . Both native siderophores and synthetic siderophore mimics have been evaluated as platforms for therapeutic development . In the clinic, the native siderophore desferrioxamine b is used for iron - chelation therapy in patients with iron overload . Several antibiotic small molecules found in nature called sideromycins provide inspiration for synthetic siderophore the sideromycins are secondary metabolites comprised of a siderophore moiety and a toxic cargo; the siderophore portion targets sideromycins to bacterial strains expressing the appropriate siderophore receptor . Antibiotic conjugate produced by a clinical isolate of klebsiella pneumoniae, is an 84-residue antibacterial peptide with a glucosylated enterobactin (ent, figure 1) derivative attached to its c - terminus that exhibits enhanced antibacterial activity against strains expressing the enterobactin receptor fepa . From the standpoints of antibacterial activity and therapeutic potential, studies of synthetic siderophore antibiotic conjugates have provided the community with mixed results, causing some skepticism about the potential of siderophore - based approaches despite the successful utilization of such molecules by nature . Structures of enterobactin (1, ent) and a generalized enterobactin cargo conjugate . Many of the failures encountered with early and recent studies of siderophore - based antibiotic delivery may be attributed, at least in part, to (i) use of non - native siderophores with relatively low fe(iii) affinities and/or compromised receptor recognition; (ii) modification of antibiotics such that the antibacterial activity is attenuated or lost completely; (iii) bridging the siderophores and antibiotics with problematic linkers, including linkers designed for drug release that are either too stable or too labile, the latter of which promotes premature release; and (iv) antibiotic resistance . Nevertheless, the lessons of many unsuccessful studies highlight the complexity of siderophore - based therapeutic development and provide a foundation for inventing improved next - generation approaches . Many of the issues described above may be overcome by careful molecular design and biological evaluation . In particular, the selection of appropriate native siderophore platforms and modification of these platforms in ways that do not compromise iron binding or receptor recognition, installation of an antibacterial cargo in such a manner that antibacterial activity is retained, and the development and application of assays that afford insight into the fate of siderophore antibiotic conjugates are critical to the overall success of this approach . Along such lines, a recent and insightful study by pfizer addressed complications associated with using relatively low - molecular - weight siderophore mimics in vivo . Their results indicate that competition between the siderophore - conjugated monobactam mb-1 and native siderophores resulted in poor in vivo efficacy against pseudomonas aeruginosa and provide support for designing and evaluating siderophore one recent and successful example based on a native siderophore platform is a mycobactin artemisinin conjugate that exhibits enhanced antibacterial activity against mycobacterium tuberculosis compared to unmodified artemisinin . Enterobactin (ent, figure 1) is a triscatecholate siderophore biosynthesized by enteric bacteria and used for iron acquisition in the vertebrate host . Motivated by the importance of ent in the host / microbe interaction as well as the decades of investigations pertaining to its (bio)synthesis, coordination chemistry, and biology, in prior work we reported a synthetic route to monofunctionalized ent platforms . Moreover, we established that the native ent platform, when monofunctionalized at the c5 position of one catecholate ring (figure 1), affords delivery of nontoxic small - molecule cargo across the outer membrane of gram - negative organisms that express ent uptake machinery (e.g., fepabcdg of escherichia coli). As described herein, this proof - of - concept study motivated us to demonstrate that ent effectively delivers antibacterial cargo to organisms that utilize ent for iron acquisition, thereby providing antibiotic targeting to specific sub - populations and a means to address antibiotic resistance that results from the gram - negative outer membrane permeability barrier . In this work, we present the syntheses and characterization of siderophore antibiotic conjugates based on the native ent platform that harbor the clinically relevant -lactam antibiotics ampicillin (amp) and amoxicillin (amx). These antibiotics block cell wall biosynthesis by inhibiting transpeptidases, also named penicillin binding proteins (pbps), located in the periplasm of e. coli . We report that the ent--lactam conjugates exhibit significantly enhanced antibacterial activity (up to 1000-fold) against pathogenic e. coli and provide more rapid cell - killing than the parent -lactams as a result of ent - mediated delivery to the periplasm . Moreover, in proof - of - concept studies for species - specific killing, these conjugates selectively kill e. coli in the presence of staphylococcus aureus, a gram - positive organism that is more susceptible to the parent -lactams . These studies support the notion that native siderophore platforms provide an effective means to target molecular cargo to siderophore - utilizing organisms and to hijack siderophore uptake machinery to deliver cargos, including antibiotics, across the outer membrane permeability barrier of gram - negative microbes . Dimethylformamide (dmf) and dichloromethane (ch2cl2) were obtained from a vac solvent purification system (vacuum atmospheres). Anhydrous dimethyl sulfoxide (dmso) was purchased from sigma - aldrich and used as received . L - ent 1, the d - enantiomer of benzyl - protected ent - co2h 2, and the benzyl - protected ent - peg3-n33 were synthesized according to previously reported procedures . 11-azido-3,6,9-trioxaundecan-1-amine was purchased from fluka . All other chemicals and solvents were purchased from sigma - aldrich or alfa aesar in the highest available purity and used as received . Benzyl - protected ent - azide 3 (80 mg, 55 mol) and pentamethylbenzene (pmb, 147 mg, 990 mol) were dissolved in 5 ml of anhydrous ch2cl2 to give a light yellow solution . This solution was cooled to 78 c in an acetone / dry ice bath under n2, and bcl3 (660 l of 1 m solution in ch2cl2, 660 mol) was added slowly along the flask wall . After the solution was stirred for 1.5 h, dipea (300 l, 1.73 mmol) was added to the flask, followed by meoh (2 ml) to quench the reaction . The reaction was then warmed to room temperature, and the solvents were removed under reduced pressure . The resulting white solid was dissolved in 5:3 meoh/1,4-dioxane and purified by preparative hplc (33% b for 5 min and 3360% b over 20 min, 10 ml / min). The product eluted at 17 min and was lyophilized to yield compound 4 as white solid (13.9 mg, 28%). H nmr (dmso - d6, 500 mhz): 3.353.57 (16h, m), 4.384.41 (3h, m), 4.634.69 (3h, m), 4.894.96 (3h, m), 6.74 (2h, dd, j = 7.5, 8.0 hz), 6.97 (2h, d, j = 7.5 hz), 7.35 (2h, d, j = 8.0 hz), 7.46 (1h, s), 7.94 (1h, s), 8.338.35 (1h, m), 9.12 (2h, d, j = 6.0 hz), 9.29 (1h, d, j = 6.0 hz), 9.44 (2h, bs), 9.76 (1h, bs), 11.6 (2h, bs), 11.9 (1h, bs). C nmr (cdcl3, 125 mhz): 50.1, 51.5, 63.6, 69.1, 69.4, 69.8, 69.8, 69.9, 69.9, 115.3, 115.4, 115.4, 117.7, 118.5, 118.7, 119.4, 125.2, 145.9, 146.3, 148.7, 148.7, 150.8, 166.0, 168.4, 169.1, 169.6, 169.7 . Ir (kbr disk, cm): 3389, 2954, 2928, 2868, 2111, 1754, 1645, 1589, 1535, 1460, 1384, 1329, 1266, 1176, 1132, 1074, 992, 846 . 11-azido-3,6,9-trioxaundecan-1-amine (36 l, 181 mol) and d - bn6ent - cooh (2, 177 mg, 142 mol) were dissolved in 5 ml of dry ch2cl2 . Pyaop (147 mg, 283 mol) and dipea (98.5 l, 568 mol) were added to give a light yellow solution . The reaction was stirred for 4 h at room temperature and concentrated, and the crude product was purified by preparative tlc (50% etoac / ch2cl2) to afford 5 as white foam (159 mg, 77%). H nmr (dmso - d6, 500 mhz): 3.33 (2h, j = 5.2 hz), 3.623.69 (14h, m), 4.024.06 (3h, m), 4.154.18 (3h, m), 4.914.94 (3h, m), 5.045.21 (12h, m), 6.96 (1h, s), 7.117.45 (36h, m), 7.657.67 (2h, m), 7.857.85 (1h, m), 7.977.97 (1h, m), 8.508.54 (3h, m). C nmr (cdcl3, 125 mhz): 25.6, 29.5, 38.8, 40.0, 45.3, 51.3, 51.4, 63.9, 64.1, 69.8, 39.8, 70.0, 70.3, 70.4, 70.4, 71.2, 71.2, 76.3, 76.3, 116.8, 117.5, 120.4, 123.0, 124.3, 125.4, 126.1, 126.2, 127.6, 127.6, 127.9, 128.2, 128.3, 128.4, 128.4, 128.5, 128.5, 128.6, 128.6, 128.8, 128.8, 128.9, 129.0, 130.1, 135.4, 135.7, 135.9, 136.0, 136.1, 146.8, 146.9, 149.1, 151.6, 151.8, 164.3, 164.9, 164.9, 165.8, 168.9, 169.0, 169.1, 176.2 . Ir (kbr disk, cm): 3357, 3062, 3032, 2958, 2923, 2859, 2104, 1751, 1551, 1576, 1515, 1455, 1375, 1345, 1299, 1264, 1204, 1126, 1082, 1040, 1018, 957, 915, 854, 811 . Hrms (esi): compound 6 was synthesized from 5 (153 mg, 105 mol) following the same procedure as for compound 4 . The crude reaction was purified by preparative hplc (33% b for 5 min and 3360% b over 20 min, 10 ml / min). The product eluted at 17.0 min and was lyophilized to yield compound 6 as white solid (31 mg, 33%). H nmr (cdcl3, 500 mhz): 3.343.56 (16h, m), 4.394.41 (3h, m), 4.614.66 (3h, m), 4.884.94 (3h, m), 6.74 (2h, dd, j = 7.8, 7.8 hz), 6.96 (2h, d, j = 7.8 hz), 7.33 (2h, d, j = 7.8 hz), 7.44 (1h, s), 7.91 (1h, s), 8.318.33 (1h, m), 9.129.13 (2h, m), 9.279.28 (1h, m), 9.50 (2h, bs), 9.84 (1h, bs), 11.6 (2h, bs), 11.9 (1h, bs). C nmr (cdcl3, 125 mhz): 50.1, 51.5, 63.6, 69.1, 69.4, 69.8, 69.8, 69.9, 69.9, 115.3, 115.4, 115.4, 117.7, 118.5, 118.7, 119.4, 125.2, 145.9, 146.3, 148.7, 148.7, 150.8, 166.0, 168.4, 169.1, 169.6, 169.7 . Ir (kbr disk, cm): 3390, 2958, 2925, 2863, 2110, 1754, 1645, 1589, 1535, 1460, 1384, 1342, 1262, 1176, 1117, 1074, 841, 800 . Hrms (esi): [m+na]m / z calcd 936.2506, found 936.2512 . 5-hexynoic acid (113 l, 1.00 mmol) and thionyl chloride (1.00 ml, 13.8 mmol) were combined and refluxed for 1 h. the reaction was cooled to room temperature and concentrated under reduced pressure, and the resulting crude acyl chloride was dissolved in acetone (0.5 ml) and carried on to the next step without purification . Ampicillin sodium salt (186 mg, 0.500 mmol) was dissolved in a solution of nahco3 (210 mg, 2.5 mmol) in 4:1 water / acetone (2.5 ml) and cooled on ice, to which the acyl chloride was added slowly with stirring . The reaction was subsequently warmed to room temperature and stirred for 1 h. water (3 ml) was added to the reaction, and the aqueous phase was washed with etoac (2 10 ml), acidified to ph 2 by addition of hcl, and extracted with etoac (20 ml). The resulting organic phase was washed with cold water (2 5 ml), dried over na2so4, and concentrated under reduced pressure . The crude reaction was triturated with hexanes, which afforded a yellow solid (180 mg, 77%). H nmr (dmso - d6, 500 mhz): 1.41 (3h, s), 1.55 (3h, s), 1.641.69 (2h, m), 2.132.16 (2h, m), 2.292.32 (2h, m), 2.772.78 (1h, m), 4.20 (1h, s), 5.39 (1h, d, j = 4.0 hz), 5.52 (1h, dd, j = 4.0, 8.0 hz), 5.70 (1h, d, j = 8.0 hz), 7.257.43 (5h, m), 8.57 (1h, d, j = 8.0 hz), 9.11 (1h, d, j = 8.0 hz). C nmr (dmso - d6, 125 mhz): 17.4, 24.4, 26.6, 30.4, 33.8, 55.5, 58.1, 63.7, 67.3, 70.3, 71.5, 84.2, 127.2, 127.6, 128.2, 138.2, 169.0, 170.2, 171.5, 173.5 . Ir (kbr disk, cm): 3297, 3058, 3023, 2970, 2937, 2863, 2626, 2526, 2120, 1780, 1688, 1518,1455, 1437, 1390, 1373, 1324, 1295, 1208, 1139, 1027, 1001, 843 . Compound 8 was synthesized as described for compound 7 except that amoxicillin (760 mg, 2.1 mmol) was used instead of ampicillin sodium salt . Compound 8 was obtained as light yellow solid (533 mg, 56%) after trituration and employed without further purification . H nmr (dmso - d6, 500 mhz): 1.42 (3h, s), 1.56 (3h, s), 1.631.68 (2h, m), 2.122.15 (2h, m), 2.252.29 (2h, m), 2.762.78 (1h, m), 4.19 (1h, s), 5.39 (1h, d, j = 4.0 hz), 5.515.54 (2h, m), 6.69 (2h, d, j = 8.5 hz), 7.19 (2h, d, j = 8.5 hz), 8.41 (1h, d, j = 8.0 hz), 8.93 (1h, d, j = 8.0 hz), 9.40 (1h, bs). C nmr (dmso - d6, 125 mhz): 17.6, 24.5, 26.8, 30.4, 34.0, 55.4, 58.2, 63.9, 71.6, 71.7, 84.4, 115.1, 128.4, 128.7, 128.8, 157.0, 169.2, 170.9, 171.7, 173.8 . Ir (kbr disk, cm): 3356, 3294, 3045, 2970, 2928, 1770, 1738, 1650, 1615, 1515, 1457, 1373, 1208, 1009, 945, 839, 815 . Hrms (esi): [m+na]m / z calcd 460.1537, found 460.1534 . Compound 7 (60 mg, 0.13 mmol) was dissolved in 1:1 h2o / mecn (5 ml), and tfa was added to a final concentration of 1% . The solution was incubated at 37 c for 24 h and purified by preparative hplc (2050% b over 25 min, 10 ml / min), which afforded a white powder (12 mg, 25%). The white powder is a diastereomeric mixture of products, and no further separation was performed . H nmr (dmso - d6, 500 mhz): (mixture of two diastereomers) 1.221.23 (3h, pair of s), 1.541.58 (3h, pair of s), 1.621.68 (2h, m), 2.122.14 (2h, m), 2.282.30 (2h, m), 2.77 (1h, s), 3.183.24 (0.5h, m), 3.303.36 (0.5h, m), 3.443.49 (0.5h, m), 3.553.60 (0.5h, m), 3.92 (0.5h, s), 4.01 (0.5h, s), 4.67 (0.5h, dd, j = 6.7, 6.5 hz), 4.78 (0.5h, dd, j = 5.2, 5.2 hz), 5.455.48 (1h, m), 7.277.40 (5h, m), 8.508.61 (2h, m). C nmr (dmso - d6, 125 mhz): (mixture of two diastereomers) 18.1, 25.0, 27.6, 28.1, 28.4, 29.5, 34.4, 42.2, 56.8, 56.8, 72.1, 72.2, 84.8, 127.9, 128.2, 128.9, 139.2, 139.4, 171.2, 171.4, 172.0 . Ir (kbr disk, cm): 3297, 3071, 3041, 2967, 2938, 2535, 2124, 1734, 1653, 1527, 1456, 1427, 1375, 1299, 1199, 1137, 1070, 1027, 836 . Hrms (esi): compound 10 was synthesized as described for compound 9 except that compound 8 was used instead of 7 (60 mg, 0.13 mmol). The product was purified by preparative hplc (2050% b over 25 min, 10 ml / min), and obtained as white powder (14.5 mg, 24%). H nmr (dmso - d6, 500 mhz): (mixture of two diastereomers) 1.26 (3h, s), 1.551.59 (3h, pair of s), 1.611.67 (2h, m), 2.122.14 (2h, m), 2.242.27 (2h, m), 2.77 (1h, s), 3.203.26 (0.5h, m), 3.343.39 (0.5h, m), 3.423.47 (0.5h, m), 3.563.60 (0.5h, m), 4.03 (0.5h, s), 4.12 (0.5h, s), 4.68 (0.5h, dd, j = 6.5, 6.5 hz), 4.79 (0.5h, dd, j = 5.5, 5.5 hz), 5.285.31 (1h, m), 6.69 (2h, d, j = 8.5 hz), 7.17 (2h, d, j = 8.5 hz), 8.368.50 (3h, m). C nmr (dmso - d6, 125 mhz): (mixture of two diastereomers) 17.5, 24.3, 26.9, 27.5, 27.7, 28.8, 33.8, 41.4, 55.7, 55.8, 71.4, 71.6, 84.2, 114.8, 115.0, 128.5, 128.7, 128.8, 156.9, 158.3, 158.6, 171.2, 171.3 . Ir (kbr disk, cm): 3301, 3071, 3028, 2973, 2928, 2548, 2111, 1737, 1662, 1606, 1593, 1515, 1435, 1377, 1197, 1139, 837 . Hrms (esi): ampicillin - alkyne 7 (120 l of an 80 mm solution in dmso, 9.6 mol) and ent - peg3-n34 (250 l of a 13 mm solution in 1,4-dioxane, 3.3 mol) were combined, and 400 l of dmso was added . Cuso4 (100 l of a 90 mm solution in water, 9.0 mol) and tris[(1-benzyl-1h-1,2,3-triazol-4-yl)methyl]amine (tbta, 200 l of a 50 mm solution in dmso, 10 mol) were combined, and 100 l of dmso was added to give a blue - green solution, to which naasc (400 l of a 90 mm solution in water, 36.0 mol) was added . This solution became light yellow and was immediately added to the alkyne / azide solution . The reaction was shaken on a benchtop rotator for 2 h at room temperature, diluted by 3- to 4-fold with 1:1 mecn / water, centrifuged (13,000 rpm 10 min, 4 c), and purified by semi - preparative hplc (20% b for 5 min and 20%50% b over 11 min, 4 ml / min; 0.005% tfa was used in the solvent system to prevent decomposition of the -lactam). The hplc fractions containing 11 were collected manually and flash frozen in liquid n2 immediately after collection to prevent -lactam decomposition . H nmr (dmso - d6, 500 mhz): 1.40 (3h, s), 1.54 (3h, s), 1.781.81 (2h, m), 2.27 (2h, t, j = 6.8 hz), 2.58 (2h, t, j = 6.5 hz), 3.48 (12h, m), 3.76 (2h, s), 4.19 (1h, s), 4.384.44 (5h, m), 4.644.66 (3h, m), 4.914.92 (3h, m), 5.39 (1h, d, j = 3.5 hz), 5.515.52 (1h, m), 5.72 (1h, d, j = 7.5 hz), 6.74 (2h, dd, j = 7.8, 7.8 hz), 6.96 (2h, d, j = 7.5 hz), 7.267.35 (5h, m), 7.427.45 (3h, m), 7.81 (1h, s), 7.92 (1h, s), 8.33 (1h, s), 8.55 (1h, d, j = 7.5 hz), 9.12 (3h, d, j = 7.0 hz), 9.29 (1h, d, j = 6.5 hz), 9.42 (2h, bs), 9.74 (1h, s), 11.6 (2h, s), 11.9 (1h, bs), 13.35 (1h, bs). Hrms (esi): [m+na]m / z calcd 1379.4021, found 1379.4046 . Compound 12 was synthesized as described for 11 except that compound 8 was used instead of compound 7 . H nmr (dmso - d6, 500 mhz): 1.40 (3h, s), 1.54 (3h, s), 1.781.80 (2h, m), 2.23 (2h, t, j = 6.5 hz), 2.57 (2h, t, j = 6.5 hz), 3.47 (12h, m), 3.76 (2h, bs), 4.18 (1h, s), 4.394.43 (5h, m), 4.634.65 (3h, m), 4.90 (3h, bs), 5.38 (1h, s), 5.525.56 (2h, m), 6.68 (2h, d, j = 8.5 hz), 6.73 (2h, dd, j = 7.8, 7.8 hz), 6.96 (2h, d, j = 7.5), 7.19 (2h, d, j = 8.5 hz), 7.33 (2h, d, j = 7.5 hz), 7.44 (1h, s), 7.80 (1h, s), 7.92 (1h, s), 8.338.39 (2h, m), 8.94 (1h, d, j = 8.0 hz), 9.119.12 (2h, m), 9.29 (1h, bs), 9.389.43 (3h, m), 9.75 (1h, s), 11.6 (2h, bs), 11.9 (1h, bs). Compound 13 was synthesized as described for 11 except that compound 6 was used instead of compound 4 . Hrms (esi): [m+na]m / z calcd 1379.4021, found 1379.4022 . Compound 14 was synthesized as described for 12 except that compound 6 was used instead of compound 4 . Hrms (esi): [m+na]m / z calcd 1395.3970, found 1395.3995 . Compound 15 was synthesized as described for 11 except that compound 9 was used instead of compound 7 . Hrms (esi): [m+h]m / z calcd 1331.4409, found 1331.4389 . Compound 16 was synthesized as described for 12 except that compound 10 was used instead of compound 8 . Hrms (esi): [m+na]m / z calcd 1369.4177, found 1369.4191 . General microbiology materials and methods, including details of ent - amp / amx stock solution preparation and storage, are provided as supporting information . Overnight cultures of the bacterial strains (table s1) were prepared in 15-ml polypropylene tubes by inoculating 5 ml of lb media with the appropriate freezer stock . The overnight cultures were incubated at 37 c for 1618 h in a tabletop incubator shaker set at 150 rpm and housing a beaker of water . Each overnight culture was diluted 1:100 into 5 ml of fresh lb media containing 200 m 2,2-dipyridyl (dp) and incubated at 37 c with shaking at 150 rpm until od600 reached 0.6 . G / l) with or without 200 m dp to achieve an od600 value of 0.001 . A 90-l aliquot of the diluted culture was combined with a 10-l aliquot of a 10 solution of the antibiotic or ent - antibiotic conjugate in a 96-well plate, and the covered plate was wrapped in parafilm and incubated at 30 c with shaking at 150 rpm for 19 h in a tabletop incubator housing a beaker of water . Bacterial growth was determined by measuring od600 (end point analysis) using a biotek synergy ht plate reader . Each well condition was prepared in duplicate and at least three independent replicates using two different synthetic batches of each conjugate were conducted on different days . The resulting mean od600 values are reported, and the error bars are the standard error of the mean (sem) obtained from the independent replicates . These assays were performed with e. coli atcc 35218 and k. pneumoniae atcc 13883 following the general procedure except that sulbactam (sb) or potassium clavulanate (pc) were mixed with ampicillin or amoxicillin and the ent - amp or ent - amx conjugates, respectively . The molar ratios of the inhibitor/-lactam mixtures were sulbactam / amp or ent - amp, 1.5:1, and potassium clavulanate / amx or ent - amx, 0.9:1 . These assays were performed with e. coli k-12 and cft073 following the general procedure except that varying concentrations (1100 m) of synthetic l - ent were mixed with ent - amp / amx . This assay is based on a published protocol and was conducted with e. coli cft073 . Each overnight culture was serially diluted into m9 minimal medium to provide 1010 cfu / ml . Lipocalin 2 was diluted in pbs to a concentration of 10 m upon arrival, aliquoted, and stored at 20 c until use . A 90-l aliquot of the diluted culture was added to each well of a 96-well plate that contained varying concentrations of lipocalin 2, ent - amp, and ent, and the final volume was adjusted to 100 l with sterile pbs . The 96-well plate was incubated at 37 c for 24 h in a tabletop incubator set at 150 rpm, and bacterial growth was determined by measuring od600 using a plate reader . Each well condition was repeated at least three times independently on different days and with different batches of lipocalin 2 . The resulting mean od600 is reported, and the error bars are the sem . A 5-ml overnight culture of e. coli k-12 or cft073 was grown in lb (vide supra) and diluted 1:100 into 5 ml of fresh lb media containing 200 m dp, and this culture was incubated at 37 c with shaking at 150 rpm in a tabletop incubator housing a beaker of water until od600 reached 0.3 . The culture was centrifuged (3000 rpm 10 min, rt), and the resulting pellet was washed twice by resuspension in 50% mhb and centrifugation (3000 rpm 10 min, rt). The resulting pellet was resuspended in 50% mhb with or without dp, and the od600 was adjusted to 0.3 . A 90-l aliquot of the resulting culture was mixed with a 10-l aliquot of a 10 solution of amp / amx or the ent - amp / amx in a 96-well plate, which was covered, wrapped in parafilm, and incubated at 37 c with shaking at 150 rpm . The od600 values were recorded at t = 0, 1, 2, and 3 h by using a plate reader . In a parallel experiment, a 10-l aliquot of the culture was taken at t = 0, 1, 2, and 3 h, serially diluted by using sterile phosphate - buffered saline (pbs), and plated on lb - agar plates for colony counting (cfu / ml). The resulting mean od600 or cfu / ml is reported, and the error bars are the sem . A 5 ml overnight culture of each bacterial strain was grown in lb, diluted 1:100 into 5 ml of fresh lb media containing 200 m dp, and incubated at 37 c with shaking at 150 rpm in a tabletop incubator housing a beaker of water until od600 reached 0.6 . Each mid - log - phase culture was diluted to 10 cfu / ml in 50% mhb with or without 200 m dp . For experiments requiring a mixture of two species, a 1:1 mixture was prepared (10 cfu / ml for each strain) in 50% mhb with or without 200 m dp from the mid - log - phase cultures . To confirm cfu / ml of each culture, the single- and double - species cultures were serially diluted by using sterile pbs, and aliquots were plated on a chrom - uti plate (starter - culture plate). For each cell - killing experiment, a 90-l aliquot of each culture was combined with a 10-l aliquot of a 10 m solution of the antibiotic or ent - antibiotic conjugate in a 96-well plate, which was covered, wrapped in parafilm, and incubated at 30 c with shaking at 150 rpm for 19 h. bacterial growth was assayed both by measuring od600 using the plate reader and by plating on chrom - uti plates after serial dilution (assay plate). Each well condition was repeated at least three times independently on different days . The resulting mean od600 is reported, and the error bars are the sem . We aimed to harness our enterobactin - mediated cargo delivery strategy to enable the transport of toxic cargo across the outer membrane of e. coli . To address this goal, we linked the -lactam antibiotics ampicillin (amp) and amoxicillin (amx) to a monofunctionalized ent scaffold where ent is derivatized at the c5 position of one catechol ring via a flexible and stable peg3 linker . We selected amp and amx as antibacterial cargo for several reasons: these molecules are commercially available and amenable to synthetic modification, retain antibacterial activity when appropriately modified, possess periplasmic targets in gram - negative bacteria and must cross the outer membrane to be active against these species, and have relatively low molecular weights . We selected low - molecular - weight antibiotics because our prior studies of ent - mediated cargo transport indicated that the ent transport machinery of e. coli k-12 imports ent - cargo conjugates harboring relatively small cargos (e.g., cyclohexane, naphthalene, phenylmethylbenzene) to the cytosol readily, whereas large cargos (e.g., vancomycin) are not transported to the cytosol . Moreover, in prior studies, various -lactams including amp and amx have been linked to simple catechols and more complex catechol - containing siderophores or mimics thereof, which provides the opportunity to compare the outcomes obtained for different siderophore - inspired design strategies . In scheme 1, we present the syntheses of ent - amp 11 and ent - amx 12, which feature installation of alkyne - modified -lactam warheads onto ent - azide 4 via copper - catalyzed azide - alkyne cycloaddition (hereafter click reaction) in the final step . The catechol moieties of benzyl - protected ent - azide 3 were deprotected by using bcl3 at 78 c to achieve ent - azide 4 as a white powder in 28% yield following purification by reverse - phase preparative hplc . Catalytic hydrogenation using hydrogen gas and a pd / c catalyst is typically employed to deprotect ent catechols; however, we observed that amp / amx decompose under these conditions and poison the pd / c catalyst . Moreover, deprotection of the enterobactin catechols prior to installing the -lactams requires preservation of the azide moiety, and we therefore employed bcl3 for this reaction . Initial attempts at assembling ent - amp / amx using standard conditions for the copper - catalyzed click reactions with ent - azide 4 and the alkyne - modified -lactams 7/8, prepared by thionyl chloride coupling of 5-hexynoic acid to the amino group of amp / amx, failed because of copper - mediated -lactam decomposition . This problem was overcome by including the metal - ion chelator tris[(1-benzyl-1h-1,2,3-triazol-4-yl)methyl]amine (tbta) in the click reactions, and ent - amp and ent - amx were obtained as white powders in high purity and yields of 66% and 76%, respectively, following semi - preparative hplc purification . It was necessary to perform hplc purification with eluents containing only 0.005% tfa to prevent decomposition of the acid - sensitive -lactam moieties . This synthetic route was likewise employed to prepare the d - enantiomers of ent - amp / amx 13 and 14 (scheme 1). Deprotected ent - azide 4 enables alkyne - functionalized molecules to be covalently linked to ent via a click reaction, including molecules that are incompatible with reaction conditions required to deprotect the ent catechols . Moreover, the deprotected ent - azide may be employed to append ent to surfaces, other materials, or biomolecules harboring alkyne groups . Indeed, very few examples employing the copper(i)-catalyzed click reaction with fused -lactams are reported in the literature, and it is likely that this paucity stems from the fact that -lactams are incompatible with standard conditions for copper - catalyzed azide - alkyne cycloaddition . The conditions defined in this work employing tbta allow for copper - catalyzed triazole formation and preserve the -lactam warhead . In the absence of fe(iii), ent - amp / amx exhibit an absorption band centered at 316 nm resulting from catecholate absorption and the solutions are colorless (meoh, rt). Addition of 1.0 equiv of fe(iii) to methanolic solutions of ent - amp and ent - amx causes the solution to immediately change from colorless to purple - red, and a broad absorption feature in the 400700 nm range appears (figure s1), confirming that both ent - amp / amx readily chelate fe(iii). To ascertain whether ent - amp / amx provide antibacterial activity against e. coli, including pathogenic strains, we preformed antimicrobial activity assays using six strains (table s1, figures 2, s2s7). E. coli atcc 25922 is a laboratory susceptibility test strain originally obtained as a clinical isolate . Coli uti89 and cft073 are both pathogens of the human urinary tract (upec).e . Coli atcc 35401 (serotype o78:h11) is an enterotoxigenic (etec) strain that was isolated from human feces . E. coli atcc 43895 (serotype o157:h7) is an enterohemorrhagic (ehec) strain that was isolated from raw hamburger meat implicated in a hemorrhagic colitis outbreak . Both etec and ehec strains produce virulence factors and toxins and cause diarrhea in humans . All e. coli strains biosynthesize ent for iron acquisition and express the ent receptor fepa . Some e. coli strains employed in this work also have the capacity to produce and utilize salmochelins, c - glucoyslated ent derivatives . These molecules are produced by salmonella spp . And pathogenic e. coli strains for iron acquisition . The iroa gene cluster (irobcden) encodes proteins required for the biosynthesis and transport of salmochelins, and iron is the outer membrane receptor for salmochelins encoded by the iroa cluster . Studies with salmonella indicate that iron has the ability to transport ent as well as its glucosylated forms . Of the strains considered in this work, e. coli cft073 and uti89 harbor the iroa gene cluster . E. coli h9049 does not produce salmochelins, and a blast search using available e. coli genomes reveals that the e. coli 43985 genome does not contain the iroa cluster . The genome for e. coli atcc 25922 is unpublished; however, this strain is reported to be sensitive to lipocalin-2 (vide infra), which suggests that its genome does not encode the iroa cluster . Lastly, it should be noted that e. coli cft073 is celebrated for having redundant iron import machineries, and this strain also harbors the iha gene, which encodes the outer membrane ent receptor iha that is distinct from fepa . Antibacterial activity of ent - amp / amx against various e. coli strains that include human pathogens . All assays were performed in 50% mhb medium supplemented with 200 m dp to provide iron - limiting conditions (mean sem, n 3). The data for assays performed in the absence of dp are presented in figures s2s7 . We performed antibacterial activity assays using a 10-fold dilution series to compare the abilities of ent - amp / amx and unmodified amp / amx to kill e. coli (figures 2, s2s7). These assays were conducted in 50% mhb and in the absence or presence of 200 m dp . The latter growth conditions provide iron limitation and result in expression of the ent uptake machinery fepabcdg . Amp / amx exhibit minimum inhibitory concentration (mic) values of 10 m against these e. coli strains regardless of the presence of dp in the growth medium (figures 2, s2s7). All six e. coli strains are more susceptible to ent - amp / amx than amp / amx under conditions of iron limitation (figure 2). Based on the 10-fold dilution series, ent - amp / amx are 100-fold more potent against e. coli 25922, uti89, and h9049 under iron - limiting conditions . Although the mic values for 35401 and 43895 are only ca . 10-fold higher than for amp / amx in this assay, a significant reduction in growth is observed at 100 nm ent - amp / amx, whereas this concentration of amp / amx affords no growth inhibition . The enhanced sensitivity of e. coli cft073 to ent - amp / amx is remarkable . This strain exhibits the greatest sensitivity to ent - amp / amx, providing a 1000-fold decreased mic value (10 nm), and growth inhibition in the presence of 1 nm of the conjugate . Moreover, in the absence of dp, e. coli cft073 exhibits the greatest susceptibility to ent - amp / amx (figures s2s7). A noteworthy characteristic of cft073 is its multiple mechanisms for iron acquisition, and we hypothesize that the presence of multiple receptors that recognize and transport ent (fepa, iron, iha) contributes to this enhanced sensitivity . Cft073 and uti89 both express iron, which may indicate that iha is responsible for the enhanced susceptibility of cft073; however, we cannot rule out the possibility that the conserved receptors function differently or exhibit different expression levels depending on the strain . Moreover, other unappreciated mechanisms may contribute to the potent bactericidal action exhibited by ent - amp / amx against cft073 and other strains . To gain further insight into the mechanism of ent - amp / amx antibacterial action, we performed a series of experiments with the standard laboratory strain e. coli k-12 . We employed the same 10-fold dilution series to compare the activities of ent - amp / amx and amp / amx against k-12 . In the absence of dp, ent - amp / amx and amp / amx exhibit comparable mic values, with complete killing observed at 10 m . At lower concentrations, ent - amp / amx exhibit slightly greater antibacterial activity than unmodified amp / amx (figure 3a). This phenomenon is most evident at a conjugate / drug concentration of 1 m, where ent - amp / amx inhibit e. coli k-12 growth to varying degrees and amp / amx do not affect bacterial growth . Under conditions of iron limitation, a 100-fold reduction in mic value (10 m to 100 nm) for ent - amp / amx is observed, and ca . 50% growth inhibition occurs at 10 nm of each conjugate (figure 3b). These trends are comparable to those observed for e. coli atcc 25922, h9049, and uti89 (figures 2, s2, s3, and s5). When the antibacterial activity assay was performed using a 1:1 ratio of native l - ent and amp / amx, no reduction of amp / amx mic values was observed (figure 3c), which suggests the conjugation between ent and the -lactams is required for the enhanced bactericidal action . Moreover, treatment of e. coli k-12 with the iron - bound forms of ent - amp / amx, obtained by pre - incubating each conjugate with 1 equiv of ferric chloride, afforded the same mic values as observed for apo ent - amp / amx . These results indicate that the enhanced antibacterial activity does not result from iron chelation in the growth media (figure 3d). In total, the data obtained for e. coli k-12 as well as the six other e. coli strains demonstrate that the antibacterial activity of ent - amp / amx against e. coli k-12 is enhanced under conditions of iron limitation and support a model of ent - mediated delivery of antibacterial cargo to the e. coli periplasm . Antibacterial activity of ent - amp / amx against wild - type and mutant e. coli k-12 . (a, b) growth inhibition of e. coli k-12 by amp / amx and ent - amp / amx in the absence (a) and presence (b) of dp . (c) growth inhibition of e. coli k-12 treated with a 1:1 molar ratio of ent / amp and ent / amx . (d) growth inhibition of e. coli k-12 treated with ferric ent - amp / amx . (e g) growth inhibition of fepa- (e), fepc- (f), and fes- (g) by amp / amx and ent - amp / amx . (h, i) growth of e. coli k-12 in the presence of 1 m ent - amp / amx (conjugate) and mixtures of ent - amp / amx (1 m) and 1, 5, or 20 equiv of exogenous ent in the absence (h) and presence (i) of dp . The * * indicates od600 <0.01 . All assays were performed in 50% mhb medium with or without 200 m dp (see panels) (mean sem, the data for additional assays performed in the absence of dp are presented in figure s8 . To evaluate transport of ent - amp / amx into e. coli, we investigated the antimicrobial activity of ent - amp / amx and amp / amx against three single - gene knockout e. coli k-12 strains obtained from the keio collection, fepa-, fepc-, and fes- (figures 3e we selected these mutants to ascertain how components of the enterobactin transport and processing machinery contribute to ent - amp / amx antibacterial activity . E. colifepa- lacks the outer membrane ent receptor fepa that allows periplasmic delivery, fepc- lacks the atpase component of the inner membrane ent permease that transports ent into the cytosol, and e. colifes- lacks the cytoplasmic esterase fes responsible for hydrolysis of the ent macrolactone for iron release . On the basis of our studies with wild - type k-12 and other e. coli strains, we hypothesized that the activity of ent - amp / amx would be attenuated for the fepa- mutant . Moreover, we questioned whether loss of fepc or fes would modulate the antimicrobial activity . Overnight cultures of fepa- and fepc- reached od600 values (0.15) similar to that observed for wild - type k-12 when grown in 50% mhb supplemented with 200 m dp . In contrast, the fes- strain exhibited a severe growth defect under these conditions (od600 0.04). Treatment of fepa- with ent - amp / amx afforded the same mic values as for amp / amx (figure 3e) and hence a 100-fold reduction in activity as compared to wild - type k-12 . Iron deprivation is deleterious to e. coli, and we contend that the growth inhibition observed at 10 m ent - amp / amx results from iron starvation rather than an antibacterial activity of the amp / amx cargo . Analysis of 50% mhb by inductively coupled plasma optical emission spectroscopy (icp - oes) revealed a total iron concentration of ca . 4 m (table s2), which can be compared to 10 m of a high - affinity extracellular iron chelator . Indeed, we previously observed similar growth inhibition of fepa- with 10 m of an ent - vancomycin conjugate (extracellular iron chelation) and also 10 m d - ent (iron chelator that cannot be used for iron acquisition) under these growth conditions . These data confirm that fepa is essential for the potent antibacterial activity of ent - amp / amx against e. coli k-12 . In contrast to fepa-, the growth of fepc- and fes- was completely inhibited with 100 nm ent - amp / amx under iron - limiting conditions (figure 3f, g), comparable to what was observed for the wild - type strain . The targets of -lactam antibiotics are penicillin binding proteins (pbps), which are located in the periplasm of gram - negative bacteria . After crossing the outer membrane through fepa, ent - amp / amx enter the periplasm where covalent capture by the pbps presumably occurs . Thus, it is reasonable that the downstream ent transport and processing steps involving fepcdg and fes do not affect the antimicrobial activity of the conjugates if they are trapped in the periplasm as a result of pbp binding . Although it is possible that the ent uptake machinery (e.g., periplasmic binding protein fepb) competes with the pbps for ent - amp / amx, no improved antibacterial activities were observed for the fepc- and fes- mutants compared to wild - type k-12 . This observation indicates that ent - amp / amx bind to pbps and are trapped in the periplasm . It should be noted that the fepb- mutant, which lacks the periplasmic binding protein, was also considered in this work; however, this strain exhibited a severe growth defect and afforded inconsistent results . To probe interaction between fepa and ent - amp / amx, we performed growth inhibition assays employing mixtures of ent - amp / amx and varying concentrations of unmodified ent (figure 3h when these assays were performed under conditions of iron limitation, the presence of exogenous ent attenuated the antibacterial activity of ent - amp / amx . A 1:1 molar ratio of ent - amx / ent afforded an od600 value comparable to that of the untreated control, whereas higher equivalents of ent were required to block the antibacterial action of ent - amp . The origins of this difference are unclear and may indicate that the hydroxyl group of amx has a negative effect on the transport efficiency of the conjugate . In total, these ent addition assays suggest that competition for ent - amp / amx and ent occurs at the receptor(s) and that the conjugates are delivered into the bacteria via the same uptake machinery as ent . L - serine is a biosynthetic building block for ent, and a role for chiral recognition in ent transport has been probed in prior studies . In one series of investigations, e. coli fepa was found to bind ferric l - ent and ferric d - ent with similar affinities (kd = 21 and 17 nm, respectively; ascertained by measuring the binding of fe - loaded siderophores to e. coli bn1071 cells). A lack of transport of ferric d - ent into e. coli bn1071 was also reported in this work . A later study probed ent uptake in bacillus subtilis, and transport of both l- and d - ent analogues was observed to occur with similar efficiency . 2457 t to hydrolyze l - ent and d - ent was evaluated, and fes did not accept d - ent as a substrate . As a result of the transport studies in b. subtilis and enzymatic activity assays with s. flexneri fes, a model in which both ent enantiomers are transported and chiral taken together, these studies suggest that the ability to transport d - ent may vary between species and even between strains of a given species, and more studies are required to address such possibilities . Based on the observation that e. coli fepa binds d - ent and that b. subtilis transports d - ent analogues, we synthesized the d - enantiomers of the ent--lactam conjugates (13 and 14, scheme 1) and evaluated the antibacterial activity of these conjugates against e. coli k-12, 25922, cft073, 35401, and 43895 (figures s9s13). D - ent - amp / amx exhibited reduced antibacterial activity relative to ent - amp / amx for k-12, 25922, cft073, and 43895 . Under conditions of iron limitation, complete growth inhibition was observed with 1 m d - ent - amp / amx for e. coli k-12, 25922, and 43895, compared to 100 nm for ent - amp / amx . Likewise, a 10-fold reduction in antibacterial activity was observed for e. coli cft073, where 100 nm d - ent was required to inhibit growth completely . In contrast, a negligible difference in antibacterial activity of the l- and d - isomers was observed for e. coli 35401 . Regardless of enantiomer, all four ent - amp / amx conjugates provide enhanced antibacterial activity against these e. coli strains relative to amp / amx . Nevertheless, these data suggest that d - ent - amp / amx are less readily transported into various e. coli strains than the l - isomers . Although this modification provides no appreciable benefit for this -lactam delivery system with periplasmic targets, it is possible that ent - antibiotic conjugates based on d - ent may be desirable for delivering cargos to the cytosol, precluding concomitant delivery of nutrient fe(iii). With support for ent - mediated delivery of ent - amp / amx to the e. coli periplasm, we sought to confirm the essentiality of the -lactam warheads in antibacterial action . We therefore designed and prepared hydrolyzed ent - amp / amx analogues 15 and 16 (scheme 2) where the -lactam structure is destroyed . Hydrolysis of the amp / amx - alkynes 7 and 8 was achieved in the presence of 1% tfa with heating at 37 c, and the decomposition products 9 and 10 were obtained as diastereomeric mixtures (scheme 2). The formation of these species followed the reported degradation pathways for ampicillin, where hydrolysis and subsequent decarboxylation occur . The diastereomeric mixtures were employed to prepare the hydrolyzed conjugates ent - hydro - amp 15 and ent - hydro - amx 16 via a copper - catalyzed click reaction . (a, b) antibacterial activity assays against e. coli k-12 (a) and cft073 (b) using ent - amp / amx and ent - hydro - amp / amx . (c) antibacterial activity assays against e. coli atcc 35218, which expresses a class a serine -lactamase, using ent - amp / amx in the absence and presence of the -lactamase inhibitors potassium clavulanate (pc) and sulbactam (sb). All assays were performed in 50% mhb supplemented with 200 m dp (mean sem, n 3). We employed ent - hydro - amp / amx in antibacterial activity assays against e. coli k-12 and observed negligible growth inhibition (figure 4a). When e. coli cft073 was treated with ent - hydro - amp / amx in the presence of dp this result indicates that ent - hydro - amp / amx are transported into the cytoplasm of e. coli cft073, where nutrient iron is released . We also performed a series of antibacterial activity assays with e. coli atcc 35218, a strain that expresses a class a serine -lactamase . Similar to unmodified amp / amx, ent - amp / amx were inactive against e. coli atcc 35218 (mic> 10 m) in the absence and presence of dp (figures 4c and s14). Slight growth inhibition was observed at 10 m under conditions of iron limitation, which may be attributed to iron chelation . The addition of -lactamase inhibitors restored the activities of ent - amp / amx and amp / amx, and the conjugates exhibited greater antibacterial activity than the parent antibiotics (figures 4c and s14). In total, the assays with ent - hydro - amp / amx and strains expressing -lactamase demonstrate that an intact -lactam is required for the antibacterial activity of ent - amp / amx . Moreover, these studies indicate that the -lactams retain their original function and inhibit pbps when conjugated to ent . The remarkable sensitivity of e. coli cft073 to ent - amp / amx (figure 2c) motivated us to investigate the relative cell - killing kinetics of ent - amp / amx and amp / amx to determine whether these conjugates kill e. coli cft073 more rapidly than the unmodified drugs . For comparison between e. coli strains, ent - amp / amx provide more rapid cell death than unmodified amp / amx (figure 5), and this behavior is most apparent for e. coli cft073, where the od600 value was almost reduced to the baseline value after 1 h incubation with 5 m ent - amp / amx, corresponding to a 2-fold log reduction in cfu / ml . In contrast, the change in od600 and cfu / ml for e. coli cft073 treated with 50 m unmodified amp / amx is negligible over this time period . The time - kill kinetics for e. coli k-12, conducted with 50 m of both unmodified and modified -lactams, indicate a slight increase in kill kinetics for ent - amp / amx relative to amp / amx, and that the kinetics of cell - killing are slower for k-12 than cft073 (figure 5). These results support a model whereby ent modification facilitates uptake of amp / amx relative to the unmodified drugs . This effect is more dramatic for e. coli cft073 than k-12, which is in accordance with the enhanced antibacterial activity observed for cft073 relative to the other e. coli strains considered in this work . Time - kill kinetic assays for treatment of e. coli k-12 (top panel) and cft073 (bottom panel) with amp / amx and ent - amp / amx . E. coli k-12 (10 cfu / ml) was treated with 50 m of amp / amx or 50 m ent - amp / amx . E. coli cft073 (10 cfu / ml) was treated with 50 m of amp / amx or 5 m ent - amp / amx . The assays were conducted in 50% mhb medium containing 200 m dp at 37 c (mean sem, n = 3). To determine whether ent - amp / amx exhibit broad - spectrum or species - selective activity, we performed antibacterial activity assays with two additional gram - negative and two gram - positive species in both the absence and presence of dp . These species include klebsiella pneumoniae atcc 13883, pseudomonas aeruginosa pao1, s. aureus atcc 25923, and bacillus cereus atcc 14579 . K. pneumoniae is a gram - negative species that biosynthesizes and utilizes ent for iron acquisition . P. aeruginosa is a gram - negative bacterium that captures ent as a xenosiderophore and expresses two ent receptors pfea and pira.s . Aureus and b. cereus are both gram - positive bacterial species, and the ability to utilize ferric ent as an iron source is reported for both species . In contrast to gram - negative bacteria, where the pbps are located in the periplasm, the targets of -lactam antibiotics are in the extracellular peptidoglycan of gram - positive organisms . K. pneumoniae atcc 13883 has a chromosomally encoded class a -lactamase (shv-1) and lacks sensitivity to amp / amx . We observed no effect of 100 m amp / amx on k. pneumoniae growth under our assay conditions (figure s15). Only 50% growth inhibition was observed when k. pneumoniae was treated with high concentrations (10 m) of ent - amp / amx in the absence of dp, and the ent - amp conjugate provided the greatest activity under conditions of iron limitation with 90% growth inhibition at 10 m . When -lactamase inhibitors were included in the assays, k. pneumoniae exhibited greater sensitivity to amp / amx (mic = 100 m) and ent - amp / amx (mic = 10 m); however, we observed some growth inhibitory activity of the -lactamase inhibitor sulbactam alone under these assay conditions (100 m sb, figure s15). Thus, the possibility of a synergistic effect from the inhibitors and conjugates cannot be ruled out completely . The lack of activity of ent - amp / amx against k. pneumoniae atcc 13883 is reminiscent of results obtained during investigations of amp / amx - functionalized tripodal triscatecholate ligands . These compounds were inactive against k. pneumoniae, and the behavior was attributed to either an inability of the k. pneumoniae iron transport machinery to import the conjugates or the development of resistance over the course of the assay . An alternative explanation is that -lactamase expression by k. pneumoniae resulted in inactivation of the -lactams . The p. aeruginosa pao1 strain employed in this work exhibited little sensitivity to both amp / amx and the conjugates under the antibacterial assay conditions (figure s16). Amp / amx exhibited no activity up to 100 m, whereas ent - amp / amx provided growth inhibition at 10 m in both the absence and presence of dp . Whether these results indicate that ent - amp / amx will be ineffective against multiple p. aeruginosa strains is unclear . P. aeruginosa strains exhibit different phenotypes, and highly variable and strain - dependent mic values have been reported for triscatecholate-lactam conjugates against p. aeruginosa . We previously reported that p. aeruginosa pao1 imports ent - cargo conjugates, and we speculate that the lack of activity observed for this strain stems from its inherent insensitivity to amp / amx . B. cereus atcc 14579 was also insensitive to amp / amx, which only afforded growth inhibition at 100 m . Some growth inhibition was observed for b. cereus treated with 10 m ent - amp / amx, which may result from iron sequestration (figure s17). S. aureus atcc 25923 is susceptible to amp / amx, with complete growth inhibition observed at 1 m . In this case, a 10-fold reduction in antibacterial activity was observed for ent - amp / amx relative to unmodified amp / amx (figure 6). Although the origins of this attenuation are unclear, we speculate that ent - amp / amx may have trouble penetrating the thick peptidoglycan of s. aureus . An alternative possibility is that recognition of ent - amp / amx by the s. aureus ent receptor diverts the -lactams from the pbps . In total, the results from these assays indicate that ent - amp / amx exhibit antibacterial activity enhancements that are species - selective, providing increased potency against e. coli strains and not for the other strains evaluated in this work . We therefore reasoned that ent - amp / amx, at low concentrations, should selectively kill e. coli in the presence of other less sensitive species . We treated co - cultures of e. coli cft073 and s. aureus with ent - amp / amx or amp / amx and analyzed the species composition following a 19-h incubation using harty - uti plates . These agar plates are employed in medical microbiology laboratories for the diagnosis of urinary tract infections and provide species identification by the colony color . When grown on harty - uti plates, s. aureus are off - white and e. coli are purple - pink . In figure 6, we present representative images of the colonies that resulted from treating co - cultures of e. coli and s. aureus with amp / amx or ent - amp / amx . In the absence of antibiotic, the cultures provide a mixture of off - white and purple - pink colonies, indicating that both e. coli and s. aureus grow when cultured together . When the co - cultures are treated with 1 m amp / amx, only purple - pink colonies are present, which reveals that only e. coli survives . In contrast, treatment of the co - cultures with 1 m ent - amp / amx results in only off - white colonies from s. aureus . These comparisons demonstrate that ent - amp / amx selectively kill e. coli in the presence of s. aureus and that the siderophore modification reverses the inherent species selectivity of the parent antibiotics . Achieving such species - selective and single - pathogen antibiotic targeting is an important goal and unmet need for pharmaceutical development that will allow for treating disease with minimal perturbation to the commensal microbiota . Ent - amp / amx selectively kill e. coli cft073 in the presence of s. aureus atcc 25923 . (a, b) antimicrobial activity assays against s. aureus atcc 25923 in the absence (a) and presence (b) of 200 m dp . (c, d) bacterial growth monitored by od600 for cultures of e. coli only, s. aureus only, and 1:1 e. coli / s . Aureus mixtures treated with amp / amx or ent - amp / amx in the absence (c) and presence (d) of 200 m dp . (e) representative photographs of colonies from mixed cultures of e. coli cft073 and s. aureus atcc 29523 treated with ent - amp / amx (1 m) or amp / amx (1 m) in the presence of 200 m dp . All assays were conducted in 50% mhb medium (t = 19 h, 30 c) (mean sem, n 3 for a d). We evaluated the cytotoxicity of ent - amp against the human t84 colon epithelial cell line . Cell survival was evaluated by mtt assay after a 24 h treatment with apo or iron - bound ent - amp, amp, or ent . The iron - bound forms were assayed to determine whether iron chelation in the growth medium is a factor . No cytoxicity was observed for amp or ent - amp, whereas apo ent itself decreased the survival of t84 cells by approximately 30% at the highest concentration evaluated . When pre - loaded with fe(iii), percent cell survival quantified by mtt assay after a 24 h treatment with apo or iron - bound ent, ent - amp, and the parent antibiotic amp in the absence and presence of 1 equiv of fe(iii) (mean sem, n = 3). Lipocalin-2 (lcn2, also known as siderocalin or ngal) is a 22-kda protein produced and released by neutrophils and epithelial cells . It has a hydrophobic binding pocket and coordinates ferric ent with sub - nanomolar affinity . By sequestering ferric ent, this host - defense protein contributes to the metal - withholding response and prevents bacterial acquisition of this essential nutrient . To determine whether lcn2 also binds ent - amp / amx and thereby blocks antibacterial activity, we performed antibacterial activity assays with e. coli cft073 in m9 minimal medium supplemented with lcn2 or bovine serum albumin (bsa). Under these conditions, up to 1 m of lcn2 had no effect on the growth of e. coli cft073 . Addition of 1 m lcn2 to the medium rescued the growth of e. coli cft073 treated with 100 nm of ent - amp (figure 8) whereas addition of 1 m bsa had no effect on ent - amp cell killing . These results suggest that lcn2 binds ent - amp and blocks its recognition and uptake . To ascertain whether lcn2 binds ent - amp in the presence of exogenous ent, we performed a series of experiments where e. coli cft073 were treated with fixed concentrations of ent - amp (100 nm) and lcn2 (1 m) and the concentration of ent was varied (0, 0.5, and 1 m). Moreover, when ent - amp was combined with a 1:1 molar ratio of ent and lcn2 at 10-fold excess over the conjugate, no e. coli growth was observed, which suggests that lcn2 preferentially binds ent . Prior work demonstrated that lcn2 cannot bind glucosylated ent, which was attributed to a steric clash between the glucose moieties and the ent binding site of the protein, and decreased hydrophobicity of the siderophore may also be a factor . Thus, our data suggest that the nature of linker attachment at c5 and the peg3 moiety of ent - amp / amx do no abrogate lcn2 binding as effectively as the glucose moieties exhibited by the salmochelins . Antibacterial activity of ent - amp against e. coli cft073 in the presence of lcn2 or bsa . (a) e. coli cft073 treated with 100 nm ent - amp and varying concentrations of lcn2 or bsa control . (b) e. coli cft073 treated with ent - amp, varying concentration of ent, and varying concentrations of lcn2 or bsa control . The assays were performed in m9 minimal medium (24 h, 37 c) (mean sem, n 3). Ent - amp / amx are two siderophore-lactam conjugates based on the native enterobactin scaffold . These molecules hijack siderophore - based iron uptake pathways and provide potent antibacterial activity against various e. coli strains, including human pathogens . Our investigations of ent - amp / amx establish the following: (i) ent - amp / amx provide up to 1000-fold enhanced antibacterial activity against e. coli strains; (ii) ent - amp / amx are transported into e. coli by fepa and potentially other catecholate siderophore receptors (iron, iha) employed by pathogenic ctf073 and uti98; (iii) ent - amp / amx are captured by pbps in the periplasm, which results in pbp inhibition and cell death; (iv) selective killing of e. coli in the presence of less susceptible organisms such as s. aureus is achieved because of the enterobactin scaffold; (v) ent - amp / amx exhibit negligible cytotoxicity to human t84 intestinal epithelial cells; and (vi) although lcn2 has the ability to bind ent - amp / amx, this siderophore - scavenging protein prefers to capture native ent . In total, these studies demonstrate that modification of antibiotic cargo with the native enterobactin platform provides many desirable features for antibiotic delivery and efficacy . The large molecular weight of the conjugates resulting from the native ent scaffold (as opposed to a smaller mimic) enhances rather than diminishes uptake for gram - negative e. coli . Moreover, we observed no evidence for the development of resistance to ent - amp / amx over the course of the antibacterial activity assays performed during these investigations . Our studies confirm that the enhanced antibacterial activity observed for ent - amp / amx requires both enterobactin recognition by outer membrane receptors and an intact -lactam moiety . The results are reminiscent of the dramatic antibacterial activity enhancements observed for albomycin, a secondary metabolite produced by actinomyces subtropicus . Albomycin is comprised of the siderophore ferrichrome and a trna synthetase inhibitor, and it exhibits antimicrobial activities that are 30,000-fold greater than those of the unmodified trna synthetase inhibitor against e. coli and s. aureus . Nonetheless, the ent - amp / amx cell - killing mechanism may be more complex than only more efficient -lactam delivery across the gram - negative outer membrane . Binding of ent - amp / amx to the pbps presumably results in accumulation of ferric enterobactin in the e. coli periplasm for some period of time, which may have deleterious consequences . A recent study of an e. colitolc- mutant revealed that enterobactin accumulation in the periplasm affords growth defects and abnormal cellular morphologies . A fascinating observation that stems from our current work is the variable susceptibilities and responses of different e. coli strains to ent - amp / amx, which contrast the effects of unmodified amp / amx . Such differences are manifest in the mic values to some degree and time - kill kinetics; however, the results presented in figure 2 indicate that mic values alone do not provide a full description of how ent - amp / amx susceptibility differs between e. coli strains . These results suggest underlying complexity in microbial physiology related to iron - uptake pathways that cannot be fully explained by the presence or absence of a gene for a particular receptor (i.e., fepa, iron). The heightened sensitivity of uropathogenic e. coli cft073 is particularly noteworthy, and it will be interesting to decipher the physiological origins of this effect as well as the differential behavior of various e. coli pathogens toward ent - antibiotic conjugates . There is a clear and unmet need for new antibacterial agents to treat bacterial infections in humans, including antibiotics that target specific bacterial sub - populations . Preventing undesirable consequences of antibiotic treatment on the commensal microbiota, which contributes to human health in beneficial ways, is a challenge that needs to be addressed . Such targeted therapeutics will be valuable not only for treating bacterial infections when the causative agent is known (e.g., urinary tract infection and e. coli, cystic fibrosis lung infection and p. aeruginosa) but also for other pathologies that involve microbial dysbiosis, such as irritable bowel disease . Our studies of ent - amp / amx provide one step toward addressing species - specific antibiotic targeting as well as overcoming gram - negative outer membrane permeability . From the standpoint of the host environment, commensal e. coli employ ent for acquiring iron in the host, and thus further elaboration of this strategy to specifically target pathogenicity and evade host responses (e.g., lcn2) is desirable.
Huntington's disease (hd) is a dominantly inherited neurodegenerative disease caused by mutations in the it-15 gene on chromosome 4, which was subsequently renamed htt . More specifically, the disease is linked to expansion of the cag repeat tract of this gene, which codes for polyglutamine repeats in the huntingtin protein . George huntington provided the classic description of the disorder in 1872 from cases in the east hampton area of long island, n.y . The best known manifestation is chorea, which consists of random, fluid involuntary hyperkinetic movements . The term is derived from the greek word o, meaning a traditional circle dance . It is important to note that, while movements in chorea may resemble a dance, they are not rhythmic or repetitive . Psychiatric manifestations may include irritability, anxiety, obsessive compulsive disorder, paranoid ideation, and psychosis . Depression is the most common psychiatric feature of hd and suicidal ideation occurs in 510% of hd patients . Other neurologic manifestations include eye movement abnormalities, cognitive disturbance, pendular knee reflexes, motor impersistence, and postural instability . We describe a mild case of adult onset hd early in the disease course along with mri and diffusion tensor imaging (dti) features . Increased fractional anisotropy (fa) of striatum the patient is a 50-year - old right - handed caucasian male who was diagnosed with hd at age 47 by an outside movement disorders specialist . The patient received a clinical diagnosis of hd given mild neurological findings in the context of genetically confirmed family history . There is a history of depression since his diagnosis, which has become worse over the past week . He was admitted for psychiatric evaluation due to suicidal ideation with plans to overdose on sleeping pills . In addition, he complains of mild cognitive disturbance for at least one year, with difficulty processing information in distracting environments . He has a history of alcohol abuse and quit alcoholics anonymous about six months ago . His medications include paroxetine 30 mg q.h.s ., aripiprazole 5 mg q.a.m ., naltrexone 50 mg q.a.m ., memantine 10 mg b.i.d ., and trazodone 200 mg q.h.s . Upon examination, he was alert and oriented to person, place, and time . Speech was fluent . Cranial nerve examination was significant for saccadic pursuit, slow saccade initiation and velocity in the horizontal and vertical directions, and motor impersistence exhibited by inability to fully protrude the tongue for five seconds . There was only mild intermittent chorea in the fingertips bilaterally and rare lower facial chorea . He improved after one week of inpatient psychotherapy and was released for outpatient psychiatric and neurologic follow - up . Images were obtained on a 3.0-tesla phillips achieva mri system . Bicaudate ratio (bcr), an imaging measure that correlates with caudate atrophy in hd, was obtained by measuring the shortest inter - caudate distance (seen as indentation of the frontal horns) on axial slice and dividing it by the distance between the outer tables of the skull along the same line, multiplied by 100 . Phillips fibertrak software was used to analyze dti values on regions of interest which were defined as caudate head bilaterally and putamen bilaterally on axial slices . 1). Axial bcr in the patient was increased compared to control at 16 versus 12, respectively . 2) was increased in the bilateral caudate and putamen versus control (table 1). Depression and suicidality were managed with inpatient psychiatry admission and psychotherapy, followed by outpatient therapy . Subtle neurologic manifestations included slowing of saccade imitation and velocity, saccadic pursuit, difficulty with tongue protrusion due to motor impersistence, pendular knee reflexes, and postural instability demonstrated by the inability to maintain tandem stance . Tetrabenazine is fda approved for the treatment of hd chorea and its efficacy is supported by clinical evidence . Caudate atrophy and ex vacuo dilatation of the frontal horns, which is typical for hd, was visualized . Increase in fa in white matter is linked to more coherent fiber orientation and normal structure, but in striatal grey matter increased fa is due to neurodegeneration . Propose that increased fa in striatal grey matter structures is due to the loss of efferent fibers which in turn leads to increased coherence . For example, in hd there is preferential loss of putamino - pallidal connections that are oriented mediolaterally as relative preservation of anterior - posterior and dorsal - ventral cortical - putaminal fibers . Hence, loss of these mediolateral fibers may lead to more uniform fiber orientation and higher fa values . In conclusion, we have presented a patient who is early in the course of huntington's disease and who already exhibits basal ganglia neurodegeneration with atrophy and changes in fa . Dr . Fekete received honoraria from medlink, inc ., and serves as a consultant for teva neuroscience, inc ., and lundbeck, llc.
Because motor weakness is one of the most serious disabling sequelae of stroke, so it is important to elucidate the motor recovery mechanism for successful rehabilitation . Many studies have attempted to elucidate the motor recovery mechanism of stroke and several motor recovery mechanisms have been suggested . However, the majority of these studies focus on cerebral infarct and relatively little is known about the motor recovery mechanism of intracerebral hemorrhage (ich). Diffusion tensor imaging (dti) can help to investigate the motor recovery mechanism of ich by enabling the direct visualization and estimation of the corticospinal tract (cst)., we report on a patient with ich who showed a change in the origin of an injured cst from the premotor cortex (pmc) to the primary motor cortex (m1). An 86-year - old woman presented with complete paralysis of the right upper and lower extremities, which occurred at the onset of spontaneous ich (motricity index (mi): 0 (full mark: 100)) (table 1). Longitudinal changes in motor function t2-weighted mr images showed a hematoma in the left fronto - parietal lobe, including m1, at 1 month after onset, that resolved at 5 months after onset (figure 1a). From 1 month to 5 months after onset, she received comprehensive rehabilitative management, which included the administration of neurotrophic drugs (ropinirole, bromocriptine, levodopa, and amantadine), movement therapy, and neuromuscular electrical stimulation of the affected finger extensors and ankle dorsiflexors . Movement therapy focused on improving the motor functions of the right upper and lower extremities and included physical and occupational therapy sessions five times per week . Weakness of her left extremities improved from an mi score of 0 point at 1 month to 27 points at 2 months and to 52 points (5 months after ich onset) after 4 months of rehabilitation . As a result, she was able to extend the affected fingers against gravity and to walk independently on even ground . Brain magnetic resonance images, diffusion tensor imaging (dti) and transcranial magnetic stimulation results of an 86-year - old female patient with a hematoma in the left fronto - parietal lobe . (a) t2-weighted magnetic resonance images showed a hematoma in the left fronto - parietal lobe including the primary motor cortex at 1 month after onset, which was resolved at 5 months after onset . Dti results showed that at 1month and 5 months after intracerebral hemorrhage, the right csts originated from the cerebral cortex, including the primary motor cortex, and then passed through the known cst pathway . In the left (affected) hemisphere, the csts originated from the left premotor cortex at 1 month after onset, and from the left primary motor cortex and premotor cortex at 5 months after onset . A motor evoked potential of low amplitude (100 v) was obtained from right abductor pollicis brevis muscle by the affected (left) hemisphere stimulation at 1 month after onset . However, motor evoked potential amplitude was increased to the normal range (3.8 mv) at 5 months after onset . Ich volume was measured on ct image and t2-weighted mri images through picture archived communication system (pacs, marotech, korea). The maximum width (x), length (y) and height (z) of lesion at the level where a hematoma was clearly seen were measured . The ich volume was calculated according to the formula: the volume of ich was 37.19 mv on the ct images at the ich onset and 26.71 mv on the mr images at 1 month after onset . Diffusion tensor images were acquired using a synergy - l sensitivity encoding (sense) head coil on a 1.5-t philips gyroscan intera system (hoffman - laroche, mijdrecht, the netherlands) using a single - shot echo planar imaging with a navigator echo . For each of the 32 noncollinear diffusion - sensitizing gradients, 60 contiguous slices (matrix = 192 192, field of vision = 240 mm, repetition time / echo time = 10 726/76 ms, b = 600 mm / s, number of excitations = 1, thickness = 2.5 mm) were acquired . Three - dimensional reconstructions of fiber tracts were obtained using the dti task card software (philips extended mr work space 2.6.3) (threshold fractional anisotropy (fa) = 0.15, angle = 45). Fiber tracts passing through both region of interests (cst portion of the anterior mid - pons and low - pons on the color map) were designated final tracts of interest . At 1 month and 5 months after ich onset, dti results showed that in the right (non - affected) hemisphere, the right cst originated from the cerebral cortex (including m1) and passed through the known cst pathway . However, in the left (affected) hemisphere, the cst originated from the left pmc at 1 month and from the left m1 and pmc at 5 months (figure 1b). Tms was performed using a magstim novametrix 200 magnetic stimulator equipped with a 9-cm mean diameter circular coil (novametrix medical systems inc, wallingford, ct, usa). The left hemisphere was stimulated by a counterclockwise current and the right hemisphere was stimulated by a clockwise current . Motor - evoked potentials (meps) were obtained from both abductor pollicis brevis muscles in a relaxed state . Excitatory threshold (et) was defined as the minimum stimulus required to elicit an mep with a peak - to - peak amplitude of 50 v or greater during two of four attempts . Each site was stimulated three times with inter - stimulus intervals of 10 seconds, and shortest latency and average peak to peak amplitude were estimated . Mep (latency: 23.5 ms; amplitude: 100 v; et: 100%) was evoked from the affected (left) hemisphere during the tms study conducted at 1 month after ich onset, but the amplitude increased to the normal range at 5 months after ich onset (latency: 19.7 ms; amplitude: 3.8 mv; et: 70%) (figure 1c). Ich volume was measured on ct image and t2-weighted mri images through picture archived communication system (pacs, marotech, korea). The maximum width (x), length (y) and height (z) of lesion at the level where a hematoma was clearly seen were measured . The ich volume was calculated according to the formula: the volume of ich was 37.19 mv on the ct images at the ich onset and 26.71 mv on the mr images at 1 month after onset . Diffusion tensor images were acquired using a synergy - l sensitivity encoding (sense) head coil on a 1.5-t philips gyroscan intera system (hoffman - laroche, mijdrecht, the netherlands) using a single - shot echo planar imaging with a navigator echo . For each of the 32 noncollinear diffusion - sensitizing gradients, 60 contiguous slices (matrix = 192 192, field of vision = 240 mm, repetition time / echo time = 10 726/76 ms, b = 600 mm / s, number of excitations = 1, thickness = 2.5 mm) were acquired . Three - dimensional reconstructions of fiber tracts were obtained using the dti task card software (philips extended mr work space 2.6.3) (threshold fractional anisotropy (fa) = 0.15, angle = 45). Fiber tracts passing through both region of interests (cst portion of the anterior mid - pons and low - pons on the color map) were designated final tracts of interest . At 1 month and 5 months after ich onset, dti results showed that in the right (non - affected) hemisphere, the right cst originated from the cerebral cortex (including m1) and passed through the known cst pathway . However, in the left (affected) hemisphere, the cst originated from the left pmc at 1 month and from the left m1 and pmc at 5 months (figure 1b). Tms was performed using a magstim novametrix 200 magnetic stimulator equipped with a 9-cm mean diameter circular coil (novametrix medical systems inc, wallingford, ct, usa). The left hemisphere was stimulated by a counterclockwise current and the right hemisphere was stimulated by a clockwise current . Motor - evoked potentials (meps) were obtained from both abductor pollicis brevis muscles in a relaxed state . Excitatory threshold (et) was defined as the minimum stimulus required to elicit an mep with a peak - to - peak amplitude of 50 v or greater during two of four attempts . Each site was stimulated three times with inter - stimulus intervals of 10 seconds, and shortest latency and average peak to peak amplitude were estimated . Mep (latency: 23.5 ms; amplitude: 100 v; et: 100%) was evoked from the affected (left) hemisphere during the tms study conducted at 1 month after ich onset, but the amplitude increased to the normal range at 5 months after ich onset (latency: 19.7 ms; amplitude: 3.8 mv; et: 70%) (figure 1c). In this patient, we investigated changes of the injured left cst and found that the injured cst seemed to recover as detected by dti, tms and clinical observation . At 1 month after ich onset, the left cst originated from the pmc by dti, and the mep obtained at the right hand showed low amplitude and a latency compatible with that of the cst . By contrast, 5-month results revealed that the left cst originated from the left m1 by dti and that mep amplitude had improved to the normal range by tms . The m1 is the major origin of the cst, although the cst is known to originate from the extensive cerebral cortex, including the pmc, the primary somatosensory cortex, and the prefrontal cortex . On the other hand, consequently, the dti and tms results of this patient suggest that the severely injured left cst had recovered over 4 months in terms of its origin and fiber number . Furthermore, clinically, the finding that the patient was able to flex and extend against gravity at 5 months after ich onset provides additional evidence of left cst recovery . As a result, right extremity motor functions seemed to have been recovered due to reorganization of the injured left cst originating from the left pmc to left m1 . It is also possible that compression of the cst into the left pmc recovered in concert with hematoma resolution . For the motor recovery mechanism in ich, several dti based studies have described recovery of an injured cst or the contribution made to recovery by the contralateral unaffected cst . During the last 5 years, a number of serial dti studies have described the recovery of injured cst after ich . Jang et al demonstrated the recovery process of an injured cst in a patient with a subcortical ich by serial dti . The patient presented with complete paralysis of right extremities at onset, but over 16 months, motor functions of affected extremities slowly recovered to nearly normal . Furthermore, dtt showed that the origin of the cst had changed from the posterior parietal cortex at 1 month to the primary somatosensory cortex at 4 months and m1 at 16 months . The authors suggested that recovery of the origin of the damaged cst was due to a process of normalization from the parietal cortex to m1 . By contrast, our patient showed a change in the origin of the injured cst from the pmc to m1 . This study described changes of an injured cst that occurred in concert with motor recovery in a patient with ich . To the best of our knowledge, this is the first longitudinal study to demonstrate a change in the origin of an injured cst from the pmc to m1 in ich . Results from this study suggest a motor recovery mechanism of ich and the important implications regarding brain plasticity and brain rehabilitation after ich . However, this study is obviously limited by case numbers, and further complementary studies involving larger case numbers are warranted.
Colorectal cancer (crc) is one of most common cancers and leading causes of mortality in the usa, accounting for approximately 136,000 new cases and 50,000 deaths per year.1 for metastatic or unresectable crc, standard first- and second - line treatments typically involve a combination of cytotoxic chemotherapies (eg, folfiri [5- fluorouracil + oxaliplatin + irinotecan])2,3 and molecular targeted agents (eg, bevacizumab, cetuximab, panitumumab),46 which can help to improve progression - free survival and overall survival . However, due to the nature of the disease, many patients will progress through guideline - recommended standard regimens while maintaining a good performance status . Regorafenib, an oral multikinase inhibitor, which targets angiogenic, stromal, and oncogenic receptor tyrosine kinases, was approved by the us food and drug administration in 2012 for the treatment of patients with metastatic crc who have been previously treated with fluoropyrimidine-, oxaliplatin-, and irinotecan - based chemotherapy, an anti vascular endothelial growth factor (vegf) therapy and, if kras wild - type, an anti epidermal growth factor receptor (egfr) therapy.7 this approval was based on the correct study, which demonstrated a median overall survival of 6.4 months with regorafenib vs 5.0 months with placebo (hazard ratio, 0.77; 95% confidence interval, 0.640.94; p=0.0052).8 among the most common adverse effects were fatigue, hand a case of an elderly patient on regorafenib who achieved stable disease for over 11 months with strategic dose modifications was presented . No ethics approval was needed for the care of this patient as all treatments were in accordance with institutional best practices . Verbal consent to appear in this case study was given by the patient to dr seery . A 73-year - old indian female with a past medical history of hypertension and hyper cholesterolemia the patient had no history of smoking, alcohol consumption, or any family history of cancer . The patient initially had symptoms of hemoptysis for approximately 2 weeks and then subsequently developed hematochezia for 3 days . Due to these symptoms, the patient underwent a colonoscopy, which revealed left - sided colon cancer . A left hemicolectomy was performed; pathology revealed a stage iiib well - differentiated, kras wild - type adenocarcinoma, with one out of 15 lymph nodes testing positive . The patient moved back to india and did not receive adjuvant chemotherapy due to patient preference . In march 2011, the patient returned to the usa with complaints of mild abdominal distention, and a subsequent positron emission tomography / computed tomography (pet / ct) scan showed evidence of recurrence, with ovarian and peritoneal metastases . The patient began treatment with capecitabine, oxaliplatin, and bevacizumab, and developed an anaphylactic reaction in the sixth cycle . She continued with single - agent capecitabine for an additional two cycles and completed therapy in june 2011 as there was no further evidence of disease on the pet / ct scans . The patient did not receive any additional therapy until early 2012 when she was restaged and was again noted to have diffuse metastatic disease in the peritoneum, with imaging consistent with moderate to extensive carcinomatosis . The patient received cetuximab and folfiri from february 2012 through august 2012 and achieved an excellent response, with the pet / ct scan showing no evidence of measurable disease, with some minimal hypodensities in the liver that were not pet avid . The patient did well until april 2013 when she experienced increased abdominal distention, bloating, and difficulty with defecation . The patient received a repeat ct scan that showed progression of the disease, with a large pleural - based mass in the right lower lobe with bilateral pleural effusions, ascites, and multiple peritoneal implants, as well as omental infiltration along with a possible l3 lytic lesion . Also, the patient s carcinoembryonic antigen (cea) values had increased to 23 . As a result, the patient was restarted on cetuximab and folfiri in may 2013; however, a pet / ct scan 2 months later revealed a mixed response, with persistent fluorodeoxyglucose - avid right pleural disease, but also some areas of decreased activity with stable nodal disease in the left axilla, retroperitoneum, and pelvis, and increased activity in the left inguinal and right retrocrural nodes . The patient continued with cetuximab and folfiri until august 2013, and a repeat pet / ct scan in september 2013 revealed progressive disease in the right pleural mass along with increase in size of the retrocrural and left inguinal lymph nodes and liver (figure 1a). The patient was started on regorafenib 160 mg daily on september 12, 2013, but required a treatment interruption due to hypertension with consistent systolic blood pressure readings in the range of 170 mm hg and diastolic blood pressure in the range of 100 mm hg, as well as grade 3 fatigue during cycle 1 . The patient was started on antihypertensive medications, and regorafenib was held for 2 weeks to resolve these adverse events . The dose was then reduced again due to grade 3 fatigue, and the patient s dose was stabilized at 80 mg daily . A repeat pet / ct scan in february 2014 showed a mixed response, with increased activity in non - target lesions noted in multiple omental, peritoneal, and pelvic lesions, but decreased activity in target lesions, specifically the thoracic and liver lesions, with new activity in the right tenth rib (figure 1b). Specifically, a right pleural - based mass had decreased from 27.6 mm to 26.7 mm, and standardized uptake value decreased from 14.4 to 3.4 in conjunction with a decrease in disease measuring from 58.0 mm to 43.6 mm in the central pelvic mesentery superior extending to the uterine fundus . The patient s cea decreased to 3.7, while eastern cooperative oncology group (ecog) performance status improved from 2 to 1, and treatment was therefore continued at 80 mg daily . The patient s most recent pet / ct scan in august 2014 showed frank progression of disease, with an increase in number and activity of multiple metastatic pulmonary and hepatic lesions along with increased activity in omental and mesenteric implants . As of august 2015, the patient has been on regorafenib for 11 months with progression; however, her ecog performance status remains at 1, and she would like to continue taking the medication as she does not have any side effects and feels her quality of life has improved . Verbal consent was obtained from the patient prior to her departing our facility for hospice care . The patient s identification has been protected and any information which could potentially be used to reveal the identity of the patient has been removed . We reported on a case of an elderly woman with kras wild - type, refractory metastatic colon cancer who has received multiple chemotherapy regimens, most recently regorafenib for 11 months with minimal side effects and palliation of symptoms . The report shows that regorafenib can provide a decrease in tumor burden and improve quality of life with prolonged survival if an appropriate dose is used in terms of balancing side effects with benefits of symptom palliation . Moreover, the report shows that continuing therapy beyond disease progression is no longer an absolute contraindication . This case report additionally demonstrates that close and frequent follow - ups by the primary oncologist is needed in order to monitor side effects and to find the effective and tolerable dose . In the correct study, the frequency of adverse events was shown to peak during the first cycle and gradually taper off with subsequent cycles.8 this report suggests that regorafenib can be a viable option for patients who still have a good performance status and have progressed through other regimens . Importantly, patient and financial support services are in place through the reach program to help patients manage the cost of this therapy.
The treatment and surgical background of gynaecomastia has history of evolution over and above 60 years . In 1928, dufourmental described a semi circular intra - areolar incision which was later documented by webster in 1946, who recognised the fact that the conspicuous scarring left by older techniques is more embarrassing than the original condition . The categories were classified by simon in 1973 and later on, liposuction was introduced in 1983 . Ultrasound - assisted liposuction remains the accepted standard that gained much popularity for addressing dense adipose tissue for the management of relatively glandular or fibrous gynaecomastia . It is significant to mention the wide range of alternatives and advancements with minimally invasive approaches developed to eliminate the condition of gynaecomastia in a more refined way . It has been observed that liposuction technique alone has its limitation with removal of adipose tissue only and unable to address the problem of tough glandular component; therefore, combining liposuction with different kinds of direct incisions was introduced to deliver the fibrous architecture of the breast by many authors . A minimally invasive approach was advocated by some authors that consist of minimal access incisions in either peri - areolar, circum - areolar or trans - areolar region, while some proposed arthroscopic shavers to break down the fibrous capsule of the glandular tissues and deliver it with liposuction . But they have few drawbacks such as less access to the fibro - glandular tissue due to the minimal incision, visible unattractive scarring at nipple areola complex left after the peri - areolar incision as shown in figure 1, dermal necrosis and subsequent scarring due to arthroscopic shavers and poor results . Scarring left after peri - areolar incision the gynaecomastia surgery is basically sought for aesthetic purpose and such operation should not leave any tell - tale signs of the procedure . There had to be a solution for the smooth delivery of fibro glandular tissues without added scarring . This article presents an innovative approach that consists of a criss - cross trans - nipple incision to retrieve the fibro - glandular tissues following liposuction method for excellent outcomes . No additional scarring is visible, even on the operation table and even in a close up view right after the procedure with this trans - nipple approach . In all the cases, the trans - nipple incision is proved to give no signs of surgery over the nipple areola complex after a recovery period of three to six months following the surgical procedure . Between the duration of january 2012 to october 2013, 28 candidates suffering from various degrees of gynaecomastia (except grade-4, which definitively required skin excision) were surgically treated by this method . After proper consultation and examination, it was concluded that a surgical approach would be needed and all of them underwent the process of routine medical investigations for the anaesthesia fitness . Right before the procedure, markings were made to limit the boundaries of treatment [figure 2]. The procedure started with liposuction to retrieve adipose tissues through a tiny hole (approx 6 - 7 mm) at both sides of the chest using number 4 liposuction canullae . After performing thorough liposuction of the marked area, a small criss - cross incision was made right on the top of the nipple with the help of no-11 blade [figure 3]. Pre - operative marking of the treatment boundaries for liposuction the criss - cross incision at the centre of the nipple through a fine curved hemostat, the tough fibro - glandular tissues were delivered while creating slight pressure with thumb and forefinger (broad pinching) over the base of nipple areola area [figure 4a and 4b]. Only tough tethering bands, which obstructed the path, were cut with a fine curved scissors [figure 4c]. Care was taken to keep the point of scissors away from the dermis of areola . An amount of 3 - 5 mm tissue beneath the areolar skin was preserved to prevent any vascular complications [video 1]. Removal fibro - glandular tissues through the incision cutting the tethering band through scissors keeping tip away from areolar skin to preserve at least 3 - 5 mm tissue beneath areolar skin the incision was then closed with 4 - 0 monocryl in a single purse - string suture manner [figure 5]. The liposuction incisions were left open to drain any collection of fluid without any freshening of margins or stitches . The compression garment was applied on the operation table right after the procedure and was replaced with new compression garment on seventh day of the surgery when the patient was called for first follow up . All the patients were advised to do proper massage of the chest area after 7 days of procedure with any moisturiser of their choice and to wear the compression garment for the next three months following the surgery . Serial medical photographs - front, lateral and semi - lateral views were taken prior to the procedure, after three, six and twelve months from the surgery to assess the final outcomes . It was observed in a close view that there was no visible scar on the nipple areola complex even on the operation table after the surgery [figure 6] and on long - term follow - up of six months and one year [figures 7 and 8]. None of the 28 patients reported any alteration of sensation in the nipple areola region after 3 to 4 months . Trans - nipple removal of fibro - glandular tissue is an easy, innovative approach in gynaecomastia surgery that removes all the tough fibro - glandular tissue without leaving any visible scars over the nipple areola complex . It produces the most rewarding results and maximum satisfaction to the patient right from their procedure . Close - up view right after the surgery showing practically no disturbance of nipple - areola complex architecture example cases showing follow - up between six months to one year after surgery . See the close up view of practically scar less nipple - areola complex after recovery gynaecosmastia usually requires no attention, unless patient feels uncomfortable or embarrassed due to aesthetic reasons . Chances of having this condition can be same in a slender patient as well as in a healthy or over - weight patient . The difference is only the degree or severity that can be noticed differently in obese patients or who are relatively fat can develop significant visibility or enlargement . Though the root cause of this problem is unknown in most of the cases and commonly idiopathic, heredity, consumption of certain drugs, obesity or hormonal changes can be one of the various other reasons . Many techniques and refined approaches have been advocated through the years to address the issue that leave visible scars . Through the recent years, the surgical techniques that have been supported by many authors advocate liposuction or ultrasound - assisted liposuction alone or in combination with direct incision of the breast tissue through a peri - areolar incision or a pull - through technique, and various other methods have been advocated to eliminate remnant tough fibro - glandular tissue of gynaecomastia and achieve more aesthetic results . It is observed that gynaecomastia causes a great mental discomfort and impairs the self - confidence of the patient . They tend to over react and sometimes become extra - conscious about their condition and start to notice even a mild elevation which is left in most cases of liposuction alone methods . This slight elevation can be acceptable for normal people but becomes great matter of mental stress for the patients suffering from this problem . It has been noticed that slight bulges are left as remnant after doing liposuction and it is evident on operation table during the surgery that significant elevation is left after doing liposuction only [figure 9a]. This elevation is formed by the leftover fibro - glandular tissues that causes visible bulges and cannot be retrieved through only liposuction . That is why liposuction alone cannot serve the purpose as it has its limitations and is only ideal for removal of adipose tissue . In most of the cases where patients are more conscious even about a mild elevation, there remains a need of removal of this fibro - glandular tissue to achieve flat chest appearance [figure 9b]. The invasive approaches along with liposuction are able to address this issue but they leave some conspicuous scarring (peri - areolar or trans - areolar incisions) or loss of sensation of the nipple areola complex, compromised vascualrity of overlying skin and subsequent complication (arthroscopic shavers or modified liposuction shaver canullae), leading to inadequate outcomes . Per - operative view showing visible bulge below nipple - areola complex just after completing liposuction due to remaining tough fibro - glandular tissue per - operative view of same case showing flat chest just after removal of tough fibro - glandular tissues through trans - nipple approach we consider a trans - areolar incision to remove these tough glandular tissues to achieve flat chest and eliminate any elevation caused by fibro glandular component over the nipple areola complex [figure 9c]. Our technique is relatively simple and retrieves any volume of the tough fibro - glandular breast tissue without leaving any visible scars on the nipple areola complex, unlike peri - areolar incision or other invasive techniques . The tough fibro - glandular tissues are removed through a criss - cross incision over the centre of nipple right after performing liposuction of the marked area . The key to prevent any complication is to cut only the tethering band coming in way of delivery of fibro - glandular tissue and preservation of 3 - 5 mm tissue beneath the areolar skin . The technique offers the perfect control over nipple position as there are absolutely no chances to re - positioning or traction on the nipple due to subsequent scar that sometimes causes nipple malpositioning or inversion in peri - areolar incision technique . On table final result right after the procedure in our study, the presented patients had different categories of gynaecomastia and patient - a [figure 10a] delivered little amount of tough fibro - glandular component (approx 50 g) and while patient - b [figure 10b] delivered huge amount (approx 250 g). Both patients were operated with the same modality of treatment, i.e., liposuction for adiposal component and trans - nipple approach for removal of tough fibro - glandular tissue to treat their condition and discharged on the same day of their operation . Both were advised to do regular massage and wear compression garment for three to four months . They both enjoyed flat chest, very faint and thin scar of liposuction incision site and practically invisible scars over the nipple areola complex after 6 months of follow up . Example patients - a (before and after) with fewer amounts of fibro - glandular tissues example patient - b (before and after) with large amount of fibro - glandular tissues trans - nipple approach is an easy, innovative and practically excellent technique to remove any amount of the residual glandular fibrous tissue after liposuction without leaving any additional visible scars on the nipple - areola complex in gynaecomastia surgery and results in best aesthetic outcomes . All the 28 patients who were operated by this approach reported very satisfactory aesthetic outcomes after their recovery . The procedure offers a promising approach for all the patients of gynaecomastia, especially for the patients who want to keep secret of having any surgical procedure done to their chest.
The natural history of acute myeloid leukamia (aml) is typically unfavorable with rapid progression leading to death in the absence of specific therapy . Spontaneous remission (sr) is a rare but well - documented event with hundreds of cases published to date . Despite reported cases and progress of conventional cytogenetic and molecular biology, ever since the generalization and rapid start of induction chemotherapy after acute myeloid leukemia diagnosis, sr has been very rarely observed . In particular, aml with monocytic differentiation (aml m5) represents the largest subgroup of aml with spontaneous remission reported in the literature, with seven cases published since 1980 17 but the existence of a link between aml m5 and sr remains elusive . For the first time, we report the cases of three patients with similar genetic characteristics diagnosed with aml m5 according to the fab classification, who underwent rapid and transient sr . Aml diagnoses were established by conventional morphologic, histologic, cytochemical, immunophenotypic, and genetic criteria . Metaphases were obtained and analyzed using standard techniques of colchicine arrest, hypotonic treatment, and 3:1 v / v methanol / acetic acid fixation . Karyotypes were reported using the international system for human cytogenetic nomenclature, based on the analysis of at least 20 metaphases when possible . Dna was extracted from bone marrow using proteinase k, followed by salt and ethanol precipitation and stored at 20c in 10 mmol / l tris cl 1 mmol / l edta (ph 8) buffer . Pcr reactions were performed on 100 ng of dna using npm1-f (5- tgg ttc ctt aac cac att tct tt-3) and npm1-r (5- ttc cat aca tac tta aaa cca agc a-3) to exon 12 of npm1 ., two fragments were amplified using pp1-f: 5-tcgccatgccgggagaactctaac-3; pp1-r: 5-ctggtaagggaagaggccggccag-3 and pp2-f: 5-ccgctggtgatcaagcagga-3; pp2-r: 5-gggcaagcctcgagatcc-3 primers . Only the hotspot region was sequenced for flt3 (flt3-f: 5-ccaggaacgtgcttgtcacccac-3 and flt3-r: 5-tcaaaaatgcaccacagtgag-3). All the pcr products were visualized on 1.5% agarose gel electrophoresis after gel red staining . All pcr - amplified samples were purified by standard methods and sequenced on abi prism 3130 genetic analyzer (life technologies, carlsbad, ca). In january 1998 a 24-year - old female patient with no medical history presented with acute febrile polyarthralgia and fatigue . Physical examination showed reduced performance status (ecog =; 2) and high grade fever (40c) but no sign of organ infiltration and no lymphadenopathy . A full - body ct scan revealed no abnormalities . Upon admission, blood cell count showed moderate pancytopenia (hb 10 g / dl, platelets 45 g / l and wbc 2 bone marrow aspirate was very poor and showed 58% of blast cells with monocytic appearance and lobulated nuclei . The bone marrow biopsy found a blast infiltration by cells of monocytic morphology expressing myeloperoxidase (mpo). An empirical broad spectrum antibiotic therapy was started and led to prompt clinical improvement with fever defervescence, normalization of hemogram values and a complete sr documented by bone marrow examination (blasts <5%). No colony - stimulating factors were administered and the patient was able to return home . Second leukemia relapse occurred 1 year later with hypercellular bone marrow aspirate showing 80%, mpo negative, esterase - positive monoblastic cells . The patient then underwent stem cell transplant with a fully hla - matched unrelated donor and standard myeloablative regimen . Npm1 mutation analysis was performed on dna extracted from a fixed cytogenetic pellet and revealed a favorable npm1 mutation in this patient (c.861_862instgtc; identified in cosmic database as cosm20861). No mutations were found in either cebpa or flt3 (neither internal duplication (it d) nor mutations in tyrosine kinase domain (tkd)). A 33-year - old male patient with no medical history was admitted in january 2013 . He had presented fever, fatigue, moderate weight loss, and anorexia for 1 month before hand . G / l with 5% of blast cells), and normal platelet count (175 bone marrow aspirate showed reduced cellularity with 10.5% of mpo positive blast cells and 14.5% of monocytes with dysgranulopoiesis . A bone marrow biopsy confirmed aml m5a diagnosis according to the fab classification with flow cytometry revealing cd68 +, cd117 +, cd15 +, cd34, cd163, and mpo blast cells . The karyotype was normal, fish analysis showed no mll rearrangements, and molecular biology identified a type j npm1 mutation (an insertion of tatg, fig . 1) without flt3-itd, flt3-tkd, or cebpa mutation . Due to the patient's septic condition, antibiotic therapy with piperacillin / tazobactam was introduced and cytostatic drugs were not immediately started . Sr was observed in the following days with normalization of complete blood count (cbc) and clearance of blasts cells in the blood and bone marrow . Blast cell morphology, cytogenetics, and molecular biology were identical to that of first diagnosis . The pulmonary aspergillosis has resolved and the patient is still alive in complete hematological remission 17 months after three courses of high - dose cytarabine . A 74-year - old woman was hospitalized in our leukemia unit in january 2014 following the discovery of anemia and leukocytosis with monocytosis in the context of febrile pneumonia . This patient had a history of high blood pressure, diabetes, and high cholesterol . Cbc revealed a hemoglobin level of 8.8 g / dl, a white blood cell count of 21 g / l with 8% of neutrophils and 15 bone marrow aspirate displayed 14% of blasts comprising granular blasts and monoblasts, and 41% of promonocytes . The karyotype was normal, fish analysis identified no mll rearrangements, and molecular biology revealed a type a npm1 mutation (duplication of tctg, fig . 2) with neither flt3-itd nor flt3-tkd, nor cebpa mutation . Antibiotic therapy with amoxicillin / clavulanic acid was started for the pneumonia and we observed a leukemia sr with normalization of cbc and disappearance of bone marrow blasts (despite persistence of approximately 15% of dystrophic monocytes). Given the sr, the patient was allowed to return home . Flow cytometry performed on the bone marrow identified a cd13 +, cd33 +, cd117 +, cd64 +, and cd14 + blast population, in favor of a monocytic differentiation . Cytogenetics and molecular biology were identical to that of the initial diagnosis . Following aml type chemotherapy, electropherogram as generated by sanger sequencing showing type a npm1 exon 12 mutation in dna extracted from bone marrow . In january 1998 a 24-year - old female patient with no medical history presented with acute febrile polyarthralgia and fatigue . Physical examination showed reduced performance status (ecog =; 2) and high grade fever (40c) but no sign of organ infiltration and no lymphadenopathy . A full - body ct scan revealed no abnormalities . Upon admission, blood cell count showed moderate pancytopenia (hb 10 g / dl, platelets 45 g / l and wbc 2 bone marrow aspirate was very poor and showed 58% of blast cells with monocytic appearance and lobulated nuclei . The bone marrow biopsy found a blast infiltration by cells of monocytic morphology expressing myeloperoxidase (mpo). An empirical broad spectrum antibiotic therapy was started and led to prompt clinical improvement with fever defervescence, normalization of hemogram values and a complete sr documented by bone marrow examination (blasts <5%). No colony - stimulating factors were administered and the patient was able to return home . Second leukemia relapse occurred 1 year later with hypercellular bone marrow aspirate showing 80%, mpo negative, esterase - positive monoblastic cells . The patient then underwent stem cell transplant with a fully hla - matched unrelated donor and standard myeloablative regimen . Npm1 mutation analysis was performed on dna extracted from a fixed cytogenetic pellet and revealed a favorable npm1 mutation in this patient (c.861_862instgtc; identified in cosmic database as cosm20861). No mutations were found in either cebpa or flt3 (neither internal duplication (it d) nor mutations in tyrosine kinase domain (tkd)). A 33-year - old male patient with no medical history was admitted in january 2013 . He had presented fever, fatigue, moderate weight loss, and anorexia for 1 month before hand . G / l with 5% of blast cells), and normal platelet count (175 bone marrow aspirate showed reduced cellularity with 10.5% of mpo positive blast cells and 14.5% of monocytes with dysgranulopoiesis . A bone marrow biopsy confirmed aml m5a diagnosis according to the fab classification with flow cytometry revealing cd68 +, cd117 +, cd15 +, cd34, cd163, and mpo blast cells . The karyotype was normal, fish analysis showed no mll rearrangements, and molecular biology identified a type j npm1 mutation (an insertion of tatg, fig . 1) without flt3-itd, flt3-tkd, or cebpa mutation . Due to the patient's septic condition, antibiotic therapy with piperacillin / tazobactam was introduced and cytostatic drugs were not immediately started . Sr was observed in the following days with normalization of complete blood count (cbc) and clearance of blasts cells in the blood and bone marrow . Blast cell morphology, cytogenetics, and molecular biology were identical to that of first diagnosis . The pulmonary aspergillosis has resolved and the patient is still alive in complete hematological remission 17 months after three courses of high - dose cytarabine . A 74-year - old woman was hospitalized in our leukemia unit in january 2014 following the discovery of anemia and leukocytosis with monocytosis in the context of febrile pneumonia . This patient had a history of high blood pressure, diabetes, and high cholesterol . Cbc revealed a hemoglobin level of 8.8 g / dl, a white blood cell count of 21 g / l with 8% of neutrophils and 15 bone marrow aspirate displayed 14% of blasts comprising granular blasts and monoblasts, and 41% of promonocytes . The karyotype was normal, fish analysis identified no mll rearrangements, and molecular biology revealed a type a npm1 mutation (duplication of tctg, fig . Antibiotic therapy with amoxicillin / clavulanic acid was started for the pneumonia and we observed a leukemia sr with normalization of cbc and disappearance of bone marrow blasts (despite persistence of approximately 15% of dystrophic monocytes). Given the sr, the patient was allowed to return home . Flow cytometry performed on the bone marrow identified a cd13 +, cd33 +, cd117 +, cd64 +, and cd14 + blast population, in favor of a monocytic differentiation . Cytogenetics and molecular biology were identical to that of the initial diagnosis . Following aml type chemotherapy, the patient is alive, in good health and in cr . Electropherogram as generated by sanger sequencing showing type a npm1 exon 12 mutation in dna extracted from bone marrow . Spontaneous remission of acute myeloid leukemia is an extremely rare and almost always transient event, with a mean duration in the literature of 7.7 months (range 136) 8 . Since 1979, approximately thirty cases of aml sr have been reported 8,9, although the underlying mechanisms involved remain unclear . A potential role of bacterial and/or fungal infections and blood transfusions was suggested in sr occurrence 10 by triggering an immune and antileukemic response . Activation of the immune system and cytokines such as tumor necrosis factor (tnf), interferon gamma (ifn-), and interleukin-2 (il-2) released during infections, in conjunction with an increase in the antibody - mediated cytotoxicity of natural killer (nk) cells and cytotoxic activity of t lymphocytes and macrophages, may play a role in the occurrence of sr 1,2,11,12 . Indeed, immune response might be a potential mechanism of prolonged disease control: in 2012, the case of a german patient who presented aml with a 10-year - long sr was reported . In this patient, it was demonstrated in vitro that nk cells displayed cytotoxic activity against the k562 myeloid leukemia cell line with upregulation of cd107a in flow cytometry 13 . In addition, infection is often accompanied by a hypergammaglobulinemia, which has been associated with aml sr in some cases 13 . The potentially beneficial effect of systemic infections in aml led to preliminary trials using bacterial extract inoculations or vaccination programs, which did not show a significant effect on survival or occurrence of remission 14,15 . Our patients did not receive any blood transfusions but all presented with infections that responded to antibiotic treatment with normalization of cbc and occurrence of sr . Unfortunately, no blood cytokine monitoring was performed in our three patients . To date, most cases of aml sr have been described in patients with normal karyotypes 3,11 . To our knowledge, this is the first report of three cases of sr in aml m5 according to the fab classification with normal karyotype and npm1 mutations . Npm1-mutated aml represents a separate entity included in the aml who classification since 2008, with a favorable prognosis in patients with normal karyotype aml (aml - nk) in the absence of the flt3-itd mutation 16 . The npm1 mutation is the most common genetic alteration found in aml - nk and is present in approximately one - third of adult aml 17 . Npm1 mutations are located in the last exon (exon 12) and consist in duplication or insertion of small nucleotide sequences . More than 50 mutations have been reported to date, leading to a npm1 protein variant with modification of the last seven residues, as well as four additional residues 13,17,18 . Physiologically, npm1 encodes a nucleocytoplasmic shuttling protein (npm1wt) involved in the regulation of ribosome genesis and centrosome duplication 19 . Npm1 mutation is associated with the aberrant cytoplasmic localization of npm1, thereby disrupting its function and perturbing multiple cellular pathways 18,20 . The potential role of npm1 mutations in the occurrence of sr is not known but it has been previously reported that patients with aml - nk and mutated npm1 had a better response rate to induction chemotherapy 17,18 which could advocate for increased chemosensitivity of blast cells with npm1 mutation . Recent findings suggest that npm1 mutations are associated with increased sensitivity to oxidative stress and that mutant npm1 is a target of arsenic trioxide - mediated oxidative stress 21,22 . Systemic infections, usually described in cases of sr, are associated with exuberant activation of the immune system and high production of reactive oxygen species (ros). Thus, we hypothesize that the three cases of sr presented herein might be linked by the presence of mutant npm1, and therefore be more susceptible to oxidative stress than cases with wild - type npm1 . Moreover, we can speculate that there is a link between sr and ros that still need to be demonstrated . Sr is an exceptional phenomenon, often transient and relatively well documented but the underlying molecular mechanisms are still unknown . Npm1-mutated aml m5 patients who developed spontaneous remission after antibiotic treatment of febrile neutropenia present at diagnosis seem to form a subtype with favorable prognosis and chemo sensitive disease . A better understanding of this rare process could uncover new potential therapeutic targets for acute leukemia.
Rare cancers are not rare at all . Despite the relatively low incidence of each type of cancer, rare cancers combined account for 20%-30% of cancer cases and deaths . Every year, more than 500,000 people are diagnosed with a rare form of cancer in the european union, and more than 4 million are living with the diagnosis of rare cancer . Survival rates for rare cancers are lower than those for common cancers (5 years; 47% vs. 65%). Therefore, rare cancers pose a significant burden and should be recognized as a public health priority . Patients and their families, as well as clinicians, researchers, and policy makers involved in cancer care are faced with the profound challenges and difficulties imposed by these forms of cancer . In fact, it is estimated that thousands of patients with these forgotten cancers are paying a high price as a result of repeated misdiagnosis and receiving inappropriate treatment . Some may need to travel long distances in order to obtain the necessary treatment, while many struggle to find information about their disease and find their consultation time insufficient . Clinicians also face unique challenges resulting from the lack of sufficient evidence, and researchers face challenges from insufficient funding or low number of research subjects . From the perspective of policy makers, the disproportionate occurrence of such forms of cancer in younger and minority populations, unequal access to quality care, and geographic variation in survival rates create additional challenges in terms of health disparities . Several workshops held in the united states and the eu promoting epidemiological research on rare and understudied cancer are notable examples of recent progress in this area . Consequently, a multi - stakeholder initiative dedicated to improving rare cancer care was launched, and several recommendations were made by professional societies . These initiatives and recommendations reflect the joint effort of research communities, clinicians, policy makers, patients, and industry partners, and workshop attendees are often advocates and supporters of proposed recommendations and thus may not represent the views of the average clinician or researcher . In addition, several recommendations lack firm scientific evidence or support, and may be up for debate . For example, it remains unclear as to whether patients with rare cancers treated in high volume centers show better survival . In addition, there is some evidence that the public do not have a societal preference for treating rare diseases over common ones, implying that the degree of rarity of a disease does not justify prioritization (e.g., the special market access status of orphan drugs). Moreover, proponents assert that incentives promoting use of orphan drugs have resulted in market failures . Experts also disagree on whether current incentives for research focusing on rare diseases are adequate . To the best of our knowledge, there are no currently available quantitative data on the experiences and opinions of physicians involved in cancer care . Therefore, in this study involving nationally representative samples of oncologists, we sought to investigate perceived difficulties with regard to rare cancer care and potential solutions endorsed by oncologists . This study was part of a nationwide survey conducted in order to explore views regarding medical care and treatments among physicians involved in cancer care . Physicians in the national cancer center and 12 government - designated regional cancer centers across korea participated in the survey . The study was approved by the institutional review board of the national cancer center, korea . Of the 901 cancer care physicians invited to participate in the study, 680 agreed (75.5% participation rate) to do so and completed the study survey . Among them, those who directly see cancer patients for diagnosis and treatment were asked to answer questions regarding rare cancer issues, and 175 physicians who provided clinical support to oncologists (e.g., radiologists, pathologists, cardiologists, rehabilitation specialists, pain specialists, and psychiatrists) were excluded from the study . In addition, 30 oncologists who did not answer the questionnaire regarding rare cancer issues and 52 physicians who reported that they do not see rare cancer patients at all were also excluded from the study . Three additional oncologists were excluded from the analyses due to their high rates of missing responses (50% of items on rare cancer questions), leaving 420 oncologists comprising the study s eventual sample . We developed a questionnaire based on themes identified from various scientific and lay literature regarding rare cancer care [4,5,7,13 - 19]. Survey questions covered: (1) the proportion of rare cancer patients in participants practices, (2) personal experiences of difficulties in treatment of rare cancers, and (3) participants endorsements of potential solutions for improving rare cancer care (appendix 1). In our instructions to study participants, we addressed the current lack of a unified definition of rare cancer to ensure that they understood the survey s content: although there is presently no clear definition of rare cancer, it is commonly defined as a cancer with an incidence of 5 per 100,000 . Please answer the following questions, supposing that they do not apply to common cancers (e.g., cancers of the stomach, lung, liver, colon, breast, cervix, thyroid, prostate, gallbladder, and pancreas) but may apply to cancers with uncommon histologies in common cancer sites . Other issues related to rare cancer care that we explored through the survey included lack of clinical experience and need for referral, availability of approved drugs and need to promote off - label treatments, reimbursement issues, lack of research evidence and guidelines, research funding and clinical trials of pharmaceutical companies, support and incentives from the government, and the need for an (inter)national registry [4,16 - 18]. When answering the questions regarding difficulties faced by oncologists and solutions for improvement of care survey physicians were allowed to choose multiple answers that apply without limitation . In addition, a blank space was provided for them to share any personal experiences or opinions regarding rare cancer treatment . Data on participants age, sex, specialty, years from board certification, and patient volume (average number of outpatients per week) were also collected . Chi - square tests were performed for comparison of responses according to participants specialties and the proportions of rare cancer patients in their practices . Correlations between responses to the items were further explored to determine associations between perceived barriers and recommendations . 12.0 (statacorp, college station, tx), and a p <0.05 was considered statistically significant . This study was part of a nationwide survey conducted in order to explore views regarding medical care and treatments among physicians involved in cancer care . Physicians in the national cancer center and 12 government - designated regional cancer centers across korea participated in the survey . The study was approved by the institutional review board of the national cancer center, korea . Of the 901 cancer care physicians invited to participate in the study, 680 agreed (75.5% participation rate) to do so and completed the study survey . Among them, those who directly see cancer patients for diagnosis and treatment were asked to answer questions regarding rare cancer issues, and 175 physicians who provided clinical support to oncologists (e.g., radiologists, pathologists, cardiologists, rehabilitation specialists, pain specialists, and psychiatrists) were excluded from the study . In addition, 30 oncologists who did not answer the questionnaire regarding rare cancer issues and 52 physicians who reported that they do not see rare cancer patients at all were also excluded from the study . Three additional oncologists were excluded from the analyses due to their high rates of missing responses (50% of items on rare cancer questions), leaving 420 oncologists comprising the study s eventual sample . We developed a questionnaire based on themes identified from various scientific and lay literature regarding rare cancer care [4,5,7,13 - 19]. Survey questions covered: (1) the proportion of rare cancer patients in participants practices, (2) personal experiences of difficulties in treatment of rare cancers, and (3) participants endorsements of potential solutions for improving rare cancer care (appendix 1). In our instructions to study participants, we addressed the current lack of a unified definition of rare cancer to ensure that they understood the survey s content: although there is presently no clear definition of rare cancer, it is commonly defined as a cancer with an incidence of 5 per 100,000 . Please answer the following questions, supposing that they do not apply to common cancers (e.g., cancers of the stomach, lung, liver, colon, breast, cervix, thyroid, prostate, gallbladder, and pancreas) but may apply to cancers with uncommon histologies in common cancer sites . Other issues related to rare cancer care that we explored through the survey included lack of clinical experience and need for referral, availability of approved drugs and need to promote off - label treatments, reimbursement issues, lack of research evidence and guidelines, research funding and clinical trials of pharmaceutical companies, support and incentives from the government, and the need for an (inter)national registry [4,16 - 18]. When answering the questions regarding difficulties faced by oncologists and solutions for improvement of care survey physicians were allowed to choose multiple answers that apply without limitation . In addition, a blank space was provided for them to share any personal experiences or opinions regarding rare cancer treatment . Data on participants age, sex, specialty, years from board certification, and patient volume (average number of outpatients per week) were also collected . Chi - square tests were performed for comparison of responses according to participants specialties and the proportions of rare cancer patients in their practices . Correlations between responses to the items were further explored to determine associations between perceived barriers and recommendations . 12.0 (statacorp, college station, tx), and a p <0.05 was considered statistically significant . The mean age of the oncologists was 43.0 years, and the average number of years since board certification was 12.0 years . The sample comprised surgical oncologists (57.1%), medical oncologists (36.2%), and radiation oncologists (6.7%) (table 1). Overall, the proportions of rare cancer patients seen by the oncologists were <1% for 38.3% and 1%-5% for 39.1% . Less than a quarter of oncologists answered that rare cancer patients constituted> 5% of the patients in their practice, except for pediatric medical oncologists, 60% of whom noted that rare cancer patients comprised the majority of their patient population (table 2). Difficulties reported by more than half of the respondents included lack of standard treatment guidelines (65.7%), insufficient personal experience with rare cancer treatments (65.2%), and lack of evidence regarding rare cancer treatments (54.1%). Relatively little clinical experience was higher for those who rarely see rare cancer patients (p <0.001). Reimbursements for drug treatments (44.5%), and unavailability of sufficient approved treatment options (39.8%) were also commonly reported, particularly by medical oncologists (p <0.001). Less than 20% of participants felt that investments from pharmaceutical companies and clinical trials were lacking (18.2%), or that more research funding from the government was needed (17.1%), although oncologists who frequently saw rare cancer patients were more likely to endorse these measures as solutions for better rare cancer care (p <0.05) (table 3). More than half of our study participants noted the need for development of clinical practice guidelines (66.0%), and more flexible reimbursement guidelines for treatment of rare cancers (52.9%). More than 30% of respondents felt that the establishment of a national registry (47.4%), a referral system to high volume centers for accumulating treatment experience (35.5%), research incentives (33.8%), and government initiative and support for research in rare cancer (30.2%) would improve patient care . Only a small proportion of respondents endorsed solutions involving off - label treatments for rare cancers (21.0%) and legislation mandating budget allocations for development of drugs for treatment of rare cancers (13.1%) (table 4). The mean age of the oncologists was 43.0 years, and the average number of years since board certification was 12.0 years . The sample comprised surgical oncologists (57.1%), medical oncologists (36.2%), and radiation oncologists (6.7%) (table 1). Overall, the proportions of rare cancer patients seen by the oncologists were <1% for 38.3% and 1%-5% for 39.1% . Less than a quarter of oncologists answered that rare cancer patients constituted> 5% of the patients in their practice, except for pediatric medical oncologists, 60% of whom noted that rare cancer patients comprised the majority of their patient population (table 2). Difficulties reported by more than half of the respondents included lack of standard treatment guidelines (65.7%), insufficient personal experience with rare cancer treatments (65.2%), and lack of evidence regarding rare cancer treatments (54.1%). Relatively little clinical experience was higher for those who rarely see rare cancer patients (p <0.001). Reimbursements for drug treatments (44.5%), and unavailability of sufficient approved treatment options (39.8%) were also commonly reported, particularly by medical oncologists (p <0.001). Less than 20% of participants felt that investments from pharmaceutical companies and clinical trials were lacking (18.2%), or that more research funding from the government was needed (17.1%), although oncologists who frequently saw rare cancer patients were more likely to endorse these measures as solutions for better rare cancer care (p <0.05) (table 3). More than half of our study participants noted the need for development of clinical practice guidelines (66.0%), and more flexible reimbursement guidelines for treatment of rare cancers (52.9%). More than 30% of respondents felt that the establishment of a national registry (47.4%), a referral system to high volume centers for accumulating treatment experience (35.5%), research incentives (33.8%), and government initiative and support for research in rare cancer (30.2%) would improve patient care . Only a small proportion of respondents endorsed solutions involving off - label treatments for rare cancers (21.0%) and legislation mandating budget allocations for development of drugs for treatment of rare cancers (13.1%) (table 4). To the best of our knowledge, our study was the first to investigate the consensus among oncologists regarding rare cancer patient care . The strengths of our study include its nationally representative samples of oncologists with various specialties and experiences in treatment of rare cancers, which enabled a comparison of responses across subgroups . The lack of standard treatment guidelines was the most common difficulty in treating rare cancers noted by oncologists, regardless of their specialty . In addition, development of clinical practice guidelines for rare cancers was the most frequently endorsed item for improvement of rare cancer care . Agencies including the national comprehensive cancer network (nccn) and national institute for health and care excellence (nice) are publishing more clinical guidelines on cancers, which currently do not yet cover most rare cancers . The lack of established guidelines for rare cancer treatment often leaves oncologists with no clear direction in treating patients with rare cancers and in making treatment decisions based on empirical evidence . Indeed, the world health organization (who) and european society for medical oncology (esmo) have suggested implementing guidelines for medical and psychosocial care . Insufficient personal experience in rare cancer treatment was the second most common difficulty faced by oncologists, and was related to endorsement of referrals to high volume centers (=0.23, p <0.001). Centralization of diagnosis and multidisciplinary treatment at specialized centers have been advocated by professional societies, government bodies, and even insurers, who assume that such measures will improve outcomes . However, among our study participants, the endorsement of referrals to high volume centers was relatively low (35.5%), particularly among those who saw a higher proportion of rare cancer patients (p=0.02). In the blank space in which they could express their views and offer suggestions, several oncologists proposed that rare cancer treatments be administered in regional cancer centers rather than in centralized venues, and suggested the need for information sharing, registry establishment, and collaborative research . Further studies are warranted regarding the optimal degree of centralization in treatment of rare cancers, and the full potential of information technology / telemedicine as a viable alternative to centralization . Insufficient approved treatment options and issues pertaining to reimbursement were also frequently reported, both of which were found to be inter - related (=0.51, data not shown). These two barriers were also strongly related to their endorsement for encouragement of off - label treatment for rare cancer treatment and relief of reimbursement guidelines for rare cancer treatment (=0.20 - 0.45, p <0.001). Medical oncologists, whose main treatment modalities were anti - cancer drugs, were more likely to perceive these as being obstacles to quality rare cancer patient care and called for necessary improvements . Clinical trials on rare disease treatment are rarely conducted due to the lack of commercial incentives . When conducted, trials are likely to be underpowered due to a small number of available patients . Therefore, off - label anti - cancer drugs are often requested by patients or family caregivers, are justified or regarded as essential in certain conditions when there is compelling biological plausibility as to their efficacy, and were supported by professional guidelines and us reimbursement policies . In our study, while the need for flexible reimbursement guidelines was acknowledged by over half of the oncologists, only about 20% agreed with the idea of promoting off - label medication use . Such seemingly discrepant attitudes were also reported in a us survey; while 61% of oncologists expressed their belief that off - protocol treatment use should be discouraged among patients, only 31% agreed that such treatments should not be available . This implies that while oncologists prefer greater flexibility in cancer drug use / treatments, they also feel uncomfortable trying unproven therapies . In fact, professional oncology societies have called for timely production of standard medical compendia enlisting off - label uses judged to be legitimate . Insufficient research evidence was also among the most commonly perceived difficulties in rare cancer care . Rare cancers typically receive little scientific attention and have suffered from difficulties in patient accrual as well as underfunding due to being low in priority . Therefore, platforms for collaborative research and sustained funding mechanisms have been advocated, including registries or research networks at the national or multinational level, federal or government funding for rare disease research projects, and financial support from industry partners for clinical research or regulatory registration activities for specific rare diseases . Among such potential solutions, the development of a national rare cancer registry was supported by nearly half of the oncologists, implying that they agree with the idea that registries constitute key instruments increasing empirical evidence on rare diseases . In contrast, only a small proportion of oncologists mentioned insufficient research funding or clinical trials as difficulties . In addition, endorsement for incentives for rare cancer research and government funding was only moderate, and oncologists generally opposed the enactment of legislation mandating pharmaceutical companies allocate funds for rare cancer treatment . However, we found no negative free statements on the idea and found many others supporting such ideas . In addition, these solutions were often endorsed by oncologists who frequently saw rare cancer patients . Therefore, we supposed that the low endorsement rates reflected low personal needs for such measures rather than objections to these ideas . First, we did not provide specific lists of rare cancers, as definitions vary from one set of guidelines to another and can differ according to the determining criterion [1 - 3,9]. Therefore, the proportion of patients with rare cancers in their practice estimated by the oncologists may not be accurate . Second, the majority of survey respondents did not frequently see rare cancer patients, and thus might not have clear opinions regarding many issues in rare cancer care . However, in the real world they would be the first physicians whom patients with rare cancers see and therefore their opinions were critical in development of national policies and strategies . Third, the experiences of oncologists in treatment of rare cancers as well as their attitudes towards treatments would be specific to each country as these are largely determined by health care delivery and reimbursement systems . In summary, oncologists faced various difficulties in treatment of patients with rare cancers, including the lack of clinical practice guidelines and personal experience, lack of approved treatment options, and reimbursement issues . They were generally supportive of recent recommendations by multi - stakeholder initiatives as well as professional societies for development of clear clinical practice guidelines, flexible reimbursement guidelines, and a national rare cancer registry . However, there was only moderate endorsement for referrals to high - volume centers or encouragement of off - label treatments . Insights into the general attitudes of oncologists gained through our nationwide survey of representative samples would be helpful in development of clinical practices and public health policies in rare cancer treatment and research.
Many problems occur while we are trying to improve the quality of life and material wealth by the development of modern society . Among them, air pollution is a serious problem in western and developing countries . Long - term exposure to air pollution has a high correlation with the incidence of cardiovascular disease, respiratory diseases, and diabetes1,2,3, threatening health . In particular, air pollution is a major cause of respiratory diseases such as asthma and allergic diseases4, 5 and it has a negative effect on the healthy growth and development of growing children and adolescents . Another problem caused by the development of modern society is the reduction in physical activity across all generations throughout the world . The time spent in physical activity by children has decreased significantly compared to before6, 7 . The time spent using visual media and watching tv has increased8, 9, and physical inactivity due to excessive academic pressure and the influence of parents10, has increased obesity, incorrect posture, and muscle weakness . In addition, energy consumption has increased due to environmental, metabolic, and genetic factors11 . Thus, children living in modern society are showing gradually decreasing physical fitness . The change in the environment due to the development of modern society is a threat to the health of growing children . According to the korea education development institute, respiratory system abnormalities have been much higher than those other systems over the last three years . Reduced basic physical fitness and reduced pulmonary functions are seriously threating the physical development of korean elementary, middle, and high school students . Although the physical fitness and lung health of growing children and adolescents is threatened, some studies have reported a relationship between air pollution and pulmonary function12, 13 . However, there has been insufficient research on the relationship between exercise and lung function . Most studies on exercise have only suggested lung function is improved by regular exercise14, 15 . The present study is to our knowledge the first to investigate the relationship between basic physical fitness and lung function . The purpose of the present study was to determine the relationship between basic physical fitness and pulmonary function in healthy korean school students to enable us to present an alternative method for improving their pulmonary function . A total 240 healthy children and adolescents aged 617 years who lived in busan in korea were recruited for the present study . The participants had a body mass index (bmi) <25 kg / m, and no respiratory system abnormalities . The sample comprised 20 healthy students (10 boys and 10 girls) of each age from 6 to 17 . The participants who did not meet the bmi criteria or who showed significantly low pulmonary test values were excluded, and replacements were recruited in order to conduct the analysis with the same numbers of subjects . The participants were divided into the early period of elementary school (68 y), the late period of elementary school (911 y), the period of middle school (1214 y), and the period of high school (1517 y) in order to investigate the rapid changes in body composition and basic physical fitness with growth . All participants gave their informed consent and the experimental protocol was approved by the ethical committee of dong - a university . Body composition including height, weight, bmi, and body fat (%) was measured using a body composition analyzer venus 5.5 (jawon medical, korea) while subjects were fully relaxed . Muscle strength, muscle power, flexibility, and balance were measured to evaluate basic physical fitness . All participants were given a full explanation about the correct posture and procedure of each measurement . Hand - grip strength was evaluated using a grip - d grip strength dynamometer (takei, japan) with 0.1 kg accuracy of both the right and left hands . Flexibility was evaluated by measuring sit and reach using a helmas iii trunk forward flexion instrument (o2run, korea) with 0.1 cm accuracy . Muscle power was evaluated by measuring the sargent jump height using a helmas iii sargent jump instrument (o2run, korea) with 0.1 cm accuracy . Balance was evaluated by measuring the eyes - closed single - leg standing time using a helmas iii blind single - leg stand instrument (o2run, korea) with 0.1 sec accuracy, and the participants performed the test using their preferred leg . Pulmonary function tests were conducted under standard laboratory conditions (temperature: 2225 c, relative humidity: 5560%). All spirometric tests were conducted by the same technician to reduce inter - observer variability and to prevent the failure of the measurement due to the young age of the subjects . The participants were given sufficient explanation about the method and instrument use, and the tests were performed in a sitting position while wearing a nose clip . The forced vital capacity (fvc) and the forced expiratory volume in one second (fev1) were measured using a quark pft (cosmed, italy). All of the pulmonary tests were conducted following the standards presented by the american thoracic society / european respiratory society16 . The data were analyzed using the statistical package for the social sciences (spss version 22.00) and the results are presented as the mean standard deviation . The significance of differences between boys and girls were examined using the independent t - test . The relationships between body composition and pulmonary function, and basic physical fitness and pulmonary function were analyzed using simple linear regression analysis . Table 1table 1.the differences in body composition between groups (gender and age) and within group (n=240)groupsage (yrs)height (cm)weight (kg)bmi (kg / m)body fat (%) malefemalemalefemalemalefemalemalefemaleperiod of early elementary school6123.7 4.43121.6 4.9624.8 4.5622.9 3.4816.1 2.0415.4 1.748.9 3.0113.0 5.067127.7 4.66126.2 3.9728.8 4.3726.2 5.0717.6 2.2016.4 2.619.6 4.4216.5 3.618133.0 4.57133.9 5.4631.7 6.6730.8 5.4117.8 2.9117.2 2.4310.5 6.1717.9 3.461,2<31<2<31<3 1,2<3period of late elementary school9137.3 4.62139.6 5.7135.9 7.7634.8 6.7718.9 3.1817.6 2.2313.1 5.9617.8 4.5110143.9 5.80147.1 9.4245.2 11.9236.8 10.1121.6 4.1216.7 2.8818.2 7.8017.0 4.7611155.5 6.65154.5 2.8648.2 11.5946.8 6.1419.9 3.9919.6 2.2014.7 3.8221.4 5.171<2<31<2<3*1<3 1,2<3*period of middle school12164.9 6.43158.3 4.9149.3 10.8347.4 6.9519.5 2.9718.9 2.5913.1 8.4720.4 4.0813165.9 3.41159.1 3.3055.1 11.1250.8 6.4520.0 3.9320.3 2.1612.8 7.6021.0 5.8114169.6 5.17158.0 3.0864.9 10.9153.2 5.4421.7 1.7921.3 2.0716.8 3.9721.1 6.401,2<3period of high school15176.5 7.30164.1 3.8160.3 7.9258.6 2.2419.3 1.4921.8 1.5311.5 4.8126.1 2.9416174.3 5.63162.5 5.8057.6 3.5453.1 5.8720.6 0.5720.0 1.4514.7 3.0024.2 2.3617172.8 4.01163.1 5.0162.4 8.1857.0 5.3220.9 2.3821.4 1.5715.5 5.8326.1 2.06values represent means sd . Bmi: body mass index . : significant difference within group ( p<0.05, * * p<0.01, * * * p<0.001) shows the differences in body composition between groups (gender and age) and within groups . Height was significantly different in the early period of elementary school (boys: p<0.01, girls: p<0.001) and the late period of elementary school (p<0.001 for both boys and girls). Weight was significantly different in the periods of early and late elementary and middle school for boys (p<0.05), and in the periods of early and late elementary school for girls (p<0.01). : significant difference within group ( p<0.05, * * p<0.01, * * * p<0.001) table 2table 2.the differences in basic physical fitness between groups (gender and age) and within group (n=240)groupsage (yrs)basic physical fitnesspulmonary functionright hand grip (kg)left hand grip (kg)sit and reach (cm)sargent jump (cm) single - leg stance (sec)fvc (l)fev1 (l)boysperiod of early elementary school68.42.508.81.954.75.2823.23.808.46.071.360.321.300.20710.62.3610.52.678.05.8326.64.5615.911.371.730.451.540.4089.32.6010.92.594.44.8226.02.5910.97.181.840.351.720.44period of late elementary school912.51.4714.13.325.66.3528.13.6925.819.002.000.381.850.411017.82.7517.02.765.66.7230.15.6544.224.672.550.442.220.321119.14.1018.73.897.99.1738.56.5933.118.672.590.532.390.531<3**1<2,3 1,2<3*1<2,3 1<3period of middle school1220.97.5221.89.112.29.6238.55.5226.317.333.430.703.190.621326.64.3029.94.385.27.6141.26.5626.222.533.540.563.530.291436.69.3937.97.137.89.1145.35.7530.813.514.270.493.840.511<2,31,2<31,2<3period of high school1531.96.1232.67.117.05.6343.64.4131.425.804.180.654.010.571636.30.3538.50.8512.12.2543.50.5052.56.504.600.724.390.661736.83.0438.32.8611.59.5147.69.8236.422.784.120.703.850.65girlsperiod of early elementary school67.71.878.52.059.43.3122.14.1411.17.901.460.261.230.2178.81.589.21.9510.83.7825.96.0916.315.781.430.271.310.2789.31.829.33.456.13.7525.03.134.32.091.660.281.570.283<1period of late elementary school910.23.0310.93.226.46.2327.94.4223.917.791.830.411.580.311012.72.6813.84.009.88.5430.04.8329.621.722.040.591.930.531116.83.5317.43.9910.87.4030.48.6735.422.812.540.392.330.341<31,2<31<3 * 1<3period of middle school1218.92.5520.01.9313.48.5333.65.8512.65.732.630.442.490.371321.74.8322.64.8310.38.7329.82.5727.820.372.610.322.580.241420.12.7021.73.6311.78.3428.44.0337.422.332.510.572.300.50period of high school1523.62.4225.53.9910.911.3129.87.8023.319.162.780.372.680.371622.23.9324.14.0415.310.6629.64.1330.215.853.010.472.850.371720.23.2323.03.4110.311.7028.95.0524.76.582.920.212.810.16values represent means sd . Fvc: forced vital capacity, fev1: forced expiratory volume in 1 second . : significant difference within group (* p<0.05, * * p<0.01, * * * p<0.001) shows the differences in basic physical fitness and pulmonary function between and within groups . For boys, right and left hand - grip strength were significantly different in the late period of elementary school (right: p<0.001, left: <0.05) and middle school (p<0.01). The sargent jump height was only significantly different in the late period of elementary school (p<0.01) for boys . In the case of girls, right and left hand - grip strength significantly different in the late period of elementary school (p<0.01), and the sit and reach distance was significantly different in the early period of elementary school (p<0.01). For boys, fvc was significantly different in the late period of elementary school and middle school (p<0.05), and fev1 was significantly different only in the late period of elementary school (p<0.05), and for girls, fvc and fev1 were significantly different in the late period of elementary school (p<0.05). Fvc: forced vital capacity, fev1: forced expiratory volume in 1 second . : significant difference within group ( p<0.05, * * p<0.01, * * * p<0.001) table 3table 3.the results of simple linear regression analysis between pulmonary function and body compositionfvcfev1rrboysheight0.827*0.758weight0.6770.658bmi0.1680.144body fat (%) 0.048 0.037girlsheight0.756*0.764weight0.7280.733bmi0.4550.462body fat (%) 0.392*0.400 * * * p<0.05, * * p<0.01, * * * p<0.001 shows the results of the simple linear regression analysis that was performed for fvc and fev1 against height, weight, bmi, and body fat (%). For boys, fvc significantly correlated with height (r=0.827, p<0.001), weight (r=0.677, p<0.001), bmi (r=0.168, p<0.001), and percent body fat (r=0.048, p<0.05) in descending order . Fev1 significantly correlated with height (r=0.758, p<0.001), weight (r=0.658, p<0.001), and bmi (r=0.144, p<0.001); however, there was no significant correlation with percent body fat (r=0.037, p=0.077). In the case of girls, fvc significantly correlated with height (r=0.756, p<0.001), weight (r=0.728, p<0.001), bmi (r=0.455, p<0.001), and percent body fat (r=0.392, p<0.001) in descending order . Fev1 significantly correlated with height (r=0.764, p<0.001), weight (r=0.733, p<0.001), bmi (r=0.462, p<0.001), and percent body fat (r=0.400, p<0.001) in descending order . * p<0.05, * * p<0.01, * * * p<0.001 the results of the comparison between boys and girls revealed that height and weight were similarly correlated . However, bmi (fvc: 0.455 vs. 0.168, fev1: 0.462 vs. 0.144) and percent body fat (fvc: 0.392 vs. 0.048, fev1: 0.400 vs. 0.037) showed higher correlations with pulmonary function in girls than in boys . Table 4table 4.the results of simple linear regression analysis between pulmonary function and basic physical fitnessvariablefvcfev1rrboysright hand grip strength 0.774*0.794left hand grip strength 0.7470.762sit and reach distance 0.0420.039sargent jump height0.5730.584single - leg standing time 0.058 0.048girlsright hand grip strength 0.6190.652left hand grip strength 0.6070.641sit and reach distance 0.0200.015sargent jump height0.1290.121single - leg standing time 0.0180.020 p<0.05, * * p<0.01, * * * p<0.001 shows the results of the simple linear regression analysis that was performed for fvc and fev1 against right hand - grip strength, left hand - grip strength, sit and reach distance, sargent jump height, and single - leg standing time . For boys, both fvc and fev1 showed high correlations with right hand - grip strength (r=0.774, r=0.794, p<0.001), left hand - grip strength (r=0.747, r=0.762, p<0.001), sargent jump height (r=0.573, r=0.584, p<0.001), and single - leg standing time (r=0.058, r=0.048, p<0.05) in descending order . In the case of girls, both fvc and fev1 showed high correlations with right hand - grip strength (r=0.619, r=0.652, p<0.001), left hand - grip strength (r=0.607, r=0.641, p<0.001), and sargent jump height (r=0.129, r=0.121, p<0.01) in descending order, whereas the sit and reach distance and single - leg standing time showed no significant correlations . * p<0.05, * * p<0.01, * * * p<0.001 the results of boys and girls were similarly in descending order . However, basic physical fitness parameters were more highly correlated with pulmonary function for boys than for girls, especially the sargent jump height (fvc: 0.573 vs. 0.129, fev1: 0.584 vs. 0.121). The purpose of this study was to determine the relationship between basic physical fitness and pulmonary function in healthy korean school students, in order to present an alternative method for improving their pulmonary function . An investigation of the development of korean students was conducted by the ministry of education in 2014 . The results show that boys grow 56 cm per year from an average height of 121.5 cm in the first grade of elementary school . The biggest growth was found among 6th grade elementary school students and 1st grade middle school students . Girls grow around 6 cm per year from an average height of 120.3 cm in the first grade of elementary school . Their degree of growth decreases rapidly after 6th grade elementary school compared to boys . Body weight of boys showed the biggest differences between the first and second grades of middle school (5.9 kg), and between the 5th and 6th grades of elementary school among girls (5.5 kg). The results of the present study show that the height of both boys and girls rapidly increased in the elementary school period . However, weight dramatically increased in the period of elementary school and middle school among boys, and in the period of elementary school among girls . The growth of children and adolescents who participated in this study seems to be representative of korean children and adolescents since the results appear to be similar to the findings of the ministry of education . However, bmi was somewhat different since we selected healthy students, excluding obese children as subjects . Basic physical fitness is closely related to health . Especially, it has a negative correlation with the prevalence of obesity17, hypertension18, and cardiovascular disease19 . In this respect, the korean government has implemented the physical activity promotion system (paps) in order to systematically measure the physique and fitness, as well as to prescribe physical activity for individuals, highlighting the need of regular exercise . However, accurate measurement, evaluation, and prescription of exercise seem insufficient . In the present study, basic physical fitness was measured using the correct postures and methods by a fully trained technician . Muscle strength increased sharply in the late period of elementary and middle school in boys, and in the late period of elementary school in girls . In addition, muscle power of boys increased sharply only in the late period of elementary school . Pulmonary function was reported as being correlated with age20, 21, in addition, height, weight, area of body surface, percent body fat, smoking status, and residential environment also have effects on spirometry22,23,24,25 . Height is a factor positively influencing lung function at all ages26, whereas age and body fat mass sometimes have an inverse correlation in accordance with the age of subjects . The present study revealed that pulmonary function increased with age for subjects during the growth period . In addition, pulmonary function was influenced by height, weight, bmi, and percent body fat, in descending order . Height, area of body surface, and weight correlate with pulmonary function in descending order27, a result which is consistent with the results of our present study . Girls showed higher correlation than boys between percent body fat and pulmonary function in the present study . However, the percent body fat showed slight or no correlation with pulmonary function for boys, a result which differs from those of previous studies . Rossi et al.28 found that body fat and lung function showed an inverse correlation in obese female adults . Gonzalez - barcala et al.29 conducted a study of children aged 6 to 18 years whose bmi was under 30 kg / m . They suggested that lung function can be difference depending on individual differences . In the present study, subjects had normal weight and bmi <25 kg / m . Park et al.30 reported that it was difficult to explain the effects of bmi, percent body fat, and muscle mass on pulmonary function . Therefore, different results of pulmonary function could be occurred depending on the body composition of the individual subjects . Thus, aerobic exercise and resistance exercise are effective ways of improving lung function . Besides the present study revealed that pulmonary function is highly correlated with right hand - grip strength, left hand - grip strength, and sargent jump height in descending order . Therefore, exercises for muscle strength and power could be effective at improving school students pulmonary function . In summary, in order to improve the pulmonary function of growing children and adolescents, aerobic exercise and exercise programs to increase muscle strength and power are needed, and they would be especially effective if they were to begin in the late period of elementary school when the muscle strength and power of students are rapidly increasing.
The idea of a constitutively expressed death machinery in each cell has given way to the notion that survival factors repress this machinery and, if such factors are unavailable, cells default into death [1, 2, 3]. This theory is supported by findings showing that many forms of programmed cell death do not require mrna or protein synthesis . In fact, mrna and protein synthesis inhibitors can induce apoptosis, suggesting that in some cases transcriptional activity might actually impede cell death [4, 5]. To identify genes that are transcriptionally regulated in cells undergoing apoptosis by survival factor deprivation gene trapping involves introduction of a reporter gene into a random collection of chromosomal sites, including transcriptionally active regions . By selecting for gene expression, recombinants are obtained in which the reporter gene is fused to the regulatory elements of an endogenous gene . Transcripts generated by these fusions faithfully reflect the activity of a disrupted cellular gene and serve as a molecular tag to clone any gene linked to a specific function [6, 7]. To identify genes that are transiently expressed during a biological process, we developed a strategy, which makes use of the site - specific recombination system cre / loxp . By combining gene trap mutagenesis with site - specific recombination, it is possible to uncouple a trapped cellular promoter from a transduced reporter gene . This enables the recovery of recombinants even in the absence of an active cellular promoter and thus allows selection for integrations into transiently expressed genes [8, 9]. We employed this strategy to isolate genes induced in hematopoietic cells (fdcp1) undergoing apoptosis by growth factor withdrawal . Briefly, the interleukin-3 (il-3)-dependent hematopoietic cells (floxil3) expressing a reporter plasmid encoding hsv - thymidine - kinase, neomycin - phosphotransferase and murine il-3, were transduced with a retroviral gene trap vector carrying coding sequences for cre recombinase (cre) in the u3 region . Activation of cre expression from integrations into active genes resulted in a permanent switching between the selectable marker genes that converted the floxil3 cells to factor independence . Selection for autonomous growth yielded recombinants in which cre sequences in the u3 region were expressed from upstream cellular promoters . As the expression of the marker genes is independent of the trapped cellular promoter, genes could be identified that were transiently induced by il-3 withdrawal (figure 1). (a) u3cre gene trap activation from integrations in genes induced by il-3 withdrawal excises the tkneo fusion gene, which is flanked by loxp sites from the reporter plasmid pgklxtkneoil3 . This places the il-3 cdna immediately downstream of the pgk promoter and enables its expression . Cre, cre recombinase; pol ii, rna polymerase ii; pgk, phosphoglycerate kinase promoter; lx, loxp target sequence for cre recombinase; tkneo, hsv - thymidine - kinase / neomycin - phosphotransferase fusion gene; pa, bovine growth hormone polyadenylation sequence; il-3, murine interleukin-3 cdna . U3cre - infected floxil3 cells were first selected in g418 to eliminate integrations into constitutively expressed genes . The g418-resistant cells were then plated into semisolid (agar) cultures in the absence of il-3 . Analysis of several recombinants obtained by this method indicated that cells upregulate survival genes before committing to cell death . As a result, cells receiving a transient apoptotic stimulus exhibit improved survival in a growth factor deficient environment . From a floxil3 gene trap integration library consisting of approximately 2 10 unique proviral integrations, we have isolated 125 individual clones that converted to factor independence upon il-3 withdrawal . Cellular sequences adjacent to u3cre integration sites (gene trap sequence tags, gtsts) were obtained from 102 clones by inverse pcr or 5'race using cre - specific primers . Ninety clones yielded informative gtsts (> 30 nucleotides), of which 15 (17%) belonged to known genes, 10 (11%) matched single ests or full cdnas with unknown function and 65 (72%) had no match within the public databases (figure 2). Interestingly, the frequency of unknown genes recovered with this method was significantly higher than that obtained with a standard gene trap protocol and a similar gene trap vector . For example, only 782 (46%) of 1,687 gtsts recovered from embryonic stem (es) cells expressing a u3 geo gene trap virus were novel . This difference is consistent with the ability of the cre / loxp approach to enrich for transiently expressed genes that are difficult to isolate by standard methodology and therefore underrepresented in the nucleotide databases . Figure 3a shows the structure of the proviral integration sites in the 15 previously characterized genes . Like earlier studies involving retroviral gene trap vectors with a reporter gene in the u3 region of the long terminal repeat (ltr), most integrations occurred in or near 5' exons, although integrations further downstream were observed occasionally . To confirm transcriptional induction by il-3 withdrawal, polyadenylated rnas were isolated from fdcp1 cells after various intervals of factor deprivation and hybridized on northern blots to gene - specific probes . (a) gene trap position in previously characterized genes . Gtsts were either obtained by 5'race or inverse genomic pcr . Exon / intron boundaries were either available from the databases or were determined by rt - pcr reactions using mrna from fdcp1 cells and exon - specific primers . Polyadenylated rnas (5 g per lane) extracted from fdcp1 cells at various intervals following il-3 withdrawal were fractionated on formaldehyde - agarose gels, blotted onto nylon filters and hybridized to p -labeled gene- or exon (net1)-specific probes . As summarized in table 1, a significant number of the trapped known genes encode proteins involved in cell growth and survival . For example, the guanine nucleotide exchange factors (gefs) for the rho - family gtpases net1 and h - pem2 (kiaa0424) [10, 11] belong to a large family of proteins that regulate cell growth and can transform cells in culture . Transformation is dependent on the highly conserved dbl homology (dh) domain and in most cases requires amino - terminal truncation of the protein [13, 14]. Interestingly, a novel splice variant of net1, net1a, which was isolated in these experiments, encodes an amino - terminally truncated protein that is transforming in its native form (table 1). Thus, when overexpressed in nih3t3 fibroblasts, net1a but not net1 produces transformed foci with typical rho morphology (f.w . And h.v.m . In addition to the dh domain, rho - gefs have a pleckstrin homology domain that connects the rho signal transduction pathway to the phosphatidylinositol-3-phosphate kinase (pi3 kinase) signal transduction pathway . The latter is responsible for mediating survival signals, particularly in hematopoietic cells, and is directly activated by il-3 . Surprisingly, two members of this pathway, the phosphatidylinositol 4/5 phosphate kinases (pip5 kinases) type i (which corresponds to human pip5 kinase type i) and type i, were induced by il-3 withdrawal (table 1). Pip5 kinase converts the lipid phosphatidylinositol-4-phosphate to phosphatidylinositol-4,5-phosphate (pip2), which enhances cell survival by at least three mechanisms . Moreover, overexpression of pip5 kinase type i in cultured cells prevents apoptosis induced by caspase 9 or tumor necrosis factor- (tnf-). Second, pip2 is phosphorylated by pl3 kinase to give phosphatidylinositol 3,4,5 phosphate (pip3), which activates the survival kinase akt . Akt prevents cell death by phosphorylating and inactivating the proapoptotic proteins bad, caspase 9 and the forkhead family transcription factor fkhrl1, which induces the expression of fas ligand [17, 18, 19, 20]. Third, pip5 kinase activates atp - dependent potassium channels and thereby helps to maintain the polarity of the cell membrane . As shown previously, apoptotic signals can cause membrane depolarization, which leads to cell death by directly inhibiting potassium channels . Summary of previously characterized genes disrupted by gene trap mutagenesis another gene with a putative survival function encodes the cell - cycle - regulated protein p38 - 2g4 (ebp1). Although less well characterized than the above proteins, p38-g4 is absent from go cells and may stimulate cell - cycle progression [23, 24]. It includes the il-12 receptor, which inhibits fas - mediated apoptosis in certain types of t lymphocytes and the transcription factor yb-1, which represses fas gene expression . Interestingly, yb-1 also activates the expression of the multidrug resistance gene (mdr1) whose product, the p glycoprotein, is an efflux pump that eliminates a variety of toxins from the cell, including proapoptotic anticancer drugs . Similarly cytoprotective is vmat2, the protein product of the vesicular monoamine transporter gene, which moves cytoplasmic monoamine transmitters into secretory vesicles and prevents cell death by sequestering proapoptotic molecules such as mpp and histamine [28, 29]. Finally, il-3 withdrawal induced transcription of several dna repair genes . These were the mouse homologs of the bacterial alkb gene and of the yeast rec8 and rad50 genes . While alkb protects dna from damage by alkylating agents, rec8 and rad50 are dna double - strand break repair proteins [31, 32, 33]. Only a few genes upregulated by il-3 withdrawal (4 out of 15) were not directly related to cell death or survival and their induction may reflect some secondary events occurring during apoptosis (table 1). However, the low frequency with which such genes were recovered underscores the specificity of the gene trap approach . To further study transcriptional regulation during growth factor starvation, we analyzed the expression patterns of a large number of previously characterized genes in fdcp1 cells undergoing apoptosis by il-3 withdrawal using atlas mouse cdna arrays (clontech). Atlas arrays contain cdna probes for 588 genes involved in development, oncogenesis, dna repair, tumor suppression and apoptosis . Figure 4a shows that of the 588 genes displayed on the arrays, only 12 were differentially expressed after factor deprivation . Differential expression of these genes was reproducible in independent array experiments and could be confirmed by northern blotting (figure 4c). (a) comparative atlas array hybridization with cdna probes derived from fdcp1 cells after 0 h (left) and 2 h (right) of il-3 deprivation . Filters were scanned with a phosphoimager (molecular dynamics) and analyzed with the atlasimage 1.5 software . (b) relative changes in gene expression on a log scale . 1, no regulation;> 1, upregulation; <1, downregulation . Note that the apparent downregulation of bcl-2 could not be confirmed on northern blots . Induction of c - kit and chop10 (asterisked) was observed only on arrays hybridized to cdna recovered from cells after 8 h of factor deprivation . (c) northern blot analysis of transcripts identified as differentially expressed by the atlasimage 1.5 software . Poly(a) rna (2.5 g) prepared after 0, 2, 4, 6 and 8 h of il-3 deprivation was fractionated on formaldehyde gels, blotted onto nylon filters and hybridized with p - labeled gene - specific probes . Similar to the trapping results, several genes with survival functions were upregulated in the absence of il-3 . Among these were the survival kinase akt, the c - kit receptor tyrosine kinase, the potent inhibitor of apoptosis flipl, and the il-3-receptor itself [34, 35, 36]. Moreover, survival functions seemed to be strengthened by the transient downregulation of the proapoptotic activin receptor [37, 38]. In confirmation of previous results, however, the expression of several survival genes appeared il-3 dependent . Thus, transcripts for antiapoptotic pim-1, c - myc and bcl - xl proteins were repressed in the absence of il-3 . Finally, only two genes with arguably proapoptotic functions (that is, jund and chop10) were upregulated in these experiments (figure 4c). Taken together, the gene trap and cdna array results suggested that the transcriptional changes at the onset of apoptosis should favor cell survival rather than cell death . The activation of protective mechanisms during the early stages of programmed cell death suggested that a transient exposure to an apoptotic stimulus might be favorable for cell survival . Accordingly, a transient factor deprivation should, by upregulating anti - apoptotic gene expression, reduce the fdcp1 cell's need for il-3 . To test this, we evaluated the ability of il-3-deprived and non - deprived cells to form colonies in agar cultures that contained suboptimal amounts of il-3 . Figure 5a shows that cells pre - exposed to factor deprivation generated significantly more colonies than their non - exposed counterparts, indicating that survival mechanisms were induced by factor deprivation . Whereas growth stimulation was highest in cultures containing il-3 at concentrations that normally fail to support colony formation, it gradually subsided with prolonged il-3 deprivation . After incubation for 0, 1, 2 and 4 h, il-3 was added at the indicated concentrations . 1 10 cells were deprived of il-3 for 1 and 2 h, respectively . After re - adding il-3 for 4 h, cells were washed and incubated without il-3 . After 6, 9 and 12 h factor deprivation, the cells were stained with annexin and the frequency of apoptotic cells was estimated by flow cytometry ., we directly tested whether cells pre - exposed to factor deprivation develop increased apoptotic tolerance to subsequent factor starvation . To this end, il-3 was initially withdrawn from fdcp1 cells for 1 or 2 hours . Subsequently, il-3 was re - added for 4 hours only to be removed again for 6, 9 and 12 hours . At these time points cells figure 5b shows that pretreated cells died significantly more slowly that their non - pretreated counterparts, indicating that apoptotic prestimulation increases resistance to factor deprivation . Like the clonal survival assays, the cell protective effect was highest after a prestimulation of 2 hours . On the basis of these results, we conclude that cell protective mechanisms are activated following growth factor withdrawal and transiently prevail prior to irreversible commitment to death . From a floxil3 gene trap integration library consisting of approximately 2 10 unique proviral integrations, we have isolated 125 individual clones that converted to factor independence upon il-3 withdrawal . Cellular sequences adjacent to u3cre integration sites (gene trap sequence tags, gtsts) were obtained from 102 clones by inverse pcr or 5'race using cre - specific primers . Ninety clones yielded informative gtsts (> 30 nucleotides), of which 15 (17%) belonged to known genes, 10 (11%) matched single ests or full cdnas with unknown function and 65 (72%) had no match within the public databases (figure 2). Interestingly, the frequency of unknown genes recovered with this method was significantly higher than that obtained with a standard gene trap protocol and a similar gene trap vector . For example, only 782 (46%) of 1,687 gtsts recovered from embryonic stem (es) cells expressing a u3 geo gene trap virus were novel . This difference is consistent with the ability of the cre / loxp approach to enrich for transiently expressed genes that are difficult to isolate by standard methodology and therefore underrepresented in the nucleotide databases . Figure 3a shows the structure of the proviral integration sites in the 15 previously characterized genes . Like earlier studies involving retroviral gene trap vectors with a reporter gene in the u3 region of the long terminal repeat (ltr), most integrations occurred in or near 5' exons, although integrations further downstream were observed occasionally . To confirm transcriptional induction by il-3 withdrawal, polyadenylated rnas were isolated from fdcp1 cells after various intervals of factor deprivation and hybridized on northern blots to gene - specific probes . (a) gene trap position in previously characterized genes . Gtsts were either obtained by 5'race or inverse genomic pcr . Exon / intron boundaries were either available from the databases or were determined by rt - pcr reactions using mrna from fdcp1 cells and exon - specific primers . Polyadenylated rnas (5 g per lane) extracted from fdcp1 cells at various intervals following il-3 withdrawal were fractionated on formaldehyde - agarose gels, blotted onto nylon filters and hybridized to p -labeled gene- or exon (net1)-specific probes . As summarized in table 1, a significant number of the trapped known genes encode proteins involved in cell growth and survival . For example, the guanine nucleotide exchange factors (gefs) for the rho - family gtpases net1 and h - pem2 (kiaa0424) [10, 11] belong to a large family of proteins that regulate cell growth and can transform cells in culture . Transformation is dependent on the highly conserved dbl homology (dh) domain and in most cases requires amino - terminal truncation of the protein [13, 14]. Interestingly, a novel splice variant of net1, net1a, which was isolated in these experiments, encodes an amino - terminally truncated protein that is transforming in its native form (table 1). Thus, when overexpressed in nih3t3 fibroblasts, net1a but not net1 produces transformed foci with typical rho morphology (f.w . And h.v.m ., unpublished data). In addition to the dh domain, rho - gefs have a pleckstrin homology domain that connects the rho signal transduction pathway to the phosphatidylinositol-3-phosphate kinase (pi3 kinase) signal transduction pathway . The latter is responsible for mediating survival signals, particularly in hematopoietic cells, and is directly activated by il-3 . Surprisingly, two members of this pathway, the phosphatidylinositol 4/5 phosphate kinases (pip5 kinases) type i (which corresponds to human pip5 kinase type i) and type i, were induced by il-3 withdrawal (table 1). Pip5 kinase converts the lipid phosphatidylinositol-4-phosphate to phosphatidylinositol-4,5-phosphate (pip2), which enhances cell survival by at least three mechanisms . Moreover, overexpression of pip5 kinase type i in cultured cells prevents apoptosis induced by caspase 9 or tumor necrosis factor- (tnf-). Second, pip2 is phosphorylated by pl3 kinase to give phosphatidylinositol 3,4,5 phosphate (pip3), which activates the survival kinase akt . Akt prevents cell death by phosphorylating and inactivating the proapoptotic proteins bad, caspase 9 and the forkhead family transcription factor fkhrl1, which induces the expression of fas ligand [17, 18, 19, 20]. Third, pip5 kinase activates atp - dependent potassium channels and thereby helps to maintain the polarity of the cell membrane . As shown previously, apoptotic signals can cause membrane depolarization, which leads to cell death by directly inhibiting potassium channels . Summary of previously characterized genes disrupted by gene trap mutagenesis another gene with a putative survival function encodes the cell - cycle - regulated protein p38 - 2g4 (ebp1). Although less well characterized than the above proteins, p38-g4 is absent from go cells and may stimulate cell - cycle progression [23, 24]. It includes the il-12 receptor, which inhibits fas - mediated apoptosis in certain types of t lymphocytes and the transcription factor yb-1, which represses fas gene expression . Interestingly, yb-1 also activates the expression of the multidrug resistance gene (mdr1) whose product, the p glycoprotein, is an efflux pump that eliminates a variety of toxins from the cell, including proapoptotic anticancer drugs . Similarly cytoprotective is vmat2, the protein product of the vesicular monoamine transporter gene, which moves cytoplasmic monoamine transmitters into secretory vesicles and prevents cell death by sequestering proapoptotic molecules such as mpp and histamine [28, 29]. Finally, il-3 withdrawal induced transcription of several dna repair genes . These were the mouse homologs of the bacterial alkb gene and of the yeast rec8 and rad50 genes . While alkb protects dna from damage by alkylating agents, rec8 and rad50 are dna double - strand break repair proteins [31, 32, 33]. Only a few genes upregulated by il-3 withdrawal (4 out of 15) were not directly related to cell death or survival and their induction may reflect some secondary events occurring during apoptosis (table 1). However, the low frequency with which such genes were recovered underscores the specificity of the gene trap approach . To further study transcriptional regulation during growth factor starvation, we analyzed the expression patterns of a large number of previously characterized genes in fdcp1 cells undergoing apoptosis by il-3 withdrawal using atlas mouse cdna arrays (clontech). Atlas arrays contain cdna probes for 588 genes involved in development, oncogenesis, dna repair, tumor suppression and apoptosis . Figure 4a shows that of the 588 genes displayed on the arrays, only 12 were differentially expressed after factor deprivation . Differential expression of these genes was reproducible in independent array experiments and could be confirmed by northern blotting (figure 4c). (a) comparative atlas array hybridization with cdna probes derived from fdcp1 cells after 0 h (left) and 2 h (right) of il-3 deprivation . Filters were scanned with a phosphoimager (molecular dynamics) and analyzed with the atlasimage 1.5 software . (b) relative changes in gene expression on a log scale . 1, no regulation;> 1, upregulation; <1, downregulation . Note that the apparent downregulation of bcl-2 could not be confirmed on northern blots . Induction of c - kit and chop10 (asterisked) was observed only on arrays hybridized to cdna recovered from cells after 8 h of factor deprivation . (c) northern blot analysis of transcripts identified as differentially expressed by the atlasimage 1.5 software . Poly(a) rna (2.5 g) prepared after 0, 2, 4, 6 and 8 h of il-3 deprivation was fractionated on formaldehyde gels, blotted onto nylon filters and hybridized with p - labeled gene - specific probes . Similar to the trapping results, several genes with survival functions were upregulated in the absence of il-3 . Among these were the survival kinase akt, the c - kit receptor tyrosine kinase, the potent inhibitor of apoptosis flipl, and the il-3-receptor itself [34, 35, 36]. Moreover, survival functions seemed to be strengthened by the transient downregulation of the proapoptotic activin receptor [37, 38]. In confirmation of previous results, however, the expression of several survival genes appeared il-3 dependent . Thus, transcripts for antiapoptotic pim-1, c - myc and bcl - xl proteins were repressed in the absence of il-3 . Finally, only two genes with arguably proapoptotic functions (that is, jund and chop10) were upregulated in these experiments (figure 4c). Taken together, the gene trap and cdna array results suggested that the transcriptional changes at the onset of apoptosis should favor cell survival rather than cell death . The activation of protective mechanisms during the early stages of programmed cell death suggested that a transient exposure to an apoptotic stimulus might be favorable for cell survival . Accordingly, a transient factor deprivation should, by upregulating anti - apoptotic gene expression, reduce the fdcp1 cell's need for il-3 . To test this, we evaluated the ability of il-3-deprived and non - deprived cells to form colonies in agar cultures that contained suboptimal amounts of il-3 . Figure 5a shows that cells pre - exposed to factor deprivation generated significantly more colonies than their non - exposed counterparts, indicating that survival mechanisms were induced by factor deprivation . Whereas growth stimulation was highest in cultures containing il-3 at concentrations that normally fail to support colony formation, it gradually subsided with prolonged il-3 deprivation . After incubation for 0, 1, 2 and 4 h, il-3 was added at the indicated concentrations . 1 10 cells were deprived of il-3 for 1 and 2 h, respectively . After re - adding il-3 for 4 h, cells were washed and incubated without il-3 . After 6, 9 and 12 h factor deprivation, the cells were stained with annexin and the frequency of apoptotic cells was estimated by flow cytometry ., we directly tested whether cells pre - exposed to factor deprivation develop increased apoptotic tolerance to subsequent factor starvation . To this end subsequently, il-3 was re - added for 4 hours only to be removed again for 6, 9 and 12 hours . At these time points cells were stained with annexin and apoptosis was quantified by flow cytometry . Figure 5b shows that pretreated cells died significantly more slowly that their non - pretreated counterparts, indicating that apoptotic prestimulation increases resistance to factor deprivation . Like the clonal survival assays, the cell protective effect was highest after a prestimulation of 2 hours . On the basis of these results, we conclude that cell protective mechanisms are activated following growth factor withdrawal and transiently prevail prior to irreversible commitment to death . A gene trap strategy was used to identify genes induced in hematopoietic cells undergoing apoptosis by growth factor withdrawal . This approach enables identification of transiently expressed genes that are difficult to isolate by standard methodology . A comparatively large proportion of unknown genes were recovered, which underscores the strategy's potential for isolating novel genes . In contrast to conventional gene trapping, which tags and disrupts random genes, the cre / loxp approach allows enrichment for genes induced by specific biological stimuli . As most regulatory genes are expressed in a temporally and spatially restricted manner, we believe that the combination of gene trap mutagenesis and site - specific recombination provides a sound alternative for functional gene analysis in the post - genomic era . As judged by the small fraction of recombinants recovered after growth factor deprivation (that is, 124 of 2 10 or 0.006%) and by the identity of the trapped known genes, the strategy seems highly specific for regulatory genes induced during programmed cell death . Accordingly, the majority of genes upregulated by il-3 withdrawal were associated with cell death and survival . Although this was similar on the cdna arrays, the differentially expressed genes recovered with the two methods were quite different . Thus, with the exception of yb1 and rad50, none of the genes displayed on the arrays were tagged by gene trap mutagenesis . Moreover, yb1 and rad50, despite being recovered in the gene trap approach as induced by il-3 withdrawal, appeared constitutively expressed or even downregulated on both arrays and northern blots, respectively (figures 3,4). First, the integration library used here covered only 25% of the genome . As only seven genes were induced on the arrays (those encoding akt, il-3 receptor, glutathione - s - transferase, flipl, c - kit, jund and chop10) their recovery from an unsaturated library was unlikely . Second, transient gene trapping as opposed to cdna hybridization has no bias towards highly expressed genes . Third, the gene trap strategy selects for real gene inductions and, unlike the arrays and northern blots, is independent of pre - existing steady - state mrna levels subjected to post - transcriptional regulation . Taken together, these considerations suggest that for the functional analysis of the mammalian genome, gene trapping effectively complements cdna - based strategies, including cdna arrays, which are unable to distinguish between transcriptional and/or post - transcriptional changes in gene expression . In terms of apoptosis whereas in most cells conflicts between prosurvival and apoptotic signals are carried out post - translationally by well characterized proteins (for example, bcl-2 family members, caspases), there is increasing evidence for transcriptional regulators of apoptosis capable of tilting the balance between the constitutively expressed pro- and anti - apoptotic proteins [20, 41]. These transcriptional regulators of apoptosis - still largely unknown - are likely to confer tissue specificity on the apoptotic process . The identity of such regulators is of considerable interest as they could provide valuable targets for prospective anti - neoplastic and/or anti - degenerative drugs . Most genes identified in this study encode cell protective and/or pro - survival functions . In line with this, the biological experiments described here have shown such functions to materialize in cells exposed to apoptotic prestimulation . Accordingly, cells receiving stimulation exhibited improved survival and reduced apoptosis in a growth factor deprived environment . Interestingly, a similar cytoprotective phenomenon known as ischemic preconditioning has been observed in animal models . In this, a short, sublethal period of ischemia induces profound resistance to subsequent ischemic events . Thus, induction of anti - apoptotic gene expression prior to a lethal stimulus seems to raise the threshold required for that stimulus to be effective . This provides an additional safety mechanism that can prevent unwanted loss of cells exposed only accidentally to apoptotic stimulation . In summary, the present experiments have shown that hematopoietic cells undergoing apoptosis by il-3 withdrawal activate survival genes that do impede cell death . This suggests that apoptosis in hematopoietic cells is the end result of a conflict between death and survival signals, rather than a simple death by default . Inverse pcr from genomic dnas was performed using the cre - specific primers described previously and a combination of the blunt end restriction enzymes sspi and hincii . 5'race was performed with 1 g of total rna using the 5'race kit from gibco - brl and the manufacturer's instructions . Amplification reactions were performed in a perkin elmer thermocycler and cloned into the p - gemt - vector (promega) as described previously . Fdcp1 cells were propagated at concentrations of 2 10 cells / ml in dulbecco's modified eagle's medium (dmem; gibco), supplemented with 10% (v / v) fetal bovine serum (boehringer - mannheim) and 5 ng / ml recombinant mouse il-3 (peprotech) unless indicated otherwise . Agar cultures were an equal volume mixture of double - strength dmem supplemented with 40% (v / v) fetal bovine serum and 0.6% (w / v) bacto - agar (difco) in double distilled water as previously described . Apoptosis was measured in a facscan flow cytometer after staining the cells with annexin using the annexin - v - fluos detection kit (roche) and the manufacturer's instructions . Poly(a) rna samples from fdcp-1 cells were reverse transcribed in the presence of p - labeled datp and hybridized to atlas mouse cdna expression array (clontech) according to the manufacturer's instructions . Filters were scanned with a phosphoimager (molecular dynamics) and analyzed with the atlasimagetm 1.5 software (clontech). For northern blots, 2.5 g of poly(a) was fractionated in 1% formaldehyde - agarose gels, transferred onto hybond n membranes (amersham) and hybridized to specific p - dctp - labeled probes produced by random priming (rediprime, amersham). Cellular sequences adjacent to proviral integrations (gene trap sequence tags; gtsts) were searched in the ncbi / nih genomic databases using the blastn algorithm . Inverse pcr from genomic dnas was performed using the cre - specific primers described previously and a combination of the blunt end restriction enzymes sspi and hincii . 5'race was performed with 1 g of total rna using the 5'race kit from gibco - brl and the manufacturer's instructions . Amplification reactions were performed in a perkin elmer thermocycler and cloned into the p - gemt - vector (promega) as described previously . Fdcp1 cells were propagated at concentrations of 2 10 cells / ml in dulbecco's modified eagle's medium (dmem; gibco), supplemented with 10% (v / v) fetal bovine serum (boehringer - mannheim) and 5 ng / ml recombinant mouse il-3 (peprotech) unless indicated otherwise . Agar cultures were an equal volume mixture of double - strength dmem supplemented with 40% (v / v) fetal bovine serum and 0.6% (w / v) bacto - agar (difco) in double distilled water as previously described . Apoptosis was measured in a facscan flow cytometer after staining the cells with annexin using the annexin - v - fluos detection kit (roche) and the manufacturer's instructions . Poly(a) rna samples from fdcp-1 cells were reverse transcribed in the presence of p - labeled datp and hybridized to atlas mouse cdna expression array (clontech) according to the manufacturer's instructions . Filters were scanned with a phosphoimager (molecular dynamics) and analyzed with the atlasimagetm 1.5 software (clontech). For northern blots, 2.5 g of poly(a) was fractionated in 1% formaldehyde - agarose gels, transferred onto hybond n membranes (amersham) and hybridized to specific p - dctp - labeled probes produced by random priming (rediprime, amersham). Cellular sequences adjacent to proviral integrations (gene trap sequence tags; gtsts) were searched in the ncbi / nih genomic databases using the blastn algorithm . This work was supported in part by grants from deutsche krebshilfe and the deutsche forschungsgemeinschaft, bonn to h.v.m and by schering ag, berlin.
Noise - induced hidden hearing loss (nihhl) refers to any functional impairment seen in subjects with noise exposing history but no permanent threshold shift (pts). This is different from the conventional definition of noise - induced hearing loss (nihl), which is based on changes in auditory sensitivity or threshold shift . Therefore, noise exposure recommendations are based on the likelihood that a particular dose of exposure will result in a pts . Physiologically, variations in auditory sensitivity following exposure to noise are largely due to the functional status of outer hair cells (ohcs) in the cochlea, which provide mechanical amplification of soft sounds [2, 3]. Noise exposures that result in only a temporary threshold shift (tts) have a reversible impact on ohc function, which is manifested by the recovery of otoacoustic emissions (oae) [46] and cochlear microphonics (cm) [711]. The functional changes in these measures parallel the recovery of hearing thresholds, as well as the repair of structures such as stereocilia and the tectorial membrane [7, 12]. By contrast, noise exposure at higher levels and/or for longer durations can cause permanent damage to, or even the death of, ohcs and, hence, lead to pts . Therefore, the ohcs and the structures surrounding them, including the tectorial membrane and the supporting cells, are considered to be the major loci of cochlear damage that result in noise - induced threshold shifts [13, 14]. Although some early reports claimed that reversible noise - induced ihc pathologies were responsible for tts [15, 16], ihcs are relatively insensitive to noise - induced cell death . However, it has long been recognized that the synapse between ihcs and primary spiral ganglion neurons (sgns) can be damaged by noise [1719]. These early studies showed that this manifests mainly as damage to the postsynaptic terminals; however, there is clear evidence from more recent studies that noise induces damage to both pre- and postsynaptic structures . More importantly, disruption of the synapses can be permanent, resulting in degenerative death of sgns . The finding that damage to ribbon synapses can occur without pts is significant because of the potential impact of such damage on hearing function . Because the physiological damage is not accompanied by a permanent shift in hearing threshold, it would likely be missed by a standard (i.e., threshold - based) hearing assessment and has thus been referred to as nihhl . Nihhl first manifests as reduced output of the auditory nerve at high sound levels, without affecting the hearing threshold . This reduction has been found in both animals [6, 2023] and human subjects with a history of noise exposure but with normal audiograms . Since the thresholds of the auditory nerve remain unchanged, the function relating compound action potentials (cap) amplitude with sound levels in nihhl animal is different from that in animals with threshold changes . Schematic curves of cap input / output functions are presented in figure 1 for a comparison across normal control and those with different pathologies . Theoretically, if the damage is restricted to ohcs, the major change in cap input / output (i / o) curve is restricted around threshold and the amplitude reaches the control value at high sound levels . In the case of nihhl, cap reduction is mainly at high sound level, with no difference at low sound level, suggesting a suprathreshold deficit . When the damage occurs at both ohcs and the ihc - sgn synapses, the reduction of cap amplitude is seen across all sound levels . As nihhl is initiated at the synapse between the ihcs and sgns, which silences the auditory nerve fibers (anfs) that extend from them, the corresponding disorder is categorized as a cochlear neuropathy (i.e., cochlear synaptopathy) [25, 26]. Presumably, the reduction in the amplitude of the auditory nerve response without threshold elevation is due to selective loss of anfs that have high thresholds, which is supported by single - unit recording studies [20, 27]. Given the important features of those low - spontaneous - rate anfs in auditory coding, the neuropathy or synaptopathy in hidden hearing loss is not simply a reduction in the number of functional anfs . Furthermore, the synaptopathy in nihhl is likely to be related to the synaptic repair after initial damage by noise, rather than a simple initial loss . In addition, the functional deficits seen in nihhl may also involve the contribution from central auditory plasticity [26, 2832]. In this review, we summarize the available data for noise - induced damage and repair around ihc sgn synapses and discuss the evidence for the contributions of cochlear malfunction and central plasticity to nihhl . Accumulated evidence has shown that the synapses between ihcs and type - i sgns are sensitive to noise and the damage to this synapse is likely to be the bases for nihhl . The synapse is characterized by presynaptic dense bodies termed ribbons [3335], which are spherical or ellipsoidal in shape, 100200 nm in diameter, and surrounded by synaptic vesicles . The ribbons are built up from ribeye protein subunits [37, 38] and anchored to the active zone of the presynaptic membrane via bassoon proteins [3941]. The functional role of ribbons has been recognized as tethering and conveying synaptic vesicles to the active zones [42, 43], where the release of neurotransmitters at these synapses is modulated by a specific l - type calcium ion channel (i.e., cav1.3) [44, 45]. Noise exposure causes damage to both the presynaptic ribbons and postsynaptic nerve terminals of the ribbon synapses [6, 22, 23, 4648]. The damaged synapses exhibit various degrees of swelling of the terminals, resulting in disruption of the synaptic connections between ihcs and sgns [20, 46, 48]. Immunohistological staining has revealed similar losses for ribbons and terminals [6, 22, 23, 49]. The mechanism for the damage to the postsynaptic terminal is glutamate - mediated excitotoxicity (reviewed in). One possible mechanism of ribbon loss is the loss of cell - cell contact that is required for the maintenance of the pre- and postsynaptic complexes [5053]. Our electron microscopy evaluations did not reveal any residual presynaptic complexes without ribbon and postsynaptic terminals . Therefore, it is likely that the entire presynaptic structure breaks down when the postsynaptic terminal is damaged . A brick assembly model, in which a ribbon is built up from multiple ribeye subunits, has been proposed for ribbon construction in retina photoreceptor cells . Moreover, the ribbons in retina sensorial cells can be partially broken down by light, but they rapidly reassemble in the dark, probably serving as a mechanism of adaption to bright light [5458]. In the retina, the ribbon size appears to be a determining factor for the quantity of neurotransmitter released . However, the dynamic disassembling / reassembling process has not been identified in the cochlea, and changes in the ribbon size and the relationship with the release of neurotransmitters have not been investigated in the cochlea . Additionally, disassembly and reassembly, as well as ribbon size, are modulated by ca signaling involving cav - channels, presynaptic ca levels and storage, and guanylate cyclase - activating protein-2 (gcap2; see the review by schmitz). Interestingly, optical stimulation of photoreceptors causes hyperpolarization of the presynaptic membrane and a decrease in [ca]i, as opposed to depolarization and the large increase in [ca]i in ihcs in response to sound . The decrease in [ca]i in photoreceptor cells is followed by a conformational change of gcap2, which results in the disassembly of the ribbons . In the cochlea, it is not known whether there is a gcap - mediated pathway that controls ribbon size . As the membrane potential of ihcs is depolarized with increasing sound levels, resulting in an influx of ca, the role of ca in ribbon assembly is unlikely to be the same as it is in the retina . The first quantitative study of noise - induced ribbon synapse damage in cba mice reported that the number of ribbon synapses was reduced to 40% compared with the control 1 day after brief noise exposure that did not lead to pts . The synapse count recovered to 50% within 1 week, but no further recovery was observed, and this 50% loss of synapses was considered permanent . Sgn death observed 2 years after the noise was found to match the 50% permanent loss of synapses . However, a study on guinea pigs carried out by the same research group found a similar loss of ribbon synapses 2 weeks after exposure to noise that did not cause pts, but this study found a much smaller final loss of sgns . This suggests that some sgns, which had originally lost their synapses with ihcs, survived and reestablished synapses with ihcs . Our studies on guinea pigs have revealed a clear recovery in the synapse count following a massive initial loss induced by noise exposure that did not lead to pts [22, 23]. Although this recovery was not complete, approximately 50% of the initial loss of paired ribbon and postsynaptic density (psd) puncta in the basal half of the cochleae was seen 1 day after noise, and the loss was recovered to <20% within 1 month . Comparing the aforementioned data from mice and guinea pigs, it appears that there may be some species difference in the ability to regenerate synapses following noise - induced hearing damage . However, a recent study of c57 mice reported that the loss of ribbon synapses induced by non - pts - inducing noise was largely reversible . This discrepancy in synapse regeneration following noise exposure requires further investigation . In a recent review, it was argued that the recovery of ctbp2/psd counts in guinea pig cochleae following noise exposure reported in our studies may be attributable to up / downregulation of the synaptic protein rather than regeneration of synaptic connections . However, there are several lines of evidence for the possibility of synapse repair following noise - induced damage . First, it has been reported that plastic changes occur in the presynaptic component, including the existence of multiple presynaptic ribbons around an active zone and the changes in the size and location of ribbons following noise exposure . Second, the change in the amplitude of the compound action potential (cap) corresponded to the changes in ribbon / psd counts: a large initial reduction in cap amplitude and synapse counts were followed by a significant recovery after the noise exposure . Third, changes in many single - anf coding activities were not seen at the time that the synapses were damaged but rather manifested later (see section 3) with the recovery in both the cap and synapse number, suggesting that those changes occurred in the anfs that connect ihcs via repaired / reestablished synapses . Further work is required to determine the mechanisms and factors that influence the repair of both pre- and postsynaptic components . Ribbon synapses exhibit spatial differences around ihcs; that is, the synapses at the modiolar side of an ihc have relatively small ribbons but larger postsynaptic terminals, whereas those at the pillar side have relatively large ribbons but smaller terminals . Liberman et al . Reported that anfs are functionally categorized by their spontaneous rate (sr), which is inversely related to the fiber's threshold and dynamic range [6365]. It is widely accepted that low - sr anfs exhibit synapses with ihcs on their modiolar side, whereas high - sr units exhibit synapses on the pillar side (this is based on data obtained using intracellular tracer injections). The low - sr units are considered critical for hearing in noisy environments due to their larger dynamic range, higher thresholds, and the ability to follow the quick change of the amplitude of acoustic signals . By contrast, high - sr units are responsible for the sensitivity to quiet sounds and are saturated by high - level background noise [26, 63, 64, 67, 68]. In nihhl, low - sr anfs appear to be more vulnerable to noise than high - sr units . Selective loss of low - sr anfs has been found following exposure to noise that did not lead to pts . Presumably, this selective loss of low - sr units should produce coding deficits, which can be predicted based on the unique features of those units . However, no coding deficits were examined and reported in this study . On the other hand, we reported a time delay in the development of coding deficits by single anfs in guinea pigs following a similar noise exposure that did not cause pts; these deficits were attributed to intensity coding and temporal coding as summarized in sections 3.1 and 3.2 . Intensity coding in the cochlea is defined as the ability of anfs to encode the sound intensity or the change of sound intensity . This ability is determined primarily by the spike rate (or the change of spike rate) of individual anf in response to sound intensity change and the number of functional anfs . Therefore, the intensity coding deficits can be evaluated in both evoked field potential and single - unit recordings . Deficits in intensity coding were first suggested by a reduction in wave i of the auditory brainstem response (abr) [6, 49], as well as a reduction in the amplitude of the cap, as this is likely due to the loss of functional anfs following synapse disruption . The fact that the reduction is more significant at higher sound levels has been considered evidence for selective damage to low - sr fibers, which have higher thresholds [25, 26, 69]. The deterioration in intensity coding following no - pts noise exposure was manifested as a reduction in the driven spike rates (peak, sustained, and total rates) of anf units that were tested only at one sound level . Such changes are significant only in low - sr anf units and are seen at a later time rather than immediately following exposure . This time delay in the development of coding deficits suggests that (1) the reduction in driven spike rates occurs in the anfs to which the synaptic connections to the ihcs are reestablished following the initial disruption and (2) the repaired synapses are functionally abnormal, with less efficient neurotransmitter release . Temporal processing ability in the cochlea as well as in the whole auditory pathway is defined as the ability to follow the quick change of acoustic signals . In human subjects, the process involves both bottom - up and top - down mechanisms; but in animal models, only bottom - up mechanisms are tested (see reviewed by). Many different tests have been used to detect the bottom - up mechanisms of temporal coding, some of them based on the peristimulatory changes of firing rate showing latency and adaptation . As reviewed above, the major function of presynaptic ribbons in ihcs is to facilitate the synaptic transmission . Indeed, such deficits were manifested as an increase in response latency of anfs in animals with nihhl . This was first demonstrated as a significant delay in cap peak latency and then further supported by the delayed latency of peak in psth (or peak latency) of anfs in our single - unit study . In another very recent report, such delay was reported in abr as the marker of cochlear synaptopathy . We also found a reduction in the ratio of peak to sustained rates in animals that were exposed to noise . This ratio is considered an index of the ability of a neuron to encode dynamic signal changes (see review by). Using a paired - click paradigm, we found that the anf response of noise - exposed animals to the second click recovered more slowly from the masking effect of the first click . These results reveal poorer coding to the transient features of acoustic signals by anfs, which were examined in previous studies to show the deterioration in of temporal coding in animals with bassoon mutation [39, 41]. Whereas an increase in peak latency was seen shortly after exposure to noise, changes in the peak rate and the peak / sustained spike ratio, as well as a slower recovery of the spike rate to the second click, were not seen until later, suggesting an association between the deficits and the synapse repair . A temporal deficit in phase - locking responses has been proposed based on selective loss of low - sr units and the functional features of this group of anfs [25, 26, 69], but it has not been tested at the single - unit level . So far, there appear to be two models for the development of coding deficits in nihhl . One model suggests that the coding deficit or synaptopathy is simply due to the loss of low - sr anfs . Since those units have unique functions in signal coding, the loss of those functions is predicted as the consequences . That is, the coding deficits are developed as the result of unhealthy synaptic repair after initial disruption . We found that the noise - induced synaptic damage in guinea pigs under nihhl is largely repairable, leaving only a small amount of synapses not being reestablished . Therefore, the coding deficits or synaptopathy cannot be simply attributed to the loss of sr units . Since the coding deficits are seen at the time when the synapse counts are largely recovered, we believe that the coding deficits likely occur in the repaired synapses (most of them innervating low - sr anfs). Studies are needed to verify which model is more likely the case in human subjects . It has long been recognized that subjects with normal audiograms may have perceptual difficulties, and this is especially true in the elderly . Age - related hearing loss with threshold elevation is termed peripheral presbycusis, whereas the perceptual difficulties seen in the elderly without threshold shift are usually termed central presbycusis . For example, temporal processing deficits and difficulties of hearing in noisy environments are two major problems experienced by the elderly . These problems were recognized long before the discovery of cochlear damage associated with nihhl and were considered to be the result of central auditory processing disorders [7175]. It was generally accepted that any perceptual deficits observed without changes to hearing thresholds and cognitive functioning can be attributed to central dysfunctions . Based on recent progress in functional deficits in cochlear coding, such separation between peripheral and central presbycusis is likely to be incorrect . The so - called central presbycusis may, at least in part, result from disorders in the auditory periphery . The coding deficits related to the loss of low - sr anfs had been described as a type of auditory neuropathy and/or synaptopathy even before any of the predicted deficits were identified . Data on changes in the sr distributions of anfs suggest the reestablishment of synapses following an initial disruption that was selective to low - sr units . Although our data revealed abnormalities in some aspects of coding in the auditory nerve, further work is required to investigate coding deficits in nihhl . Such studies cannot be replaced by speculation based on the selective loss of low - sr fibers; for example, one cannot be certain how the auditory nerve changes its response to amplitude modulation until it is measured at the single - unit level . Two possibilities must be considered: (1) the surviving anfs may change their function and (2) the initially lost low - sr fibers may be repaired but with changed function . It should be noted that there is now a tendency in the literature to consider nihhl to be a purely peripheral issue, a result of the overcorrection of the central presbycusis . However, despite the strong evidence for a peripheral contribution, the central contribution to the problems seen in nihhl should not be neglected . In other words, it may be more constructive to assume that there are both peripheral and central contributions to nihhl . It is well known that hearing loss (with elevated threshold) can induce central changes, which can result in deteriorations in signal processing . Studies aiming to distinguish the role of central plasticity from that of ribbon synapse damage are rare . One such report found that an increase in central gain was responsible for tinnitus in human subjects with typical damage seen in nihhl (i.e., reduced auditory nerve input to the brain (measured as a smaller abr wave i)) but normal hearing threshold . In an earlier study in rats, tinnitus was found 6 months after exposure to noise that caused minimal loss of hair cells and pts but significant loss of anfs . One of the central impacts of hearing loss due to damage to peripheral auditory organ is imbalance between excitation and inhibition, resulting in hyperactivity and/or hyperresponsiveness in the central auditory system (see reviews in [7780]). The types of hearing loss producing such central enhancement include cochlear ablation, drug- and noise - induced damage . While direct effect of drugs and noise on central neurons needs to be differentiated, a similarity across those hearing loss models is the reduction of cochlea output to the auditory brain, which may be the main initial factor causing the imbalance between excitation and inhibition . In this sense, while most of studies in central plasticity using nihl model correlated the central enhancement with the amount of threshold shifts [29, 8185], at least one study has reported central enhancement in mice exposed briefly to noise at a moderate level that did not cause pts, presumably producing only nihhl . Unfortunately, the reduction in auditory input from the cochlea was not quantified in this study . Taken together, available data suggest that cochlear damage, with or without threshold elevation, can lead to central plasticity by reducing input from the auditory nerve . Further work is required to establish the central contribution to coding / perception difficulties in nihhl, and previous studies on central processing disorders in subjects with nihl should be reevaluated to differentiate the central contributions from the peripheral ones . In a brief summary, we use figure 2 to summarize the available data for the mechanisms of perception difficulty experienced by subjects with history of noise exposure but normal or near normal thresholds . In this schematic diagram, we include the two potential models of noise - induced synaptopathy in cochleae . In model 1, the coding deficits are speculated based on the role of low - sr anfs in signal coding . Both models result in a reduction in the cochlear output to the auditory brain, which in turn will result in plastic reorganization of the brain . Auditory signal processing disorders experienced by subjects with long - term nihhl should include what are inherited from the coding deficits developed in the auditory peripheral and those associated with the plastic changes of auditory brain . Although more studies on the impact of noise on human hearing showing no changes in auditory sensitivity are required, evidence suggesting the occurrence of nihhl in human subjects is being accumulated . This is supported by thorough research on the signal perception deficits experienced by subjects with a history of noise exposure but normal thresholds . Since the deficits are demonstrated at suprathreshold levels, it is clear that normal hearing thresholds do not guarantee normal hearing functions, especially in subjects with history of noise exposure [24, 86]. The second line of evidence is the reduction in the output of the auditory nerve in subjects with a history of exposure to noise . Interestingly, the combination of a reduction in wave i and an increase in wave v / i ratio may be considered evidence of increased central gain and is likely responsible for the generation of tinnitus in hidden hearing loss [32, 87, 88]. The third line of evidence comes from the age - related sgn degeneration seen in the examination of human temporal bones . Unfortunately, there is, as yet, no clear human evidence that degeneration of sgns is expedited by exposure to noise that does not cause threshold elevation . The clinical implications of nihhl are manifested by the fact that noise exposure causing nihhl occurs frequently in daily life and impacts much more general population . Such noise exposure has been generally considered to be safe according to current safety standards for exposure to noise . The evidence from the studies reviewed here indicates that the resulting damage to the ribbon synapses from noise that did not induce pts can be repaired even though the repair is incomplete . More importantly, the signal coding deficits are developed in association with the synapse repair . Since the damage and repair occur repeatedly, the damage on signal coding can be accumulated during aging and likely contributes to the perceptual difficulties experienced by the elderly . This impact of noise exposure on signal coding is obviously different from the contribution made by the hearing loss defined by threshold shifts . In future, the coding deficits and related synaptic repair in nihhl should be further investigated in a laboratory setting . Since the ribbon synapse is the first gating point for temporal processing in auditory pathway, the observed coding deficits suggest a clear peripheral origin for the decline in temporal processing and perceptual difficulties during aging . Whether and how the synaptic damage will impact the central auditory processing need to be investigated in a manner that is clearly differentiated from the impact of hearing threshold shift . Moreover, the coding function of anfs should be observed over a long period of time following exposure to noise to determine whether the coding deficits are temporary or persistent . We are currently collecting data using electron microscopy, as well as conducting an analysis on the potential changes of the molecular structures of ribbons and psds, in an attempt to elucidate the morphological / molecular mechanisms responsible for functional changes of repaired ribbon synapses . It would also be interesting to understand the reasons for the extreme sensitivity of low - sr synapses to noise, as well as elucidate possible methods to prevent damage . Laboratory studies should also aim to explore the mechanisms of synaptic repair in the cochlea, as well as reveal the factors that influence repair in order to promote it . To translate the knowledge to clinic, investigation is needed to establish good measures for detecting nihhl in human subjects . Although abr wave i is useful for evaluating synaptopathy caused by noise that does not induce pts, its reliability and sensitivity are questionable in human subjects where the abr amplitude is small, and other methods should be explored . A very recent report suggests the use of abr latency as the marker of nihhl . The study tested human subjects with normal hearing thresholds and reported a big variation in the threshold of envelope interaural timing difference, which was negatively correlated with the shift of abr wave v latency by background noise: the higher the threshold (poorer sensitivity), the smaller the shift . The observation of the latency shift with masking is supported by the fact that the low - sr anfs have longer latency than high - sr fibers and are resistant to background noise [91, 92]. It is not clear why the study did not report the change in wave v amplitude by masking . Theoretically, the masking should produce greater reduction in wave v amplitude in subjects with selective loss of low - sr units . Moreover, no information about the history of noise exposure was reported and it is not clear whether the poorer performance in temporal cue detection was due to noise - induced synaptopathy or other reasons . To date, the most promising methods for diagnosing cochlear synaptopathy are related to selective loss of low - sr anfs, the subcortical steady state responses (sssr) [93, 94]. Based on the animal studies, this test should be carried out using amplitude - modulated signals at relatively high intensity and a shallow modulation depth . The input intensity of the driving signal should fall within the saturation range of the high - sr fibers . High frequency carrier waves with a high intensity and with shallow amplitude modulation are especially useful for evaluating the function of low - sr fibers . This is supported by modeling the loss of low - sr fibers . To differentiate the sssr contribution from the auditory nerve from that of central neurons a recent mouse study found that the modulation frequency close to 1 khz was optimum with a high frequency carrier without concern of modulation depth . However, a recent human study reported a successful detection of the low - sr unit loss using off - frequency maskers and a shallow modulation depth.
Total talar dislocations are uncommon injuries and usually seen following high velocity injuries . Total talar dislocations we hereby report a closed total talar dislocation in a 25 year old male without an associated fracture around ankle . A 25 year old male presented to orthopaedic causality with injury to right ankle following a road traffic accident . Patient complained of severe pain and deformity of ankle following injury . On examination ankle was deformed and swollen . Plain radiographs of right ankle joint revealed total anterolateral dislocation of talus without any accompanying ankle fracture . Total talar dislocations are very rare injuries and should be treated as impending open fractures . There is no consensus on treatment of such complex injuries as very few cases have been reported in literature . Talar fracture dislocations are very rare injuries accounting for 0.06% of all dislocation and 2% of all talar injuries and are usually associated with malleolar fracture or a talar fracture itself[1 - 2]. Total dislocation of the talus bone from all of its three joints (tibiotalar, subtalar, and talonavicular) without a fracture is a extremely rare injury[3 - 5]. These type of injuries are usually caused by high velocity injury with disruption of almost all ligaments and capsular attachments of the talus . It usually leads to degenerative changes in neighbouring joints and frequently avascular necrosis is a predictable outcome . We present a case of total talus dislocation without any fracture which was treated by closed reduction and immobilization for six weeks . A 25 years old male presented to orthopaedic causality with injury to right ankle following a road traffic accident . Patient complained of severe pain and deformity of ankle following injury . On examination ankle was deformed and swollen with very tense skin . Plain radiographs revealed total anterolateral i dislocation of talus without accompanying ankle fracture [fig.3, 4], ct scan with 3d reconstruction also confirmed our radiographic findings [fig.5]. Under spinal anaesthesia and fluoroscopic guidance, closed reduction was performed by longitudinal traction and pushing the talus in the posteromedial direction . Following a 3 mm k - wire was passed from calcaneum to tibia through talus [fig.6, 7]. Plain radiographs at one year follow up showed no signs of avascular necrosis of talus or arthritis of subtalar joint . Pre- operative clinical photograph showing talar dislocation pre operative clinical photograph showing abrasion over lateral malleolus . Proposed treatments for total talus dislocation varied from primary talectomy or arthodesis to closed reduction and below knee cast . The few case reports found in the literature and the non - existent guidelines add to the confusion regarding the best method of treatment . Depending on whether the foot comes to supination or pronation the dislocation can be either anterolateral (the most usual) or posteromedial . This type of injury is supposed to be in continuation of subtalar dislocation when the force magnifies and continues . Dislocation of subtalar joint is the first stage of the injury . When the force progress, talonavicular joint dislocates and finally tibiotalar joint dislocation occurs . Because of all capsular and ligamentous attachments of the talus are ruptured in this injury and dependence of talus bone vascularization on these ligaments, avascular necrosis of the talus is mostly predictable . There is a deference of opinion regarding best method of treatment for closed talar dislocations . Most of the authors suggest open reduction for open talar dislocations [table 1]. Ritsema et al suggested open reduction as the best choice of treatment for closed injuries . Anterolateral incision is preferred approach for open reduction as it gives an excellent approach to ankle joint . Taymaz and gunal reported a case of closed total talus dislocation treated by closed means with excellent result . Hadji et al . Also reported good result after closed reduction of a complete talus dislocation on a three year follow - up . Total talar dislocations are very rare injuries seen following a high velocity injuries and are associated with ankle fractures . Closed reduction at the earliest is the best method of treatment for closed talar dislocations.
They have been suggested as the main cause of time off from work, reduced school performance, and low quality of life1,2,3 . Furthermore, they have led to personal, familial and societal burdens, and significant healthcare problems globally4, 5 . The prevalence of headaches is estimated at 13% of the united states population6, 20% of the australians1, and migraines are estimated at 11%, with tension - type headaches at 78% of the population world - wide5, 7 . According to the international headache society, headaches can generally be divided into two categories which are primary and secondary headaches on the basis of the underlying pathology8 . Primary headaches are not associated with pre - existing medical conditions and there are three types: migraines, tension - headaches and cluster - headaches6 . The remaining headache - sufferers discontinue medications due to adverse side - effects or excessive use of abortive medications . These can lead to a refractory condition of medication overuse headache, which means a consequent worsening of the headaches10 . As a result of these shortcomings, complementary and alternative medicine has recently become common practice in current headache management6, 10,11,12,13 . Yoga exercises are considered to be complementary and alternative medicine and are practiced by approximately 5% of the adult population in the united states and 12% of australians for alleviating headaches14 . Yoga has been reported as a safe and cost - effective intervention for managing pain1, 14 . Evidence for the efficacy of yoga exercise for a number of conditions is emerging . A growing body of evidence also supports the belief that yoga benefits physical and psychosocial health through the mechanisms of down - regulation of the hypothalamic - pituitary - adrenal axis and the sympathetic nervous system15,16,17 . As a result, yoga plays an important role in reducing sympathetic activity, increasing parasympathetic activity, improving quality of life, and decreasing pain levels18, 19 . As stated, there is evidence of the benefit of yoga in reducing pain20, 21 . However, rigorous methodology and quality of the evidence needs to be examined to establish whether or not we can assert yoga can be used as a complementary and alternative therapy for sufferers of headaches22, 23 . Therefore, the aim of this review was to assess the evidence for the effectiveness of yoga exercises in the management of primary headaches . The review was planned and conducted in accordance with the preferred reporting items for systematic reviews and meta - analyses (prisma) guidelines24, and the consolidated standards of reporting trials (consort) guidelines for reporting parallel group randomized trials25 . The cochrane library, cinahl, embase, psycinfo, pubmed, and koreamed electronic databases were searched to identify rcts published between 1966 and january 2015 . All potentially eligible studies were retrieved and the full texts of the articles were reviewed to determine whether they met the following selection criteria . To be eligible, studies had to meet the following conditions . 1) population: participants in the trials had to meet diagnostic criteria according to the international classification of headache disorders, 3rd edition (beta version) published by the international headache society 20138; primary headaches . 2) intervention: randomized controlled trials were included that used yoga as an intervention to review or reduce symptoms associated with headaches or migraines compared with no yoga . 3) outcomes: primary outcomes were headache intensity, frequency, and duration; secondary outcomes were anxiety and depression scores, and symptomatic medication use . Quality assessment of the articles was conducted using the critical appraisal, cochrane risk of bias tool for rcts, which was recommended by the cochrane handbook for systematic reviews of interventions26 . The cochrane risk of bias tool is a six - item list designed to assess sequence generation, allocation concealment, blinding, incomplete outcome data, selective outcome reporting, and other potential sources of bias . Each item is rated as yes, no, or unclear . According to the cochrane handbook, the quality of clinical trials can be divided into three levels27 . When the study design fully meets the preceding six criteria, it is considered a level, which means a low risk of bias . B level is assigned when one or more criteria are partly met, and when one or more criteria are not met, the study is defined as c level, implying high risk of bias . A total of 179 titles related to the search terms were screened . Among these, there were 32 potential trials identified from chinal, 52 from koreamed, 43 from psycinfo and 52 from the pubmed databases . After the titles had been retrieved a total of 121 studies were excluded either because they were duplicates or they were case studies, commentaries, review articles, or had no target concepts, which means no headaches or migraines . Thirty - four potential trials were identified in the search conducted in january 2015 . Thirty - four potentially relevant papers were retrieved for evaluation of the full text . After evaluation of the 34 full texts, 33 studies were excluded, because 30 studies had no randomized trials and 3 studies had no full text of rct . . The characteristics of the included study are also presented (table 1table 1.characteristics of included randomized controlled trialsauthor, year, location participantsinterventionsoutcome measuresmain resultsadverse events limitationsexperimental groupcontrol grouppopulation sample sizen (n; eg / cg)n mean age (years; eg / cg)n drop out n (%) interventions delivery method duration interventionistjohn et al.,2013, india/ rajasthangeneral person72 (36/36)34.2 (34.3/34.2)7 (9.7)yoga postures, pranayama, kriya 60 min per day, 5 days per week 3 months yoga therapistself - care education once a month 3 months handouts headache intensityheadache frequencyheadache durationanxiety - depression scoressymptomatic medication use(p<0.001)(p<0.001)(p<0.001)(p<0.001)noneabsence of a placebo groups.no blinding.all subjective outcome measures.no long - term follow - up dataeg: experimental group; cg: control group; n: number). Flowchart of included studies through the literature searches eg: experimental group; cg: control group; participants were recruited from a headache clinic of the nmp medical research institute by advertising in local newspapers . The participants mean age was 34.2 years, and they had primary headaches with migraines . The yoga program comprised yoga postures, breathing and pranayama, and kriya etc . Yoga postures included physical exercises such the stretching of the neck, shoulder and back muscles, followed by relaxation, toning, strengthening, and flexibility . Breathing and pranayama means conscious breathing, kriya was practiced as a jalaneti (nasal water cleansing) and kapalbhanti (forced exhalations). Program length, frequency, and duration of one trial was 60 minutes a day, 5 days per week for 3 months . One trial was identified that compared a control group with a yoga intervention group and evaluated the effect on headaches . Headache intensity (p<0.001), headache frequency (p<0.001), anxiety and depression scores (p<0.001), and symptomatic medication use (p<0.001) were significantly lower in the intervention group then in the control group (table 1). Neither included trial reported data on adverse effects of treatment (table 1). Assessments of each methodological quality item of the one included trial are described (table 2table 2.methodological quality summary of included trialsstudy, yearrandom allocationallocation concealmentblindingincomplete outcomeselective reportingother biasquality leveljohn et al . The quality of the one trial was level b. +: criteria met; -: criteria not met;? : unclear whether criteria were met the purpose of this review was to assess the evidence for the effectiveness of yoga interventions for primary headaches when compared to no yoga . A meta - analysis combining results from all the trials was not possible because only one study was identified . Its interventions included yoga poses, pranayama, and kriya to manage headaches or migraines . One trial reported a significant decrease in headache intensity, headache frequency, anxiety and depression scores, and symptomatic medication use in the trained group . If required, participants were allowed to take acute medication prescribed by neurologists during the trial . The effects of the medication could have diminished the efficacy of yoga exercises for alleviating headaches . In spite of both groups having received medication, reduction in the outcome of the yoga group was significantly higher than that of the control group . As stated in previous studies, these results support yoga practice as a means of evidence - based positive management of headaches or migraines20, 22, 28, 29 . The quality rating of the trial included in this review had a moderate methodological quality, and the trial did not mention blinding . However, no strong conclusion can be made due to the number of small trials and other methodological considerations . Major strengths of this group of studies include the study, the use of randomization, and the quality of measurement tools . Second, the trial did not mention blinding, lack of which may have threatened the internal validity of the trial . Third, all the outcome measurements were questioner - based and subjective; objective parameters were lacking . Therefore, evidence - based research employing objective outcome parameters is needed to identify the efficacy of integrated yoga therapy for headaches . Fourth, the trial had no long - term follow - up data concerning the durability of the treatment effect . Finally, the generalizability of the findings was limited due to the number of small trials and their partially limited quality . However, this one trial does provide a strong basis for future studies and suggests that yoga exercises could provide a safe, cost - effective therapy for the growing public health issue of headaches . Furthermore, this review contributes to the development of knowledge in physical therapy about how sufferers with primary headaches can manage themselves . In conclusion, although this review retrieved only a limited number of small trials, of partially limited quality, its findings suggest that yoga practice can effectively alleviate symptoms associated with primary headaches . However further rigorous methodological and high - quality rcts are needed to confirm and further comprehend the effects of standardized yoga programs aiming to control pain intensity and frequency, symptoms, and medication use etc.
Staphylococcus (s.) intermedius and s. pseudintermedius are both staphylococcus intermedius group (sig) members . These microorganisms are opportunistic pathogens that cause skin and nosocomial infections in dogs and cats . S. pseudintermedius is the predominant sig species in korea and until recently was misclassified as s. intermedius . Increased identification of methicillin - resistant sig (mrsig) isolates has emphasized the importance of public health precautions for veterinary staff and pet owners . Many current studies have evaluated methicillin - resistant characteristics and the staphylococcal cassette chromosome mec (sccmec) of sig isolates, particularly s. pseudintermedius . Although type i to type viii sccmec types have been identified, there is no significant studies have been conducted to examine sccmec types isolated from veterinary hospitals located nationwide in korea . Sccmec types may produce staphylococcal exotoxins including staphylococcal enterotoxins (ses), exfoliative toxins (ets) including s. intermedius exfoliative toxin (siet), and toxic shock syndrome toxin 1 (tsst 1); these toxins are associated with atopic dermatitis, mastitis, food poisoning, canine pyoderma, and chronic otits . Siet (encoded by the siet gene), first described by terauchi et al ., plays a potential role in the pathogenesis of canine pyoderma and chronic otitis . There may also be a greater chance of sig isolates to colonization and/or expand in veterinary staff, companion animals, and hospital environments . Therefore, this study is focused on the genetic identification of staphylococcal exotoxins, sccmec types, and genetic relatedness by pulsed - field gel electrophoresis (pfge) of the sig isolates in korea . Samples of s. pseudintermedius (n = 167) and s. intermedius (n = 11) were isolated; their identity was confirmed by gram - staining and biochemical testing such as coagulase, dnase production and hydrolysis . Polymerase chain reaction (pcr) was carried out with primers targeting s. intermedius nuclease and 16s rrna genes . To differentiate s. pseudintermedius from s. intermedius and s. delphini, pcr - restriction fragment length polymorphism analysis was performed using pta gene - specific primers (pta_f1:5'-aaa gac aaa ctt tca ggt aa-3', and pta_r1: 5'-gca taa aca agc att gta ccg-3'), and the restriction enzyme mboi (new england biolabs, usa). Samples were collected from eight veterinary hospitals from four different regions in korea between november 2006 and january 2010 as previously described . Pcr analysis to detect the methicillin - resistance gene (meca) pcr was also performed to amplify genes encoding sea (sea), seb (seb), sec (sec), sed (sed), see (see), seg (seg), seh (seh), sei (sei), tsst 1(tsst-1) and ets (eta, etb, etd, and siet) using primers and pcr conditions used in other previous studies . Fri913, fri 361, fri 472, fri 569, mnhoch, and rn4220 strains were used as either positive or negative controls for superantigen genes . A superantigen gene obtained in this study was further analyzed by dna sequencing using the vector nti align x program (invitrogen, usa). All mrsig isolates including 49 methicillin - resistant s. pseudintermedius (mrsp) and three methicillin - resistant s. intermedius (mrsi) were subsequently classified as sccmec type i to viii by either single or multiplex pcr assays using protocols in other previous studies including primers and pcr conditions . Genomic dna plug kits (bio - rad, usa) with smai (new england biolabs, usa) digestion were used for the pfge analysis of 39 s. pseudintermedius isolates from a private referral veterinary hospital collected at different times (eight isolates from november 2006, 24 isolates from april 2008, four isolates from june 2009, and three isolates from october 2009) and six s. intermedius isolates (all collected during april 2008). Pfge was performed according to protocol used in previous study and the manufacture's instruction (bio - rad, usa) except for an initial pulse time of 5 sec and final time of 40 sec for 21 h . Analysis of the sig isolates (n = 178) for ses and tsst 1 genes showed that only a single s. pseudintermedius isolate from a veterinary staff member (h1 - 23) harbored the enterotoxin c (sec) gene (fig . 2) revealed that the amplified sec gene the canine type c se gene (seccaine). A total of 166 out of 178 sig isolates (155 s. pseudintermedius and 11 s. intermedius) isolated from veterinary staff members (n = 38, 95%), companion animals (n = 107, 93%), and veterinary hospitals (n = 21, 91.3%) harbored siet originally detected in s. intermedius . However, other et genes such as eta, etb, and etd, known to originate from s. aureus, were not identified . Sccmec typing of the 49 mrsp isolates identified 38 type v isolates, one type iv isolate, and 10 non - identifiable isolates . Pfge analysis showed that sig isolates from a private referral animal hospital (collected between 2006 and 2009) recovered from veterinary staff, companion animals, and the environment had the same band patterns (fig . 3). Six s. pseudintermedius isolates (two from companion animals collected in september 2006, three from veterinary staff members, and one from companion animals collected in april 2008) and two s. pseudintermedius isolates (one from a veterinary hospital environment isolated in april 2008, and one from a companion animal isolated in june 2009) showed the same band patterns (fig . Since sig is closely related to s. aureus, studies have been performed to determine whether sig has adapted the eta, etb and etd toxins from s. aureus by pcr test with specific primers . However, 166 out of 178 (93.3%) sig isolates harbored the siet toxin originating from s. intermedius . This result and those of other studies imply that the majority of sig isolates harbor the siet gene . Although the siet gene was present in 93.3% of the sig isolates (from 108 dogs and two cats) in this study, only 14 dogs had a history of various skin disease including allergy and prolonged inflammation lesion in skin (n = 13) or otitis (n = 1) (data not shown). Therefore, other factors such as the general health of an animal and existence of other sig virulence factors may play an important role in outbreaks of various kinds of skin disease or otitis . The sec gene was detected in a single isolate in the present study, which was identified as seccanine by dna sequencing . However, this isolate was isolated from a veterinary staff member, and no additional seccanine isolates were identified in the veterinary hospital where this individual worked . This suggested that the isolate might be transmitted from an in- or outgoing companion animal with which the veterinary staff was in contact . The low incidence of toxins in this study could be secondary to the small number of isolates collected from companion animals that a history of skin disease or otitis . Only 11.3% of s. pseudintermedius in a previous study had exotoxins although all samples were taken from patients diagnosed with pyoderma or chronic otitis and referred to a veterinary teaching hospital . Although the majority of sccmec types were type v (78.9%), one isolate was type iv . In a previous study, 23 isolates (85.2%), three isolates (11.1%) and one isolate (3.7%) from veterinarians, staff, students, companion animals and environment in the veterinary hospitals were determined to be an mrsp hybrid sccmec type i~ii, type v, or non - identifiable, respectively . A previous european and north american study identified 75 hybrid sccmec type ii~iii isolates (72.8%), two type iii isolates (1.9%), six type iv isolates (5.8%), 14 type v isolates (13.6%), four type vii isolates (3.9%), and two non - identifiable isolates (1.9%) from diseased and healthy dogs in veterinary diagnostic laboratories of different countries . This demonstrated that the majority of mrsig isolates in korea harbor the sccmec type v whereas the hybrid type ii~iii is the main sccmec type found in veterinary hospitals in japan, europe, and north america . In the present study, pfge analysis of the 39 s. pseudintermedius and six s. intermedius isolates from a private referral veterinary hospital (collected during november 2006, april 2008, june 2009, and october 2009) showed that 20 isolates (lines 1~6, lines 7~9, lines 10~15, lines 16~18, and lines 19~20) had the same band patterns . Moreover, some isolates obtained on different sampling dates showed the same band patterns (six isolates from lines 12~17, and two isolates from lines 21~22). These results suggest potential contamination or expansion of s. pseudintermedius and s. intermedus isolates among veterinary staff, companion animals, and veterinary hospital environments, and colonization by these specific strains for more than 13 or 18 months in the same hospital . In conclusion, eta, etb, etd genes were not detected but siet toxin was found in 166 isolates in the current study . Pfge analysis results of isolated from h animal hospital showed that s. pseudintermedius isolates collected in over a period of 13 and 18 months from veterinary staff, companion animals, and the hospital environment had the same band patterns . S. pseudintermedius infections in humans, spread of mrsp populations, and association of sig with canine pyoderma and chronic otitis has been previously reported . Therefore, sig, especially mrsig, may have significant clinical implications for companion animals with skin infections or chronic otitis that is of concern for veterinary staff, companion animal owners, and healthy companion animals.
Increasing life expectancy has been accompanied by improvements in the health and quality of life of the middle - aged and elderly and knowing that women live one third of their lives during menopause . Apart from age, decreased well being, increased symptomatology, and natural menopause contribute to significant reduction in sexual activity . Physical changes, such as urogenital ageing and musculoskeletal changes which occur just before or after menopause, affect sexual functioning . On the other hand, some studies have shown that having sexual activity may have positive effect on health improvement and protection of the natural physique of postmenopausal women . In a global study, nicolosi et al ., found that 39% of women were affected by at least one sexual dysfunction, while in a survey, only 14% of americans aged 4080 said that they d been asked by their physicians about sexual difficulties during the past 3 year . Beliefs and practices that influence a woman's perception of menopause can also influence her sexual function in many societies . There is a belief that older women become sexually retired after menopause and are, therefore, expected to be less sexually active . On the other hand, some women may feel freedom because of the end of the reproductive age and also less responsibility about children . In our society, lots of women are embarrassed to ask questions about their sexual function because of some cultural taboos . No information exists on sexual functioning of postmenopausal women who are living in amol, a city in iran . Therefore, we have conducted a research to determine the frequency of sexual activity, satisfaction and sexual dysfunction among postmenopausal women . This is a cross - sectional study that has been done on 280 postmenopausal women . Exclusion criteria include known disease that have effect on sexual function, such as thyroid disease, heart - pulmonary diseases, supra renal disorders, diabetes, infectious diseases, genitalia injury, use of some medications such as antidepressants, antiblood pressure, opioids, hrt (hormonal replacement therapy), surgery resulting in menopause, stress experience or living without spouse, sexual dysfunction in spouse, etc . A semi structured questionnaire in the persian language was designed to obtain information on the sexual functioning of postmenopausal women . The development and design of the questionnaire was done following extensive literature review of related studies and fsfi (female sexual function index) to ascertain face validity of the questionnaire . The content of the questionnaire was reviewed by 5 academic faculty members (psychologist, nursing, and midwifery department faculty members). Reliability co - efficient (cronbach's alpha) was 0.82, which indicated that the instrument has a good reliability . The questionnaire had two parts, demographic characters and sexual function . To assess the desire, arousal, orgasm, sexual pain, lubrication of vagina, their satisfaction, and spouse's satisfaction about these women's sexual function, two questions were considered and each question was scored from 0 (no problem) to 4 (worst problem). Total score was from 048 and based on total score, subjects were classified into three groups: 024 normal, 2536 borderline, and 3748 severe . The mean age of the subjects and their spouses in this study was (55.9 6.02, 61.1 7.83) years and average time from their last date of menstruation was 7.5 5.43 years . 90% of the subjects had primary school education, and majority of them mentioned their economic status as good [table 1]. Mean of some socio demographic factors among selected subjects in our study 56.4% of subjects had sexual dysfunction in different phases . More than half of them had severe dysfunction based on our scoring system, one third were facing borderline dysfunction . It can be seen from the figure that desire and arousal dysfunction are predominant in females . Based on these observations it is obvious that that menopause women are high risk for sexual dysfunction . Sexual dysfunction is a broad collection of concerns that occur in menopausal women and can be effective in their quality of life . 70% reported decrease in frequency of intercourse after menopause.41.8% of women revealed that their spouses sexual interest decreased, while frequency of sexual desire decrease among women was twice more than their spouse; more than half of the subjects reported that spouses sexual interest was more than their interest.52.6% of them had noticed changes in vaginal stretching for full penetration and half of them reported some problems with their sexual function . Menopausal changes suggest that most mid - life women report a range of somatic, emotional, and vasomotor conditions including irritability and vaginal dryness . The research on whether these conditions are caused by hormonal fluctuations due to menopause, aging, or life stressors of midlife is inconclusive.31.9% of respondents revealed that their spouses were unsatisfactory with their sexual function, and 52.5% mentioned that sexual problems had an effect on their relationship to their husbands.55.6% of subjects had painful intercourse after menopause . More than half of them had severe dysfunction based on our scoring system, one third were facing borderline dysfunction . It can be seen from the figure that desire and arousal dysfunction are predominant in females . Based on these observations it is obvious that that menopause women are high risk for sexual dysfunction . Sexual dysfunction is a broad collection of concerns that occur in menopausal women and can be effective in their quality of life . 41.8% of women revealed that their spouses sexual interest decreased, while frequency of sexual desire decrease among women was twice more than their spouse; more than half of the subjects reported that spouses sexual interest was more than their interest . 52.6% of them had noticed changes in vaginal stretching for full penetration and half of them reported some problems with their sexual function . Menopausal changes suggest that most mid - life women report a range of somatic, emotional, and vasomotor conditions including irritability and vaginal dryness . The research on whether these conditions are caused by hormonal fluctuations due to menopause, aging, or life stressors of midlife is inconclusive . 31.9% of respondents revealed that their spouses were unsatisfactory with their sexual function, and 52.5% mentioned that sexual problems had an effect on their relationship to their husbands . In this study, 70.3% of subjects reported a decrease in sexual activity after menopause . This incidence is somewhat similar to park's study in korea and dhillon's study in malaysia . It is, however, different from dennerstein's study in australia where 31% of respondents reported a decrease in sexual activity with nearly two - third reporting no change . The reason for the differences between the two studies may be related to the age range of the study population and it seems that in our study subject's mean age are higher than dennerstein's study . In this study, 56.4% of subjects had sexual dysfunction but according to arman, et al ., the frequency of sexual dysfunction in isfahan was 72.4%, while the frequency of sexual dysfunction among middle - aged and elder women in addis's study was 45% and in bonnie's study was 20% . Possible explanations for the higher frequencies in our study include sociocultural differences, genetic (racial) differences and religions differences . A study in australia revealed all types of sexual dysfunction increased significantly with age and apart from age, sexual dysfunction significantly increased with advancing menopausal years . Also other studies showed that natural sexual activity will be reduced on becoming older . On the other hand women reported either an absent or decreased sexual desire that is the same as kaboody's study which was done in 2002 in kermanshah (70%). Hayes et al ., found the proportion of european women with low desire increased from 11% amongst women aged 2029 years to 53% amongst women aged 6070 years ., found women aged 6170 years had a 2.8-fold higher risk for sexual desire disorder as compared to those aged 1230 years . Although we did not find any association between dyspareunia and number of sexual intercourses, frequencies showed women with dyspareunia had a lower intercourse frequency . In a study in malaysia relationship between number of intercourse and dyspareunia was significant . In our study, frequency of dyspareunia was 55.6% and it is higher than studies that have been done in other parts of the world, such as australia 12%, taiwan32%, pakistan 16.9%, and turkey 16%. [21719] reason for this high difference is unknown . Decrease in vaginal lubrication during sexual activity or menopausal atrophic changes can be reasons for pain during sexual intercourse . Prevalence of vaginal dryness in singapore was 20.7%, thailand 20.7%, and 23.6% among taiwanese women . One of the reasons for high incidence in our study could be due to the age range of our study population . Recent evidence suggests that the declining vaginal secretion might also be due to the effect of age and hypoandrogenism . A negative correlation between age and the density of androgen receptor expression has been reported, suggesting that androgen might also be involved in the regulation of vaginal blood flow and secretions . In this study, we found significant relationship between sexual desire loss and number of sexual intercourses during the last year (p=0.000). We found association between the number of sexual intercourse with some variables, such as subject's education level and their spouse's education level (p=0.000, p=0.006) and these findings were similar to addis et al ., findings . A negative correlation was noticed between the age of spouse and the number of sexual intercourse (p=0.000) and also between age of the spouse and the sexual desire of the women (p<0.01) similar to the kelantanese women in malaysia . There were no association between sexual dysfunction and occupation, income, number of children . Arousal problems were 80% and it was similar to another study in iran that was done by arman et al ., in their study arousal problems was 75.3% . 52.5% of the subjects mentioned that sexual problems had an effect on their relationship with their husbands, also nicolosi et al ., study found that 76% of the women agreed that satisfactory sex is essential to maintain relationships . Iranian traditional sexual beliefs persist and moreover the meaning of sexuality in iranian society is based on religious minds, which makes them feel themselves passive in sexual activities . These women avoid showing their sexual emotion because of lack of knowledge and traditional beliefs . Nowadays promotion of quality of life in elder women is one of the most important issues . So it seems to need more investigations because sexual activity and satisfaction is one of the important aspect of human life which is largely ignored, especially in developing countries.
The tubularized incised plate urethroplasty (tipu) repair is the procedure of choice for distal and mid - shaft hypospadias and is increasingly being used for proximal hypospadias and redo repairs . Post - operatively, urethrocutaneous fistula is the most common complication . To decrease fistula rate, an additional tissue cover after neourethra reconstruction has been described using transverse island dorsal subcutaneous flap, de - epithelialized skin flaps, lateral dartos flap, double dartos flaps, double breasted de - epithelialized penile skin flap, tunica vaginalis, and corpus spongiosum . The use of corpus spongiosum as an intervening layer over the neourethra is less frequent . We evaluated the efficacy of spongioplasty alone after mobilization of corpus spongiosum in prevention of urethral fistula in patients undergoing tip repair for hypospadias . A prospective clinical study was carried out at our institution between june 2010 and march 2012 . The parents (for minors) and patients were informed about the merits and complications of the procedure and a written informed consent was obtained . Intra - operatively, we divided the corpus spongiosum into three categories depending on the appearance and vascularity . Poorly developed - thin spongiosal tissue with decreased vascularity; the diameter of the neourethra covered by spongiosum after spongioplasty was less than the proximal healthy urethra [figure 1]. Poorly developed corpus spongiosum (a) mobilized spongiosum on left side with minimal spongiosum tissue (b) mobilized spongiosum on right with minimal spongiosum tissue (c) hypoplastic urethra with mobilized spongiosum on both sides with minimal spongiosum tissue (d) after completion of spongioplasty, proximal normal urethra bulkier than distal neourethra with spongioplasty moderately developed - average thickness and vascularity of spongiosal tissue; the diameter of the neourethra covered by spongiosum after spongioplasty was almost equal to that of the proximal healthy urethra [figure 2]. Moderately developed corpus spongiosum (a and b) both side mobilized spongiosum with moderate spongiosum tissue (c and d) spongioplasty of mobilized spongiosum well - developed - robust, thick spongiosum with good vascularity; the diameter of the neourethra covered by spongiosum was greater than that of the proximal healthy urethra [figure 3]. Well - developed corpus spongiosum (a - c) both side mobilized spongiosum with robust healthy spongiosum tissue (d) spongioplasty of mobilized spongiosum an u - shaped incision is made encircling the meatus up to the corona, preserving the urethral plate and then extended circumferentially around the corona . Penile de - gloving is done when required, up to the root of the penis by creating a plane at the level of buck's fascia . A gittes test is carried out to evaluate the chordee after penile de - gloving . Starting well proximal to the hypospadiac meatus, a plane is created between the buck's fascia and tunica albuginea [figure 4] and the dissection is carried out on the penile shaft just lateral to the margin of the corpus spongiosum . From lateral to medial, the spongiosum is dissected off the underlying corpora cavernosa . Mobilization is done well beneath the true urethral plate on both sides, lifting off the midline spongiosum and urethral plate [figure 5] from the corpora cavernosa when required, to correct chordee . Dissection is performed with care, avoiding damage to the corpus spongiosum or corpus cavernosum . The proximal urethra is mobilized according to the severity of ventral curvature up to the bulbar region in case of persistent chordee . The spongiosal pillars spread beneath the glans wings on each side, care must be taken while mobilizing the glans wings distally with an oblique incision at about 45 degrees, which leaves healthy, thick glans wings yet intact spongiosum when done correctly [figure 6]. The urethral plate is then tubularized with a subcuticular 7 - 0 polydioxanone (pds) suture after a dorsal incision to create a new urethra . The mobilized spongiosal pillars are then approximated in the midline with 7 - 0 pds continuous sutures covering the entire neourethra right up to the glans [figure 2d]. A 6-to 10 fr urethral catheter, depending on patient age no additional cover of dartos, tunica vaginalis or de - epithelialized skin was used . Skin is sutured with 7 - 0 pds interrupted suture . Showing creation of plane proximal to meatus between tunica and buck's fascia to mobilize the corpus spongiosum mobilization of the corpus spongiosum and urethral plate mobilization of the corpus spongiosum and urethral plate into the glans cephalosporins with analgesic and anti - inflammatory drugs are given for 7-to 10 days till the catheter is in situ . Patients were evaluated at follow - up visits after 1, 3, 6, 12 months and then yearly . Results were evaluated in terms of patients, parents and surgeon's satisfaction, keeping in mind the stream, cosmesis and lack of complications . If at 3 months follow - up the stream was narrow, we did a meatal / urethral calibration with a 6 - 10 fr infant feeding tube . An u - shaped incision is made encircling the meatus up to the corona, preserving the urethral plate and then extended circumferentially around the corona . Penile de - gloving is done when required, up to the root of the penis by creating a plane at the level of buck's fascia . A gittes test is carried out to evaluate the chordee after penile de - gloving . Starting well proximal to the hypospadiac meatus, a plane is created between the buck's fascia and tunica albuginea [figure 4] and the dissection is carried out on the penile shaft just lateral to the margin of the corpus spongiosum . From lateral to medial, the spongiosum is dissected off the underlying corpora cavernosa . Mobilization is done well beneath the true urethral plate on both sides, lifting off the midline spongiosum and urethral plate [figure 5] from the corpora cavernosa when required, to correct chordee . Dissection is performed with care, avoiding damage to the corpus spongiosum or corpus cavernosum . The proximal urethra is mobilized according to the severity of ventral curvature up to the bulbar region in case of persistent chordee . Adequacy of curvature correction is confirmed by gittes test . Since the spongiosal pillars spread beneath the glans wings on each side, care must be taken while mobilizing the glans wings distally with an oblique incision at about 45 degrees, which leaves healthy, thick glans wings yet intact spongiosum when done correctly [figure 6]. The urethral plate is then tubularized with a subcuticular 7 - 0 polydioxanone (pds) suture after a dorsal incision to create a new urethra . The mobilized spongiosal pillars are then approximated in the midline with 7 - 0 pds continuous sutures covering the entire neourethra right up to the glans [figure 2d]. A 6-to 10 fr urethral catheter, depending on patient age no additional cover of dartos, tunica vaginalis or de - epithelialized skin was used . Showing creation of plane proximal to meatus between tunica and buck's fascia to mobilize the corpus spongiosum mobilization of the corpus spongiosum and urethral plate mobilization of the corpus spongiosum and urethral plate into the glans cephalosporins with analgesic and anti - inflammatory drugs are given for 7-to 10 days till the catheter is in situ . Patients were evaluated at follow - up visits after 1, 3, 6, 12 months and then yearly . Results were evaluated in terms of patients, parents and surgeon's satisfaction, keeping in mind the stream, cosmesis and lack of complications . If at 3 months follow - up the stream was narrow, we did a meatal / urethral calibration with a 6 - 10 fr infant feeding tube . An u - shaped incision is made encircling the meatus up to the corona, preserving the urethral plate and then extended circumferentially around the corona . Penile de - gloving is done when required, up to the root of the penis by creating a plane at the level of buck's fascia . A gittes test is carried out to evaluate the chordee after penile de - gloving . Starting well proximal to the hypospadiac meatus, a plane is created between the buck's fascia and tunica albuginea [figure 4] and the dissection is carried out on the penile shaft just lateral to the margin of the corpus spongiosum . From lateral to medial, the spongiosum is dissected off the underlying corpora cavernosa . Mobilization is done well beneath the true urethral plate on both sides, lifting off the midline spongiosum and urethral plate [figure 5] from the corpora cavernosa when required, to correct chordee . Dissection is performed with care, avoiding damage to the corpus spongiosum or corpus cavernosum . The proximal urethra is mobilized according to the severity of ventral curvature up to the bulbar region in case of persistent chordee . Adequacy of curvature correction is confirmed by gittes test . Since the spongiosal pillars spread beneath the glans wings on each side, care must be taken while mobilizing the glans wings distally with an oblique incision at about 45 degrees, which leaves healthy, thick glans wings yet intact spongiosum when done correctly [figure 6]. The urethral plate is then tubularized with a subcuticular 7 - 0 polydioxanone (pds) suture after a dorsal incision to create a new urethra . The mobilized spongiosal pillars are then approximated in the midline with 7 - 0 pds continuous sutures covering the entire neourethra right up to the glans [figure 2d]. A 6-to 10 fr urethral catheter, depending on patient age no additional cover of dartos, tunica vaginalis or de - epithelialized skin was used . Showing creation of plane proximal to meatus between tunica and buck's fascia to mobilize the corpus spongiosum mobilization of the corpus spongiosum and urethral plate mobilization of the corpus spongiosum and urethral plate into the glans cephalosporins with analgesic and anti - inflammatory drugs are given for 7-to 10 days till the catheter is in situ . Patients were evaluated at follow - up visits after 1, 3, 6, 12 months and then yearly . Results were evaluated in terms of patients, parents and surgeon's satisfaction, keeping in mind the stream, cosmesis and lack of complications . If at 3 months follow - up the stream was narrow, we did a meatal / urethral calibration with a 6 - 10 fr infant feeding tube . The age range of the patients was 4 months to 38 years (mean 11.53 years). The hospital stay ranged from 8 - 10 days and period of follow - up was 6 months to 2 years . The hypospadias was distal, mid and proximal in 81, 12, and 20 cases respectively . Correction of chordee was carried out by penile de - gloving alone in 5, mobilization of urethral plate with spongiosum in 22 and combination of both in 45 cases . On correlating the type of hypospadias with development of spongiosum; more proximal hypospadias was associated with poorer development of the spongiosum (p = 0.05) [table 1]. Two patients had meatal stenosis with fistula and seven patients had urethral fistula (clavien - dindo grade 3b) alone with an overall complication rate of 7.96% . Fistulas occurred in 2 cases each of distal and mid hypospadias and 5 cases of proximal hypospadias . Complications were higher in proximal hypospadias as compared to distal (p = 0.0019) [table 1]. Correlation of type of hypospadias, development of spongiosum and complications three (one each in distal, mid and proximal) out of thirteen (23.03%) cases with poorly developed spongiosum and six (distal 1, mid 1 and proximal 4) out of 53 (11.32%) cases with moderately developed spongiosum developed a fistula while none of the cases with well - developed spongiosum developed a fistula . One out of 53 (1.88%) cases with moderately developed spongiosum and 1/13 (7.69%) cases with poorly developed spongiosum developed meatal stenosis . Thus, poorer spongiosum was associated with greater number of complications (p = 0.011) [table 1]. None of the patients developed a stricture urethra and all patients / parents were satisfied with cosmesis . All nine patients with urethral fistula were operated upon 6 - 12 months later and the fistulae repaired with no further sequelae . The male urethra develops from the urethral folds as they fuse in the midline after the 6 week of fetal life . The endodermally derived distal solid urethral plate must canalize to form the distal most glanular part of the urethra . The urethra forms sequentially after the proximal urethral folds fuse and the mesenchyme within the urethral folds forms the corpus spongiosum . According to the classic theory, embryologically, the last step in urethra formation is the joining of the main endodermal urethral channel with the ectoderm as it invades the glans . The normal and hypospadiac penises differ in the development of glans and urethra, especially their vascularity . In hypospadias, there is failed urethral fold fusion and ectodermal intrusion, histologically characterized by an extremely vascular area with large endothelial lined sinuses around the abortive urethral spongiosum and abnormal glans . The degree of hypoplasia of corpus spongiosum depends upon the severity of hypospadias and proliferation of mesodermal tissue . We classified the spongiosum according to its development and vascularity as poorly developed, moderately developed and well - developed . Further, it may act as a guide to new surgeons attempting hypospadias about the feasibility of spongioplasty in a particular case and whether an additional layer may be required . None of the patients with well - developed spongiosum in distal hypospadias had any complication . Complications in proximal hypospadias with well - developed spongiosum were also very low as compared to poorly developed spongiosum cases . Most importantly, poorer was the spongiosum, greater was the number of complications (p = 0.011). On analysis of our results, we found that more proximal meatus was associated with poorer development of the spongiosum (p = 0.05) and proximal hypospadias was associated with greater incidence of complications (p = 0.0019). However, not all hypospadias with poorly developed spongiosum had complications, suggesting that other factors like development and width of urethral plate also play a role in the outcome of the repair . Since none of the patients of distal hypospadias with well - developed spongiosum had fistula, dorsal dartos cover in such patients may be omitted and the prepuce can be utilized for preputioplasty . However in patients with poorly developed spongiosa, an additional interposing layer like dartos or tunica should be used . Since its first description by snodgrass for repair of hypospadias in 1994, tip has gained widespread acceptance due to its versatility, low complication rate and reliable creation of vertically oriented meatus, with excellent cosmetic outcome . Some authors recommend tipu as the technique to be considered for primary and re - operative repair of distal and some midshaft hypospadias cases with selected proximal ones . The common complications reported after tipu repair include fistulae, urethral stricture, meatal stenosis, persistent chordee, infections and wound dehiscence with development of urethrocutaneous fistula being the most common . The causes of fistulae are local infection, ischemia, an inadequate procedure, poor tissue healing and distal obstruction due to meatal stenosis / encrustation which promotes fistulae formation . To decrease the incidence of post - operative fistulae, many modifications in the tip repair have been tried . However, no single surgical technique can be considered to be a clear winner to prevent the formation of fistulae . One of the most important factors in reducing fistula formation with the tip technique is the introduction of a protective intermediate layer between the neourethra and the skin . Dorsal, lateral, single or double dartos flaps, ventral based dartos flap, scrotal dartos, de - epithelized local penile skin, preputial flap, paraurethral tissue, spongioplasty, or tunica vaginalis flaps have all been used for the same . Out of all these, the dorsal dartos flap is used by most surgeons, but there is still no consensus over the ideal interposing tissue in tipu . Because of edema, necrosis of skin, hematoma and torque with mobilization of the dorsal dartos flap, its role as an interposing tissue has been questioned . The corpus spongiosum provides a well - vascularized, spongy protective covering to the normal urethra . It may aid in the natural propulsion of urine and semen . Using this natural, locally available tissue to cover spongioplasty also decreases the tension on the suture line of urethroplasty in the midline, especially during erections . The use of spongiosal tissue as an intermediate layer between the urethra and skin was described in 2000 by yerkes and beaudoin in separate studies . Yerkes reported no fistulae in any of her patients for whom spongioplasty was implemented in a number of urethral repair techniques, whereas beaudoin described the anatomical characteristics of the spongiosal layer in the hypospadic penis and implemented spongioplasty in patients who underwent urethral tubularization . In 2003, dodat et al . Showed no fistula formation in any of the 51 patients who underwent tipu and spongioplasty . El - sherbiny et al . Reported that covering the neourethra with a flap or spongioplasty minimized the risk of fistula formation . In a series of 500 cases, sarhan et al . Used a dartos flap, spongioplasty, or a combination of these techniques in addition to tip . They observed a statistically significant decrease in fistula formation in cases in which spongioplasty was implemented compared with cases in which it was not . In the present series we had an acceptable overall fistula rate of 7.96% with no complications in distal hypospadias with well - developed spongiosum where we did not use dorsal / ventral dartos or any other tissue in addition with spongioplasty . These results are comparable with other studies where dorsal dartos has been used with or without spongioplasty . Since the results of well - developed spongioplasty alone in distal hypospadias were very good, additional cover with dorsal dartos can be omitted . Report that various studies using dartos based flaps, tunica vaginalis and subcutaneous tissue flaps have a fistula rate of 2 - 33% . When spongioplasty alone was used as an interposing layer, in various series complications ranged from 4% to 40% with 14.2% complication rate in 197 cases whereas in the present series they were seen in 7.96% cases [table 2]. Series using spongioplasty alone for hypospadias repair our technique of spongioplasty is similar to those mentioned earlier in that we mobilize the urethral plate with the spongiosum completely off the corpora cavernosa starting laterally and moving medially . Furthermore we mobilized the spongiosal pillars into the glans obliquely at an angle of 45 almost up to the tip of the glans . The mobilization of urethral plate with spongiosum have added advantages of correction of torsion and ventral curvature, urethral plate can be tubularized without tension on suture line and many a times without incising the urethral plate . Complete spongioplasty if done properly as outlined above restores a near normal urethra and provides good support . It is a healthy, vascular cover which probably decreases tension on the midline sutures especially during erections . Better is the spongiosum, lesser are the chances of post - operative fistulae formation . The strengths of our study are that it is prospective, cases belonged to similar socio - economic status, procedures were performed by a single surgeon in similar circumstances and with adequate follow - up . The weaknesses are that the criteria for development of spongiosum are subjective so there are chances of error in judgment, especially in well and moderately developed spongiosum . To the best of our knowledge, this is the only series in current literature with such a large number of patients undergoing spongioplasty alone with tip repair . A more proximal hypospadias is associated with poorer spongiosum and this is likely to increase the chances of complications . Better developed and thicker spongiosum results in lower incidence of fistula formation after tipu . Spongioplasty reconstructs a near normal urethra as per anatomical principles of surgery with least complications . Finally, it adds an extra layer of locally available healthy vascular tissue avoiding dissection for local flaps . We recommend spongioplasty be incorporated as an essential step in all patients undergoing tip repair for hypospadias.
Premenstrual syndrome (pms) is a common disorder of young and middle - aged women, characterized by emotional and physical symptoms that consistently recur in a cyclic manner during the luteal phase of the ms and typically abate by menopause . The symptoms which exacerbate during the premenstrual period may be present in about 20 - 40% of women, though some studies quote higher figures . Premenstrual dysphoric disorder (pmdd) is characterized by a constellation of affective and somatic symptoms manifested during late luteal phase and resolve shortly after the onset of menses . Unlike other mood disorders, the mood disturbances associated with pmdd are cyclical and tightly linked to the menstrual cycle (ms); hence, the occurrence of symptoms ceases during pregnancy and after menopause . Up to 85% of menstruating women more than 200 symptoms have been associated with pms, but irritability, tension, and dysphoria are the most prominent and consistently described . Symptoms relieve within 4 days of the onset of menses and do not recur until at least day 13 of the ms . During premenstrual period, onset of a depressive episode may be observed . Significant behavioral symptoms (depression, aggression, agitated depression, irritability and so on) occur during pms and do interfere with personal, social, and occupational functioning . Overall, little data are available on the presence of pms and pmdd in indian population which houses about a sixth of the world female population and the largest adolescent female population . Moreover, studies have not dealt with rural setting, although 70% of women in india reside in rural areas . There is a suggestion that differences exist in the premenstrual symptoms and severity thereof in the urban and rural areas . Therefore, this study was undertaken in order to evaluate the presence of pms and pmdd and find their correlation with depression in a female population primarily comprising nursing students and staff in a rural setting . The current prospective observational study was carried out at a rural tertiary care medical college and hospital located in wardha, vidharbha region of maharashtra, by the department of psychiatry . Female nursing students or staff aged between 18 and 40 years, who were likely to stay within the institution for the study period was included . Those subjects with diagnosed chronic illnesses like epilepsy, migraine, anemia, thyroid disease, polycystic ovarian disease; or those using oral contraceptive pills, any hormonal drugs, propranolol or antihypertensives the students and the staff members (total: 118) were divided into batches of 25 and were given a short sensitizing lecture focusing on the symptomatology and identification of pms, pmdd, and depressive disorder . The participants rated 17 symptoms of penn daily symptom report scoring sheets on a daily basis . The menstrual period (day 1 to end of menstrual flow) and premenstrual period (5 days prior to 1 day prior to start of menstrual flow) were marked on the scoring sheet . Weekly mobile short message service and workplace / classroom reminders were sent to the participants . Subjects with features suggestive of pmdd were further investigated for the presence of pmdd using diagnostic and statistical manual of mental disorders, 4 edition, text revision (dsm - iv tr) diagnostic research criteria . Information was gathered through a structured clinical interview by one of the authors (rr). At the end of the study period, the participants were asked to rate their depressive symptoms if any, on the primary care evaluation of mental disorders (prime md) depression screen . The dsm - iv tr criteria for depression were applied to those scoring above the cutoff on prime md, and hamilton depression rating scale was applied to assess the severity of depression . Penn daily symptom report: is a self - administered checklist enquiring 17 symptoms that are rated on a likert scale of 0 (not at all) to 4 (very severe). The scale is short and reliable, valid with good internal consistency and used in a primary care setting . Primary care evaluation of mental disorders is a self - reported screening instrument for depression designed to facilitate the recognition and diagnosis of the most common mental disorders in primary care patients . The depression screen has 9 questions with 4 possible responses (not at all to every day). If the response to five or more questions is yes, with one of the first two questions being answered as affirmative, a diagnosis of depression can be considered . Hamilton rating scale for depression (ham - d): has 17 items with a total score ranging from 0 to 50, is widely used to assess the symptoms of depression . Ratings are made on the basis of the clinical interview, plus any additional available information . Diagnostic and statistical manual of mental disorders, 4 edition, text revision research criteria for pmdd require the presence of symptoms during the last week of most menstrual cycles of the preceding year which begin to remit after the onset of the follicular phase . For a diagnosis, 5 out of 11 symptoms should be present and with at least one of them being markedly depressed mood, hopelessness or self - deprecating thoughts; marked anxiety, tension or feelings of being keyed up or on the edge, marked affective lability; or persistent and marked anger, irritability or increased interpersonal conflicts . Symptoms should be associated with socio - occupational impairment, not merely be an exacerbation of another disorder, and the criteria should be confirmed by prospective ratings of at least 2 months . Using spss 19.0, descriptive statistics were applied for analysis of demographic and clinical variables; inferential statistics to access relationships between the demographic and clinical variables and pmdd and pms symptoms . The current prospective observational study was carried out at a rural tertiary care medical college and hospital located in wardha, vidharbha region of maharashtra, by the department of psychiatry . Female nursing students or staff aged between 18 and 40 years, who were likely to stay within the institution for the study period was included . Those subjects with diagnosed chronic illnesses like epilepsy, migraine, anemia, thyroid disease, polycystic ovarian disease; or those using oral contraceptive pills, any hormonal drugs, propranolol or antihypertensives the students and the staff members (total: 118) were divided into batches of 25 and were given a short sensitizing lecture focusing on the symptomatology and identification of pms, pmdd, and depressive disorder . The participants rated 17 symptoms of penn daily symptom report scoring sheets on a daily basis . The menstrual period (day 1 to end of menstrual flow) and premenstrual period (5 days prior to 1 day prior to start of menstrual flow) were marked on the scoring sheet . The days not ascribed to menstrual or premenstrual period was considered as other period . Weekly mobile short message service and workplace / classroom reminders were sent to the participants . Subjects with features suggestive of pmdd were further investigated for the presence of pmdd using diagnostic and statistical manual of mental disorders, 4 edition, text revision (dsm - iv tr) diagnostic research criteria . Information was gathered through a structured clinical interview by one of the authors (rr). At the end of the study period, the participants were asked to rate their depressive symptoms if any, on the primary care evaluation of mental disorders (prime md) depression screen . The dsm - iv tr criteria for depression were applied to those scoring above the cutoff on prime md, and hamilton depression rating scale was applied to assess the severity of depression . Penn daily symptom report: is a self - administered checklist enquiring 17 symptoms that are rated on a likert scale of 0 (not at all) to 4 (very severe). The scale is short and reliable, valid with good internal consistency and used in a primary care setting . Primary care evaluation of mental disorders is a self - reported screening instrument for depression designed to facilitate the recognition and diagnosis of the most common mental disorders in primary care patients . The depression screen has 9 questions with 4 possible responses (not at all to every day). If the response to five or more questions is yes, with one of the first two questions being answered as affirmative, a diagnosis of depression can be considered . Hamilton rating scale for depression (ham - d): has 17 items with a total score ranging from 0 to 50, is widely used to assess the symptoms of depression . Ratings are made on the basis of the clinical interview, plus any additional available information . Diagnostic and statistical manual of mental disorders, 4 edition, text revision research criteria for pmdd require the presence of symptoms during the last week of most menstrual cycles of the preceding year which begin to remit after the onset of the follicular phase . For a diagnosis, 5 out of 11 symptoms should be present and with at least one of them being markedly depressed mood, hopelessness or self - deprecating thoughts; marked anxiety, tension or feelings of being keyed up or on the edge, marked affective lability; or persistent and marked anger, irritability or increased interpersonal conflicts . Symptoms should be associated with socio - occupational impairment, not merely be an exacerbation of another disorder, and the criteria should be confirmed by prospective ratings of at least 2 months . Using spss 19.0, descriptive statistics were applied for analysis of demographic and clinical variables; inferential statistics to access relationships between the demographic and clinical variables and pmdd and pms symptoms . A total of 118 subjects was approached, of which 100 consented to the study and 18 refused consent . The mean age of the sample was 21.83 years (4.85) with a range of 18 - 37 years . The mss were regular in most of the participants, and their duration ranged from 26 to 30 days . Demographic and clinical details the phenomenology in relation to the ms is shown in table 2 . Overall, fatigue, mood swings, and irritability were the most common symptoms in the premenstrual phase; fatigue, cramps, and body aches were the most common symptoms in the menstrual phase and concentration difficulties, headache, and fatigue were the most common symptoms in the other periods . When compared to premenstrual and menstrual phase, the lesser number of symptoms were present in the other time periods . Phenomenology of symptoms during premenstrual and menstrual phase and other times according to penn daily symptom report as per the criteria of penn daily symptom report, the majority of females had some premenstrual symptoms (67 participants). Of these, 26 participants had some premenstrual distress, 20 had mild pms, 10 had moderate pms, 8 had severe pms, and 3 had very severe pms . The diagnosis of pmdd according to dsm - iv criteria was present in 10 participants . The diagnosis of pmdd according to dsm - iv tr and premenstrual symptom severity (measured by penn daily symptom report) concurred with each other (mann - whitney u = 12.5, p <0.001). The severity of premenstrual symptoms did not have significant correlation with age, socioeconomic status or cycle length (kendall tau b of 0.005, 0.039, and 0.089 with p = 0.950, 0.669 and 0.264, respectively). However, premenstrual symptom severity and the presence of pmdd diagnosis were associated with lesser age of menarche (kendall tau b = 0.170, p = 0.038 and mann - whitney u = 139.5, p <0.001, respectively). Major depressive disorder according to dsm - iv tr criteria was present in 13 of them (i.e., 46.4% of those with depressive symptoms), and the remaining 15 had subsyndromal depression . Among the 13 with major depressive disorder, 4 had mild depression (ham d scores 7 - 17), 5 had moderate depression (ham score 18 - 24), and 4 had severe depression (ham d score of> 24). Severity of premenstrual symptoms and depressive symptoms the relationship of depressive symptoms to that of premenstrual symptoms is shown in table 4 . Significant relationship was found between the diagnosis of pmdd and the presence of depression according to prime md as well as the presence of dsm - iv tr diagnosis of depression (= 5.644, p = 0.027 and = 7.162, p = 0.024, respectively). Furthermore, there was a significant relationship between the severity of premenstrual symptoms and the presence of a diagnosis of depression according to dsm - iv tr (mann - whitney u = 315, p = 0.008). Some pms symptoms were observed in 67%; diagnosis of pmdd in 10%; depressive symptoms in 28% of the sample . The diagnosis of major depression was significantly associated with the severity of pms symptoms as well as the presence of pmdd . Study takes a systematic look at the occurrence of premenstrual symptoms and pmdd in a rural area (usually neglected population in any country). Some of the key demographic variables have been looked into, and the association with a diagnosis of major depression has been explored . The sensitization lecture imparted as a part of the methodology could itself be indirect means of creating awareness of the same in the community . Highly selective sample comprising rural nursing students and staff from one academic institute that limits the generalizability of the findings; small sample size; longitudinal relations between potential confounders such as examination stress and outcome were not assessed; and the presence of medical and gynecological disorders were not systematically evaluated or ruled out which may have influenced the symptom profile encountered with pms . The diagnosis of depression was based upon clinical interview, and structured instruments like mini international neuropsychiatric interview were not used . The recruitment was through purposive sampling and only those women who consented to participate were enrolled . This study in a relatively young female population finds that distressing premenstrual symptoms are fairly common and present in about two - thirds of the studied population . A cross - cultural study comprising 14 culturally different areas in 10 countries reported prevalence of 23 - 34% in non - western cultures; 71 - 73% in western countries . However, recent studies from asian countries including egypt, saudi arabia, and japan show a high proportion of women do suffer from premenstrual symptoms . The differences in reported rates of premenstrual symptoms depend to some extent on the definitions used methods of data collection, sampling technique, and the type of study population . The occurrence of pmdd in our study (10%) is in line with published literature . Pmdd, as per the dsm - iv criteria, is found to occur in 3 - 8% of women and these figures have been closely replicated in several epidemiological studies and surveys . Gehlert and hartlage found that prevalence rates for pmdd, even when dsm - iv criteria are used, significantly vary depending upon the method of measuring symptom change . The restrictive nature of the dsm - iv pmdd criteria, particularly the requirement of an arbitrary cutoff point of at least 5 severe symptoms, remains controversial . A question arises as to whether there are a substantial proportion of symptomatic women in the general population who have premenstrual impairment and distress, and may need treatment but do not meet dsm - iv criteria for pmdd due to lesser number of symptoms reported . In this wittchen et al . Found 35.3% of women had four or more premenstrual symptoms, and prevalence of sub - threshold pmdd was 18.6% . Looking at studies from india, pmdd has been reported in about 10% of the population . Mchichi alami et al . Remarked on the high rates of depressive symptoms such as low mood, fatigue, and sleep abnormalities in those women with pmdd . Similar results of increased rates of depression in those suffering from pms have been found by other authors . Typically, irritable and depressed moods increased in the late luteal phase . Our results concur with previous findings as we observed presence of depressive symptoms to be associated with premenstrual symptoms, as well as pmdd . Though a wide range of symptoms have been associated with premenstrual complaints, we evaluated a specific set of a - priori 17 symptoms . We found that increased appetite, sleep disorders (insomnia and hypersomnia), fatigue, feeling of lack of energy, and decrease of interest for everyday activity were quite common . Some other studies have reported different frequencies and rank order of symptoms with breast tenderness and muscle pain as predominant complaints . The reason why different symptoms are reported even when using scales of similar items may be partially attributable to cultural differences and prior bias toward a set of symptomatology due to preconceived notions about premenstrual problems and symptoms . Some pms symptoms were observed in 67%; diagnosis of pmdd in 10%; depressive symptoms in 28% of the sample . The diagnosis of major depression was significantly associated with the severity of pms symptoms as well as the presence of pmdd . Study takes a systematic look at the occurrence of premenstrual symptoms and pmdd in a rural area (usually neglected population in any country). Some of the key demographic variables have been looked into, and the association with a diagnosis of major depression has been explored . The sensitization lecture imparted as a part of the methodology could itself be indirect means of creating awareness of the same in the community . Highly selective sample comprising rural nursing students and staff from one academic institute that limits the generalizability of the findings; small sample size; longitudinal relations between potential confounders such as examination stress and outcome were not assessed; and the presence of medical and gynecological disorders were not systematically evaluated or ruled out which may have influenced the symptom profile encountered with pms . The diagnosis of depression was based upon clinical interview, and structured instruments like mini international neuropsychiatric interview were not used . The recruitment was through purposive sampling and only those women who consented to participate were enrolled . This study in a relatively young female population finds that distressing premenstrual symptoms are fairly common and present in about two - thirds of the studied population . A cross - cultural study comprising 14 culturally different areas in 10 countries reported prevalence of 23 - 34% in non - western cultures; 71 - 73% in western countries . However, recent studies from asian countries including egypt, saudi arabia, and japan show a high proportion of women do suffer from premenstrual symptoms . The differences in reported rates of premenstrual symptoms depend to some extent on the definitions used methods of data collection, sampling technique, and the type of study population . The occurrence of pmdd in our study (10%) is in line with published literature . Pmdd, as per the dsm - iv criteria, is found to occur in 3 - 8% of women and these figures have been closely replicated in several epidemiological studies and surveys . Gehlert and hartlage found that prevalence rates for pmdd, even when dsm - iv criteria are used, significantly vary depending upon the method of measuring symptom change . The restrictive nature of the dsm - iv pmdd criteria, particularly the requirement of an arbitrary cutoff point of at least 5 severe symptoms, remains controversial . A question arises as to whether there are a substantial proportion of symptomatic women in the general population who have premenstrual impairment and distress, and may need treatment but do not meet dsm - iv criteria for pmdd due to lesser number of symptoms reported . In this regard, chawla et al . Reported that 12.6% of women met full pmdd criteria for one cycle . Found 35.3% of women had four or more premenstrual symptoms, and prevalence of sub - threshold pmdd was 18.6% . Looking at studies from india, pmdd has been reported in about 10% of the population . Mchichi alami et al . Remarked on the high rates of depressive symptoms such as low mood, fatigue, and sleep abnormalities in those women with pmdd . Similar results of increased rates of depression in those suffering from pms have been found by other authors . Typically, irritable and depressed moods increased in the late luteal phase . Our results concur with previous findings as we observed presence of depressive symptoms to be associated with premenstrual symptoms, as well as pmdd . Though a wide range of symptoms have been associated with premenstrual complaints, we evaluated a specific set of a - priori 17 symptoms . We found that increased appetite, sleep disorders (insomnia and hypersomnia), fatigue, feeling of lack of energy, and decrease of interest for everyday activity were quite common . Some other studies have reported different frequencies and rank order of symptoms with breast tenderness and muscle pain as predominant complaints . The reason why different symptoms are reported even when using scales of similar items may be partially attributable to cultural differences and prior bias toward a set of symptomatology due to preconceived notions about premenstrual problems and symptoms . Concurrent depression is increased by the severity of pms symptoms and the presence of pmdd . Gynecologist needs to screen such subjects for depression and refer to mental - health professional early, in routine clinical practice . There is a need for conducting larger prospective studies focusing on pmdd and its relation with depressive disorder.
Benign neoplasms of the salivary glands are frequently encountered in dental practice . These account for 3% of the tumors involving the head and neck . The majority of them occur in the parotid gland, and 80% of them are benign . Of these benign neoplasms, 50 - 80% are pleomorphic adenomas and 5 - 20% are warthin's tumors (wt). However, warthin's tumor is the most frequent monomorphic adenoma of the major salivary glands . This is a curious benign neoplasm with its intimidating histological name, papillary cyst adenoma lymphomatosum . However, the eponym wt has been extensively used ever since aldred warthin reported two cases of this tumor in 1929 . Earlier in the literature this was also referred to as adeno - lymphoma, papillary cyst adenoma, cystadeno - lymphoma, and epitheliolymphoid cyst . Wt is generally a disease of elderly men, with the highest incidence in the sixth and seventh decades and the male: female ratio is 4.6:1 . The typical features on cytology of wt include oncocytic cells in cohesive, monolayered sheets; background lymphocytes; and amorphous, cystic debris . Histopathologically, it has a cystic appearance with a double layer of oncocytes surrounding a lymphoid stroma . The following case presentation deals with wt of the left parotid gland and highlights its clinicopathologic concepts along with its therapeutic management . A 65-year - old male patient visited the department of oral medicine, with the chief complaint of swelling below the left ear lobe since six years . Patient was a known smoker since the past 25 years and there was no history of alcohol consumption . On examination, the lesion extended from the left ear lobule to the lower border of the ramus of the mandible superoinferiorly and also extended behind the left ear [figure 1]. It was approximately 5 cm in greatest dimensions; smooth contoured, was firm in consistency and had well - defined borders . Stimulation of the parotid glands yielded normal salivary flow with normal consistency, quantity and color . Based on the history and clinical examination, a provisional diagnosis of warthin's tumor was given . A differential diagnosis of pleomorphic adenoma, a low - grade parotid malignancy, lipoma and neurofibroma arising in the salivary gland were included . The investigatory workup included complete hemogram, extra - oral radiograph, ultrasonography, computed tomography and excisional biopsy of the lesion . Ultrasonographic finding showed a well - defined hypoechoic mass in the lower pole of the left parotid gland . The rest of the parotid gland parenchyma was normal and there was no evidence of ductal dilatation . Computed tomography examination revealed a rounded and well - defined cystic lesion involving the superficial lobe of the left parotid gland [figure 3]. Ultrasonograph showing well - defined hypoechoic mass computed tomography examination showing the lesion later, excisional biopsy of the lesion was planned using partial parotidectomy as the technique of choice [figure 4]. The tissue obtained was fixed in 10% of neutral buffered formalin, and processed routinely . The sections stained with hematoxylin and eosin revealed cystic spaces lined by a papillary epithelial proliferation which was bilayered . The cells of the epithelial lining appeared intensely eosinophilic . At the core of papillary projections a variable amount of lymphoid tissue with mature lymphocytes was observed [figure 5]. Tumor after superficial parotidectomy microscopic picture (10) the patient did not present with any post - surgical complications . The most accepted hypothesis about the origin of wt is that it develops from salivary duct inclusions in the lymph nodes, after the embryonic development of the parotid gland . This hypothesis is further supported by the frequent detection of salivary gland tissue in the peri- and intraparotidal lymph nodes . In the parotid region, lymph nodes the tumors presenting epithelial differentiations similar to those observed in wt develop outside lymph nodes and have no lymphoid stromal component . Benign tumors have only rarely been associated with cigarette smoking, which focuses attention on the nature of the underlying neoplastic process and how it may differ from other benign tumors . Although generally believed to be an adenoma, wt, as suggested by allegra, may be a delayed hypersensitivity reaction . An interesting fact that caught the attention of the pathologists is that a decline in the incidence in men and a concurrent increased incidence in women has been observed in recent years . The change is probably due to decline in the smoking habit in men and a reverse trend in women . The increased frequency of adenolymphoma has been ascribed to the association of adenolymphoma with smoking and the proportional increase in female smokers . Studies conducted among atomic bomb survivors suggest that radiation may also be implicated in the tumorigenesis barr virus (ebv), because of the ebv dna found in tumor cells in some studies has not been substantiated . Clinically, wt occurs almost exclusively in the parotid glands, in its superficial lobe and rarely in the deeper lobe (10%). The other preferred locations include the buccal mucosa, submaxillary gland, lip and palate . The patients can be asymptomatic or can have facial pain, rarely, facial nerve palsy may be seen in tumors associated with inflammation and fibrosis, which can be mistaken for malignant tumor . Ipsilateral earache, tinnitus and deafness are uncommon ear symptoms that might be seen in some patients . The size is variable, from a few millimeters to centimeters, averaging 2 to 4 cm in diameter, with a preferred location in the lower pole of the gland (in the jaw angle). It has been reported predominantly in whites, less frequently in orientals, and rarely in blacks . The incidence rate is higher than that of salivary gland cancer but is lower than that of benign mixed tumors (pleomorphic adenoma). Macroscopically wt presents as a spherical or ovoid mass, with a dense fibrous capsule and displaying multiple cystic compartments filled with a viscous yellow or dull brown material . However, eveson and cawson found 77% cases with an incomplete capsule, a full capsule in 8% and 16% tumors in which there was no evidence of capsule . The cytological smears in our case showed variable amounts of cellularity, ranging from barely optimum cellularity to occasional hypercellularity . There was an admixture of epithelial fragments, occasional single epithelial cells, and abundant lymphocytes noted in a granular cystic background . The epithelial cells were oncocytic in appearance with large nuclei, prominent nucleoli, and moderately abundant granular cytoplasm . Since wt can be multifocal, a preoperative diagnosis by means of fine needle aspiration biopsy is mandatory and complete bilateral screening of the gland by mri is needed to program surgery . . An experienced cytopathologist can reliably distinguish malignant salivary pathologies from benign, but a histological classification based on only aspiration is an unrealistic goal . Computerized tomography and magnetic resonance imaging enable accurate assessment of tumor extension, compression or infiltration of adjacent structures, presence of nodal metastases and better planning of the therapeutic approach . Dynamic dual - phase scinti - scanning with technetium-99, a recognized method of identifying adenolymphoma, could be used more frequently in these selected patient groups . Lesion vascularity on initial power doppler examination is often relatively sparse, but wt that did contain areas of vascularity on initial examination showed a reduction in this vascularity as the tumor size reduced . With regard to luminal cells of the tumor lining the lymphoid stroma the cells reveal a similar aspect to the striated ducts of the normal salivary glands and have numerous mitochondria . These cells, called oxifile or oncocytic cells are swollen epithelial cells, with abundant eosinophilic granular cytoplasm, rich in mitochondria and enzymes . An increased number of oncocytic cells are also observed in the normal salivary glands once the person gets older . The diffuse proliferation of the oncocytes without other changes has no pathologic significance and is called oncocytosis or oncocytic metaplasia . The epithelial cells, the oncocytes, are disposed on two layers, a luminal layer of oncocytic columnar cells, supported by a discontinuous layer of oncocytic basal cells . The nuclei of the luminal cells appear uniform and display palisading towards the free surface . The basal cells possess round to oval nuclei, centrally located, small, with conspicuous nucleoli . The lumen of the cysts contains thick proteinaceous secretions, cellular debris, cholesterol crystals, and sometimes, laminated bodies that resemble corpora amylacea . Seifert recognizes four subtypes: subtype 1 (classic wt) is 50% epithelial (77% of all wt); subtype 2 (stroma - poor) is 70 - 80% epithelial (14% cases); subtype 3 (stroma - rich) is only 20 - 30% epithelial (2%); and subtype 4 is characterized by extensive squamous metaplasia . However, presence of cellular atypia and a pseudoinfiltrative appearance of the metaplastic squamous epithelium in the residual tumor often can be mistaken for squamous cell or mucoepidermoid carcinoma . Squamous metaplasia of wt usually lacks keratinization, which is seen in most squamous cell carcinoma . In contrast to low - grade mucoepidermoid carcinoma, there is no definite infiltrative growth and the tumor cells appear more frankly squamous . A differential diagnosis must be made also with a variant of papillary thyroid carcinoma recently reported as warthin - like . The microscopic characteristic is a prominent lymphoid stroma and oncocytic metaplasia of the epithelium, but the nuclei have chromatin clearing, inclusion and groove - formation and the epithelial cells show immunohistochemical expression of thyroglobulin . The differential diagnosis of this malignancy should be performed preferably with pleomorphic adenoma and cystoadenoma . The anatomico - pathological diagnosis is generally easy, but it also should be distinguished from canalicular adenoma, sialadenoma as well as from branchial cyst when involving the parotid gland . Sunardhi - widyaputra and van darmne in 1993 immunohistochemically studied the presence of tenacin, a molecule in the mesenchyme of salivary glands believed to play a role in the embryogenesis and development of tumors, in papillary cystadenoma lymphomatosum and in oncocytoma . They found the protein to be abundant in papillary cystadenoma lymphomatosum, prominent in the proximity of the basement membrane, beneath the oncocytic epithelial components . Tenacin staining in oncocytoma was focal although oncocytes are the actively proliferating cells in this tumor . The presence of oncocytic myoepithelial cells both in papillary cystadenoma lymphomatosum and in oncocytoma surrounded by tenacin suggested that both tumors may arise from stem cells that are capable of differentiating into aberrant epithelial cells (oncocytes), myoepithelial cells in variable proportion or both . Recent molecular studies have shown that the epithelial component is polyclonal and does not exhibit clonal allelic losses, suggesting that this tumor is not a true neoplasm . Recent studies have also reported the presence of b - cells (cd20), nk (cd56) and t (cd3), including helper subtypes (cd4) and suppressor (cd8) in the tumor's stroma, something similar to that of normal or reactive lymph nodes . Also, it was found that cd20-positive b - lymphocytes were located in the germ centers and peripheral b - area while cd3-positive t - lymphocytes are located interfollicularly . Surgeons are traditionalists, and the early experience of our peers has colored current surgical opinion and slowed the introduction of conservative surgery for the benign parotid lump . This situation is now changing, and centers with experience of treating parotid tumors increasingly recognize that benign tumors can be removed safely by techniques much less invasive than a formal parotidectomy . This surgical modality is based on meticulous dissection immediately outside the tumor capsule with preservation of the facial nerves . In view of the possible association of wt with extra - salivary neoplasms, extensive workup of the patients harboring multiple wt is, therefore, indicated and long - term follow - up is mandatory, due to the possible occurrence of metachronous salivary and extra - salivary tumors even after prolonged time intervals . Rarely, either the epithelial or lymphoid component of wt can undergo malignant transformation with an estimated incidence of less than 0.1% . In order of frequency, the commonest carcinomas are squamous cell carcinoma, oncocytic carcinoma, adenocarcinoma, undifferentiated carcinoma, mucoepidermoid carcinoma and merkel cell carcinoma . Complications must be unusual and of low frequency for the surgical resection of a wt, including some complications considered of minor importance, such as paresis of the ear lobe resulting from manipulation and/or section of the auricularis magnus branch of the superficial cervical plexus . The auricularis magnus nerve, in its path toward the ear lobe, may pass through the tumor, hampering the dissection . Another complication of lesser importance is the change of facial contour due to resection of a large portion of the parotid gland.
Patients with diabetes are at increased risk of morbidity and mortality from cardiovascular disease (cvd) compared to normoglycemic controls.1 several additional factors may be associated with this increased risk, including persistent hyperglycemia, untreated hypertension, and dyslipidemia characterized by raised serum triglycerides (tg) and low concentrations of high - density lipoprotein cholesterol (hdl - c).2 the reduction in cardiovascular events produced by statins in patients with diabetes is broadly consistent with that in normoglycemic, dyslipidemic controls . In the collaborative atorvastatin diabetes study (cards), a primary prevention study in type 2 diabetes mellitus (t2 dm), atorvastatin reduced coronary heart disease events by 36%, stroke by 48%, and mortality by 27%.3 in a meta - analysis of secondary prevention studies in t2 dm, statins reduced cardiovascular events by 28% and strokes by 32%.4 despite this efficacy, a considerable residual cardiovascular risk remains among patients receiving statin therapy.2,5 hypertriglyceridemia (> 1.7 mmol / l) alters the composition of atherogenic particles and may make an important pathophysiological contribution to the residual cardiovascular risk.6 indeed, the current european atherosclerosis society consensus statement recommends treating hypertriglyceridemia of 1.7 mmol / l.6 statins, as well as lowering ldl - c, reduce blood triglyceride concentrations by up to 40%.7,8 only a few previous studies have evaluated hypertriglyceridemia in statin - treated patients with t2dm.9,10 however, there is little data evaluating these patients in primary care in the uk . Therefore, this audit assessed the prevalence of residual hyperlipidemia and the potential need for additional lipid - lowering therapy in individuals with t2 dm treated with statins in primary care in the uk . This cross - sectional, observational study audited all patients with diabetes from the first 40 primary care practices that agreed to participate from an existing panel established by an independent clinical research organization (quintiles commercial uk limited, bracknell, uk). Prescriptions issued were assessed during the preceding 4 months and repeat prescriptions were included . After excluding patients with forms of diabetes other than t2 dm, the patients were stratified according to the european society of cardiology (esc) feasible treatment targets in patients with diabetes: total cholesterol (tc) of <4 mmol / l, ldl - c of <2.0 mmol / l, hdl - c of> 1.0 mmol / l (male) or hdl - c> 1.2 mmol / l (female), and fasting tg of <1.7 mmol / l.11 due to the relatively small numbers of patients receiving combination therapy or monotherapy with lipid - lowering drugs other than statins, this analysis focused on target attainment among patients taking statin monotherapy . Descriptive statistics were performed using microsoft excel 2010 (microsoft, redmond, wa). Due to limitations in the demographic data (see discussion), inferential and comparative statistical analyses were not performed . The audit captured data on 14,652 patients with diagnosed diabetes from 40 practices . On average, 89.5% (n = 13,108) had t2 dm (median age: 66 years old, range: 17106; 44.7% female). Approximately one - fifth (22.2%; 2916) of the total t2 dm cohort had not received a prescription for lipid - lowering therapy during the 4 months before the audit . For example, only around one - fifth were within the target values for total cholesterol and ldl - c . Of patients with t2 dm who received lipid - lowering therapy, 94.7% (9647) approximately half of the patients who received statin monotherapy attained the esc treatment targets for total cholesterol, ldl - c, hdl - c, or tg (table 3). Indeed, irrespective of the individual statin used, around half of patients receiving statin monotherapy attained the esc treatment targets (table 2). Table 4 summarizes the dose distribution of each statin in t2 dm patients taking monotherapy . The audit captured data on 14,652 patients with diagnosed diabetes from 40 practices . On average, 89.5% (n = 13,108) had t2 dm (median age: 66 years old, range: 17106; 44.7% female). Approximately one - fifth (22.2%; 2916) of the total t2 dm cohort had not received a prescription for lipid - lowering therapy during the 4 months before the audit . For example, only around one - fifth were within the target values for total cholesterol and ldl - c . Of patients with t2 dm who received lipid - lowering therapy, 94.7% (9647) received statin monotherapy, which was usually simvastatin or atorvastatin (table 2). Approximately half of the patients who received statin monotherapy attained the esc treatment targets for total cholesterol, ldl - c, hdl - c, or tg (table 3). Indeed, irrespective of the individual statin used, around half of patients receiving statin monotherapy attained the esc treatment targets (table 2). Table 4 summarizes the dose distribution of each statin in t2 dm patients taking monotherapy . This large, cross - sectional, observational, systematic audit showed that, first, dyslipidemia is common among people with t2 dm in the uk and, secondly, that lipid concentrations generally and tg in particular remain elevated despite statin monotherapy . Indeed, just over a fifth of t2 dm patients were not receiving lipid - lowering therapy, despite up to approximately 80% of these subjects showing evidence of dyslipidemia (table 1). Persistent hypertriglyceridemia was common, occurring in half of those on statin monotherapy (table 2). This audit is, as far as the authors are aware, the first large study performed solely from the perspective of primary care in the uk that assesses triglyceride concentrations in patients with t2 dm receiving statin monotherapy . The results confirm the findings of the dyslipidemia international study (dysis), which stratified patients according to the presence or absence of cvd . In patients with diagnosed cvd, 37.5% did not reach the ldl - c target, 38.3% were not at the hdl - c target, and 49.1% did not achieve the triglyceride target . Among patients without cvd, the proportions were 45.0%, 30.4%, and 44.8% respectively . Only around a quarter (25.2% and 25.8% with and without cvd, respectively) achieved all three targets.9 in another study, 20.9% of 182 patients with t2 dm taking statins showed hypertriglyceridemia (2.3 <2.5 mmol / l (13.7%), ldl - c <2.0 mmol / l (8.8%), and hdl - c 0.9 mmol / l (6.0%).10 in the present study, simvastatin 40 mg was the most frequently prescribed statin dose . However, 35.2% of simvastatin users took 20 mg or less, 24.7% of atorvastatin users took 10 mg, 28.2% of pravastatin users took 20 mg, and 72.2% of rosuvastatin users took 10 mg or less (table 4). For example, simvastatin was the most widely prescribed statin in the dysis, in which the most common regimen was 2040 mg / day simvastatin equivalent.9 in a swedish study, 76% of simvastatin users took 20 mg or less, 48% of atorvastatin users took 10 mg, 55% of pravastatin users took 20 mg, and 76% of rosuvastatin users took 10 mg or less.12 the cross - sectional nature of the present audit and lack of clinical detail about each patient precluded an examination of any dose response relationship with statins or ascertaining of whether the low dose was clinically justified (eg, due to concurrent conditions or adverse events). Nevertheless, the data and previous studies suggest that a considerable proportion of patients receives low doses of statin and, as a result, may be undertreated . As such, there may be scope to intensify statin regimens in many t2 dm patients with dyslipidemia who are managed in primary care . The major limitation of this study is the absence of any information regarding the possible contribution made by potential confounders (such as coexisting obesity, dietary issues, secondary hypertriglyceridemia, impaired renal and hepatic function, and suboptimal glycemic control) to the suboptimal management of triglyceride levels . For example, more potent statins (atorvastatin, rosuvastatin, and pitavastatin) produce a robust lowering of tg, especially at high doses and in patients with hypertriglyceridemia.13 however, in this audit, a numerically smaller proportion of patients receiving more potent statins (atorvastatin and rosuvastatin) attained the treatment targets than those taking simvastatin . Similarly, the nature of the audit meant that we could not ascertain whether the subjects had relatively treatment - resistant dyslipidemia or were in the titration phase, or whether there were any differences between patients receiving statins and the group that did not receive any lipid - lowering medication . Moreover, we could not correlate hba1c levels or compliance with hypoglycemic medication with use of statins or triglyceride levels . However, the present study is based on data from a large number of patients and, therefore, probably accurately represents the proportions of people with t2 dm who attain lipid targets currently in uk primary care . As such, the audit underscores the need to ascertain the reasons why patients failed to attain the treatment targets and to consider intensified intervention . The esc guidelines specify treatment targets based on fasting triglyceride levels . However, the audit was unable to ascertain whether triglyceride levels were measured in the fed or fasting state . However, postprandial lipids, partially hydrolyzed chylomicron, and very - ldl remnants apparently accelerate atherogenesis by exacerbating endothelial dysfunction, augmenting numbers of atherogenic small ldl particles, and promoting thrombosis and inflammation . Arguably, therefore, postprandial tg more accurately predict vascular risk than fasting levels.14 as a result, both fed and fasting hypertriglyceridemia are associated with increased cvd risk . The high prevalence of hypertriglyceridemia identified in this audit suggests that tg are often undertreated . Numerous secondary causes including hypothyroidism, renal impairment, hepatic inflammation, and poor glycemic control may increase tg and should be diagnosed and managed.15 however, in this study, the number of patients who had secondary hypertriglyceridemia and the extent to which concurrent conditions influenced the likelihood of attaining the treatment targets could not be assessed . Despite these caveats, the results of the present study have implications for management . For example, clinicians should continue to emphasize that all patients should follow dietary advice and exercise regularly to improve glycemic control and lipid profiles, as well as implementing interventions to optimize compliance with lifestyle changes, and lipid - lowering and other medications . The audit showed that relatively few patients with t2 dm received statins in combination with ezetimibe, fibrate, omega 3 polyunsaturated fatty acids, or nicotinic acid . As many patients show dyslipidemic profiles that are associated with an increased risk of cvd despite statin treatment, clinicians need to consider intensifying statin regimens, offering additional lipid - modifying therapies generally, or regimens specifically aimed at lowering tg levels (such as pharmaceutical grade, highly purified omega 3-acid ethyl esters), particularly to improve the management of diabetic dyslipidemia . T2 dm patients managed in uk primary care commonly show persistent lipid abnormalities . In this study, over a fifth of t2 dm patients were found to be not receiving lipid - lowering therapy . Clinicians need to implement interventions to optimize compliance with lipid - lowering and other medications . Clinicians also need to consider intensifying statin regimens, prescribing additional lipid - modifying therapies, and specific treatments aimed at triglyceride lowering to improve dyslipidemia control in statin - treated patients with t2 dm.
Approximately 95% occur in the neck and axilla, while the remaining 5% are found in the chest and abdomen . Lymphangiomas of the abdomen are rare (accounting for 1 per 100,000 hospital admissions), but have been reported in the mesentery, retroperitoneum, gastrointestinal tract and intra - abdominal solid viscera . The clinical presentation of lymphangiomas varies from incidental discovery on imaging to presenting with an acute abdomen . Mesenteric lymphangiomas, in particular, can result in complications such as intestinal obstruction or volvulus leading to infarction . This case report documents a lymphangioma of the small bowel mesentery, incidentally discovered on pelvic ultrasound and initially thought to represent a complex cystic adnexal mass . A 42-year - old female presented with a history of chronic iron deficiency anemia and menorrhagia for several years . Ultrasound was technically difficult, but demonstrated multiple fibroids [figure 1] and a simple cyst within the left ovary . The right ovary was not visualized, but ultrasound demonstrated a complex cystic right adnexal mass measuring 9.7 5.2 6.3 cm that was presumed to originate from the right ovary [figure 2]. 42-year - old female with a history of chronic iron deficiency anemia and menorrhagia for several years and later diagnosed with cystic lymphangioma of the small bowel mesentery . Transabdominal ultrasound (a) sagittal and (b) transverse scans demonstrate a bulky uterus with a subserosal fibroid (white arrows) seen in both sagittal and transverse planes . 42-year - old female with a history of chronic iron deficiency anemia and menorrhagia for several years and later diagnosed with cystic lymphangioma of the small bowel mesentery underwent pelvic ultrasound for investigation of menorrhagia and was found to have a complex cystic right adnexal mass . Transabdominal ultrasound demonstrates a cystic lesion with numerous thin septae (curved arrows) and good through transmission (straight arrows). This confirmed multiple uterine fibroids, t1 bright foci within the left ovary suggestive of an ovarian endometrioma, as well as a dilated left fallopian tube with t1 high signal consistent with a hematosalpinx . Was only partially visualized on the pelvic mri, but was deemed to originate from the abdomen rather than the ovary, which was tethered to the uterus [figure 3]. 42-year - old female with a history of chronic iron deficiency anemia and menorrhagia for several years and later diagnosed with cystic lymphangioma of the small bowel mesentery . Axial t2 images (a and b) demonstrate tethering of right ovary (arrows) to the uterus posteriorly . This study demonstrated a cystic mesenteric lesion with high t2 signal and low t1 signal and measuring 17.6 6.8 8.7 cm . The lesion insinuated around adjacent vessels within the small bowel mesentery and demonstrated multiple thin internal septations [figure 4a]. Mass effect was notably absent, and the cisterna chyli and thoracic duct were not enlarged . The lesion was considered to represent a mesenteric lymphangioma . 42-year - old female with a history of chronic iron deficiency anemia and menorrhagia for several years and later diagnosed with cystic lymphangioma of the small bowel mesentery . (a) coronal fast imaging with steady - state precession (fisp) demonstrates a multiloculated cystic lesion with numerous septae (straight arrows), lacking mass effect . (b) sagittal t2 (13 months later) of lesion, shows increase in size of lesion with thin - walled septae (straight arrows) and insinuating around the mesenteric vessels (curved arrow). A 13-month follow - up mri examination showed that the lymphangioma had increased in size compared to the initial examination, measuring 17.6 8.4 11.4 cm and extending from the uncinate process of the pancreas to the uterine fundus [figure 4b]. The increase in size prompted a surgical referral . Furthermore, the cystic fluid of the lymphangioma demonstrated signal loss on the t1 out - of - phase sequence compared to the t1 in - phase sequence, consistent with microscopic lipid content . The signal intensity on t1 in - phase was 78.4 and on t1 out - of - phase was 56.2, thus denoting a 28.3% signal intensity loss between phases . This finding is compatible with the presence of chylous fluid and is a very suggestive feature of a lymphangioma [figure 5]. 42-year - old female with a history of chronic iron deficiency anemia and menorrhagia for several years and later diagnosed with cystic lymphangioma of the small bowel mesentery . Corresponding axial t1 (a) in - phase and (b) out - of - phase mr images demonstrate the cystic lymphangioma (straight arrows) with signal intensity loss on the out - of - phase image (b) compared to the in - phase image, indicative of microscopic lipid content of chylous fluid . Lymphangiomas are low - flow vascular malformations that develop as a result of failure of communication of lymph sacs with the venous drainage system . It is postulated that these lesions arise secondary to inflammatory processes, surgery, or radiotherapy that lead to lymphatic obstruction . Lymphangiomas are histologically divided into several types: simple capillary, cavernous, and cystic lymphangiomas . Cysts may contain serous, chylous, bloody fluid or even purulent fluid in the case of secondary infection . The presence of emulsified fats within chylous fluid most likely explains the signal loss on the t1 out - of - phase mr sequence compared to the t1 in - phase mr sequence in our study . Intra - abdominal lymphangiomas mostly occur in the mesentery followed by the omentum, mesocolon, and retroperitoneum . Involvement of solid viscera has been described in the literature in the liver, spleen, pancreas, adrenals, kidneys, bladder, and the ovaries . The clinical symptoms of a mesenteric lymphangioma are non - specific and include abdominal pain, vomiting, and constipation, making a specific diagnosis practically impossible on clinical grounds . The majority of lymphangiomas are discovered incidentally on imaging for the investigation of unrelated clinical indications . The differential diagnosis includes a wide range of cystic intra - abdominal lesions, ranging from pancreatic pseudocysts to abdominal tuberculosis, hydatid disease, or malignancies such as mucinous carcinomatosis . The patient did not have a history of previous pancreatitis to suggest pseudocysts or risk factors for pancreatitis such as gallstones or alcohol use . Abdominal tuberculosis and malignancy were excluded largely on clinical grounds, as the patient was relatively healthy with no clinical complaints other than menorrhagia . Hydatid disease was unlikely, given the absence of daughter cysts and calcifications, and the insinuating nature of the lesion lacking mass effect despite its size . The absence of aggressive features on imaging, such as invasion or solid component, made malignancy unlikely . A combination of imaging modalities may be helpful in confirming the diagnosis, with mri being the preferred examination of choice . Typical imaging findings are those of a thin - walled, multiloculated cystic lesion lacking solid components or mural nodularity . Lymphangiomas typically show t2 high signal fluid, while signal loss may be visualized on chemical shift t1-weighted imaging due to microscopic fat content from chylous fluid this is a very suggestive finding for a lymphangioma occurring in 2030% of cases, although rarely it may be found in other limited differentials such as lymphoceles, lymphoepithelial cysts, and pancreatic pseudocysts . They typically show little in the way of mass effect and generally insinuate around adjacent structures . The imaging algorithm for the work - up of lymphangiomas would depend on the local resources and availability of imaging modalities such as us, ct, and mri . In our institution, cystic lesions that are either indeterminate or suspicious for lymphangiomas on us or ct are referred to mri for further characterization . On mri, the presence of a thin - walled, multiloculated cystic lesion in the abdomen, without mass effect on adjacent structures, is suggestive of the diagnosis after relevant differential diagnoses have been excluded on clinical, laboratory, and imaging grounds . The presence of signal loss on chemical shift imaging is very suggestive of the diagnosis . If definitive confirmation is required, imaging - guided diagnostic aspiration of the cyst fluid could be performed . Secondary infection, bowel obstruction, volvulus, and rupture with hemorrhage are a few recognized complications . So, to date, radical excision remains the definitive treatment, in particular, to prevent recurrence . In conclusion, intra - abdominal lymphangiomas are rare entities, which although are typically asymptomatic, can result in life - threatening complications . In our case, the lesion was discovered incidentally on imaging for an unrelated clinical indication and demonstrated interval growth at 1 year warranting a surgical referral . As of the present time, the surgical consultation has not yet been conducted and our diagnosis is based on imaging . Our findings were those of a thin - walled multiloculated cystic lesion, lacking both mass effect and enhancement, and which insinuated around vessels within the small bowel mesentery . The cyst fluid showed signal loss on chemical shift t1-weighted mr imaging due to the microscopic fat content of chylous fluid a very suggestive finding for a lymphangioma that helps to differentiate it from other etiologies of cystic lesions in the abdomen.
Broomrapes (orobanche spp .) Are fully parasitic flowering plants that lack chlorophyll; hence they penetrate roots of susceptible hosts, removing water, minerals and sugars . (egyptian broomrape) attacks dicotyledonous crops cultivated around the mediterranean, causing massive yield losses . Broomrape attached to the host by means of tubercle, a swollen organ which may be simple or composite . With the exception of the case of transgenic target - site, herbicide - resistant host plants, meant to be a temporary measure until other effective control means are found . Orobanche generally maintains a close relationship with the host and so it is unreasonable to attack it using herbicides because the latter may adversely affect the nontarget host . Despite research on orobanche spp . For over three decades, yield losses still abound because there is no sustainable method for controlling the parasite . Fusarium compactum (wollenw .) The inundative biocontrol approach, with repeated applications of the biocontrol agent, generates a state of equilibrium with a very low level of weed density as a result of the artificial inoculation of the biocontrol agent but the fungi are not sufficiently virulent for field release, regardless of the amount used . A series of experiments were conducted using f. compactum, a biological control agent that infects orobanche without affecting the roots of tomato . Pectins are complex polysaccharides and are one of the major components of the plant cell wall of dicotyledonous plants, where they control the ionic status, cell expansion, and separation . Some cellulase producing fungi includes acremonium sp, aspergillus spp and fusarium sp,, trichoderma spp . [6, 7], zymomonas, and mutant penicillium . In this paper, a hydrolytic enzyme plays an important role in the pathogenicity of plants by facilitating fungal penetration through the host cell wall [10, 11]. Experiments using mycoherbicidal organisms plus pectinase (ec 3.2.1.15) or cellulase (ec 3.2.1.4) indicate that enzyme enhances the weed control of pathogenic fungi . Here, pectinolytic and cellulolytic enzymes have been used to enhance the virulence of f. compactum on tomato plants infested with broomrape . A semiaxenic polyethylene bag system was used that allowed easy visual observation of the fungal infection of the tubercles . This study reports that the addition of pectinase and cellulase alone or in mixtures enhanced the virulence of f. compactum on broomrape . F. compactum was cultured on potato glucose agar (pda, pronadisa) in petri dishes incubated at 25c . Subcultures were grown in 100 ml potato glucose broth (pdb, pronadisa) in 250 ml erlenmeyer flasks . The cultures were left on a rotary shaker (brunswick scientific) at 150 rpm for 48 h. f. compactum mycelia were harvested on miracloth (calbiochem, la jolla, ca), rinsed with distilled water to remove remaining spores and excess medium, and harvested by vacuum filtration . The washed hyphae were chopped at 6,000 rpm for 2 min with a homogenizer (ika t18 basic ultra - turrax usa), resuspended in sterile water, and the propagule concentrations of chopped mycelia were estimated after serial dilution and plating . Surface sterilizing seeds ensure that the fungal infection on the seeds is from deliberate infection . Thus, about 13 mg seeds in small bags formed of miracloth were wetted and surface sterilized in 80% ethanol for 1 min and in a mixture of 1% sodium hypochlorite in 0.01% aqueous tween 20 for 10 min . Tomato transplant plugs at the two to three leaf stages in speedling insert trays were purchased from hishtil inc, ashkelon, israel . The pathogenicity of f. compactum was tested in the semiaxenic polyethylene bag system; briefly about 13 mg of dry surface - disinfected seeds (up to 1,500) were sprinkled on wet whatman gf / a glass - fiber sheets (whatman int . The broomrape seeds were conditioned for a 7 d period on the wet glass - fiber sheets . A tomato seedling with three or four expanded leaves and washed roots was fixed inside each polyethylene bag containing conditioned broomrape seeds . The plant roots in each bag were moistened by capillary action with forty ml of modified hoagland's solution in the base of each bag . Modified hoagland's solution fourteen - hour photoperiods were provided by a photosynthetically active light intensity of 65 e / m / s (li - cor, inc ., photometer, model li-188b) produced by six 40 w cool white fluorescent tubes suspended 35 cm above the benches . Two ml of 5-g ml gr-24 (synthetic germination stimulant) were added to each bag with a pipette to augment the tomato root exudates . The broomrape seeds germinated, attached to tomato roots, and formed small tubercles during the following 2 weeks . Allocation of treatment to orobanche - infested tomato plants were in such a way that the tubercle numbers and sizes were almost the same . The virulence of the fungus was determined with and without various concentrations of either pectinase (ex fungal origin, 1.1 u mg, sigma) and/or cellulase (cellulysin, ex trichoderma viride, 10 u mg calbiochem - behring corp ., la jolla, ca 92037). The effect of cellulase concentration (10 to 20 u ml) on tubercle death was similarly determined at a constant inoculum level . Thereafter, the virulence of the fungus with the two enzymes was determined in combination at varying ratios . Control plants were mock - inoculated with either sterile distilled water containing 0.01% tween 20 or 4 to 20 u ml of single or combined enzyme preparations but without fungal mycelia . Tubercles on the tomato plants infested with broomrape were counted and the diameters were measured with a ruler, with the assumption that the tubercles are perfectly spherical . The treatments consisted of f. compactum or f. compactum plus cellulase (4 to 20 u ml). One ml of aqueous fungal cellulase samples (10 units mg), freshly made for each experiment as a solution containing 10 u ml, and was checked for pectinase activity . Pectinase activity was measured in a reaction mixture consisting of 533 l of 1% polygalacturonic acid (pectin), 400 l of 50 mm sodium acetate buffer at ph 5.0 and 67 l of the cellulase . The mixture was incubated at 37c for 10 min as outlined by tonukari et al . . A 100-l aliquot of the reaction mixture was mixed with 1.5 ml of 1% 4-hydroxybenzhydrazide (fluka, fluorescence grade) in 0.5 m naoh . The mixture was heated at 100c for 10 min, and cooled on ice water . Absorbance was measured at 410 nm against the zero time blank as outlined by lever, using a spectrophotometer with a versamax tunable microplate reader . Pectinase activity of the cellulase was calculated from a standard curve prepared with a d - galacturonic acid (sigma). One unit of enzyme forms 1 mol of galacturonic acid from polygalacturonic acid in 1 min under the conditions of the assay . Broomrape tubercle deaths were recorded at 24 h intervals after fungal inoculation, for 8 to 11 d. tubercles were visually scored as healthy (translucent, dense, and intact), infected (diseased), or dead (black and soft). The levels of infection caused by f. compactum plus pectinase in both experiments were always better than those achieved by the fungus without pectinase (figures 1(a) and 1(b)). The numbers of broomrape tubercles infected continuously increased over time throughout the period of observation . This was not only due to inoculum buildup in the tomato root system but also to the enzyme action . This study wanted to ascertain whether the added pectinase would have an effect on suboptimal f. compactum inoculum . Therefore, subthreshold levels of inoculum (10 propagules ml) were investigated in the presence of pectinase . F. compactum alone killed 30% of broomrape tubercles at the suboptimal inoculum levels (figure 1(a)). At the lowest inoculation density, f. compactum (3.84 10 propagules ml) combined with 11 u ml pectinase killed more broomrape tubercles than the f. compactum alone (figures 1(a) and 1(b)). The killing of broomrape tubercles indicated that pectinase enhanced the virulence of f. compactum (figure 2). Since both enzymes separately enhanced the severity of tubercle infection, it was ascertained whether the pathogenicity of f. compactum (1.4 10 propagules ml) on broomrape tubercles could be synergistic through the joint action of the enzymes (10 u ml of each enzyme) (figure 3). The broomrape tubercles on the tomato roots were large and healthy in the absence of fungal treatment but, at times, a few naturally brown tubercles could be seen (figure 3(a)). F. compactum alone infected all the inoculated tubercles but did not kill any significant number (figure 3(b)). Infested tomato roots that were inoculated with mycelia plus pectinase (20 u ml) (figure 3(c)) had over 50% tubercles dead one week after treatment . Those inoculated with mycelia plus cellulase (20 u ml) (figure 3(d)) had above 60% mortality . A mixture of both enzymes with f. compactum increased fungal infection of broomrape by f. compactum (figure 3(e)). The response of the infected tubercles varied from 100% kill to mild infection, depending on tubercle size . Mycelia plus cellulase (20 u ml) mix provoked about the same level of infection as mycelia in solution with cellulase and pectinase (10 u ml of each enzyme) (figure 3(f)). It is logical to think collective mixing of pectinase and cellulase to f. compactum will effectively enhance the fungal biocontrol potential: thus, it was tested . At various enzyme compositions, the f. compactum treatment (1.05 10 propagules ml) that caused only a hypersensitive reaction (9% death) on the tubercles caused about 35 to 85% tubercle death when the tubercles were treated with mixtures that contained mycelia, pectinase, and cellulase in various ratios (figure 4). Higher tubercle infection by f. compactum was observed with a high ratio of cellulase to pectinase . Beginning from day 4 after f. compactum inoculation on broomrape infested tomato plants; statistically significant differences among treatments were observed (figure 4). Broomrape infested roots coinoculated with chopped mycelia plus pectinase and cellulase had substantial tubercle infection and subsequent large numbers of dead tubercles (3585%) (figure 4). This system ensures easy examination of seedling attachment to the tomato roots and easy observation of tubercle infection in the course of the experiment . The activity catalyzed by an enzyme is a measure of the amount of enzyme present . Values pooled from three sets of experiment showed that in a 1 mg (10 u mg) cellulase preparation, 0.055 mg pectinase (1.1 u mg) is present . This means the 10 u cellulase has a 0.06 u of detectable pectinase . The basis for this study stems from previous studies which examines single enzyme - assisted effect on fungi pathogenicity . The orobanche were at three main phases of their life cycle (seed, germination, and parasitic phases) at fungal inoculation . Tubercles are usually present at the germination phase and later become parasitic . In this study, in apparently healthy tubercles, it may be possible to find diseased tubercles (as depicted in figure 2); such tubercles deaths were induced by accidental infestation . In total, these results demonstrate that exogenous pectinase as well as cellulase can contribute to pathogenicity . The mycelia plus enzyme mix killed some broomrape tubercles, thus they could not form new seeds to replenish the orobanche seed bank . The rationale of approach is also applicable to soil grown tubercles where the presence of enzyme may be exogenous . This is well taken care of in nature because the tubercles are borne on the roots and roots are mostly below the soil surface . The temperature in the growth chamber was kept at 25c to induce infection by f. compactum . As demonstrated in this study, descriptions of actions of cell wall degrading enzymes are given by wanjiru et al . . F. compactum can infect the tubercle by degrading the cell wall components and invading the tissues and cells . Pectinolytic and cellulolytic activities of the enzymes (in this case of fungal origin) result in a loss of structural integrity in the tubercle and characteristic damage . The enzyme substrate range and mechanism of action could partly explain the interaction between f. compactum and the enzymes on broomrape tubercles . The pathogenicity of f. compactum plus the enzyme mix observed could rarely be observed without pectinase and cellulase at the inoculum level used . Besides, neither pectinase nor cellulase was ordinarily able to injure and kill broomrape at the concentration used (max . 20 u ml), demonstrating that the broomrape tubercle infection and death observed are due to the addition of a mycoherbicidal organism . The role of pectinase and cellulase in the degradation of cell wall material as observed in this study corresponds with the reports of many authors [10, 11, 18, 19]. In the previous paper, the results reported by sasaki and nagayama showed that the fungi pathogenicity was not always proportional to the enzyme activity . It is known that the level of -glucosidase in an enzyme preparation may affect the result of cellulase assays, for example, for the estimation of activities of extracellular cellulase enzymes produced by trichoderma . As pointed out by kumpoun and motomura, the pectinase used in their study is mainly pectinase, but some glycosidases were also present . The presence of -glucosidase or other enzymes, such as cellobiose phosphorylase, which are required for cellobiose metabolism and to enhance cellulose hydrolysis but which are not, strictly speaking, cellulases or rhamnosidase and -glucosidase activities in pectinase could further complicates research findings . Care must be taken in interpreting the results of pathogenicity tests as the enzyme preparation may contain a battery of enzymes . This result may suggest the need to study the presence of other enzymes in an enzyme of interest even if the enzyme of interest is from a commercial source . Contrary to some of the findings associated with enhanced f. compactum infection on broomrape, f. oxysporum pathogenicity on broomrape was not enhanced by pectinase and/or cellulase . Thus, although f. oxysporum and f. compactum belong to the same genus, they may have different mycoherbicidal mechanisms . Mycelia of f. compactum did not penetrate nor show apparent damage to the tomato roots as also observed in this study . In conclusion, pectinolytic and cellulolytic activities are widely exhibited by bacteria and fungi . The enzymatic activities can predispose broomrape tubercles to infection by fungi, in this case, f. compactum . The latter fact adds to the commercial value of f. compactum as a potential mycoherbicide when sufficiently virulent.
Isotopic signatures of environmental plutonium are generally used to assess the origin of the material . While decay counting is restricted to pu, pu, and pu, all mass spectrometric methods can in principle detect the isotopes pu (t1/2 = 24.1 ka), pu (6.65 ka), pu (14.4 a), pu (373 ka), and pu (80.8 ma). However, there are several publications available concerning isotopic concentrations of the minor plutonium isotopes pu, pu, and pu in environmental samples . Has assessed the global distribution of pu and pu, while uses pu to distinguish chernobyl plutonium from global fallout . In we have shown how the isotopic vector of pu (pu, pu, pu, pu) can be measured by combining the respective best suited method for each isotope . Due to the strong interference of u in mass spectrometers, pu can generally be measured with alpha - spectrometry only . Also, while the combined activity of pu and pu can be determined efficiently by decay counting, their similar -energies are not readily separated, thus a mass spectrometer is usually required to determine the isotopic ratio pu / pu . The short half - life of pu renders -decay counting by lsc (liquid scintillation counting) the most efficient method . For the long lived isotopes pu and pu only mass spectrometric methods are suitable . Among the mass spectrometric methods, ams provides the most sensitive measurements, probably since in most cases the limit is imposed by background from u rather than by detection efficiency . The destruction of molecules by stripping and the combination of several spectrometers, permits ams to suppress background better than other methods . If only ams is used, pu cannot be measured, and a lower efficiency is achieved for pu . However, since no additional sample processing is required, the measurement of pu can be done at very little additional effort if the concentration of pu in the sample is high enough . During the last few years, we have carried out several studies on environmental pu [57]. In most cases, the measurement of the pu / pu ratio was the main interest . However, if we encountered samples with sufficiently high count rates for these isotopes, we additionally performed measurements of the minor pu isotopes pu, pu, and in some cases even pu . Dating with plutonium can be done very precisely by using the ratio am / pu (e.g.). The parent nuclide pu (t1/2 = 14.4 yr) and the daughter am are different chemical elements and thus this ratio yields the date of either the irradiation or that of the last chemical separation . While this is a useful method for applications in the scope of nuclear safeguards, chemical fractionation occurring while the sample resides in the environment compromises its use for environmental studies . Without the presence of the daughter isotope am in the sample, the age since the irradiation can be assessed only if the initial isotopic abundance of pu is known . This abundance, however, depends strongly on the production process, and thus is generally ambiguous . We propose to use the isotopic ratios of the other plutonium isotopes to estimate the initial pu content . To check the accuracy of this estimate, we have applied the method to measured literature values for a thermonuclear weapons test, and for the chernobyl accident where we adopt the numbers obtained by given by . Additionally, we have simulated thermal neutron irradiation of u. to demonstrate the practical applicability, we have measured environmental samples of different origin . The short lived nuclide pu (t1/2 = 5 h) decays in a reactor before pu is produced, which results in cm being the isotope of mass 244 produced in a reactor . In a nuclear explosion, however, in which all nuclear reactions happen on a time scale of microseconds, pu can be produced directly via neutron captures on plutonium isotopes, and also by captures on uranium and subsequent -decays after the explosion . Results have been published for the thermonuclear weapons test ivy mike (pu / pu = (11.8 0.7) 10) and for the low - yield test (pu / pu = (2.3 0.4) 10). Local weapons test fallout in bikini atoll sediments and soils has been measured with ams recently by, yielding pu / pu ratios between 2.8 10 and 5.7 10 . In the outer layer of a deep - sea manganese nodule a pu / pu ratio of approximately 1 10 has been observed (taken from fig . 3 of), which the authors attribute to a global fallout . Samples were obtained from three different projects . In depth profiles of pu and pu (among others) were obtained from the region of nassfeld (salzburg, austria). While cs and sr in this region stem from the reactor accident in chernobyl, pu / pu and am / pu activity ratios as well as the pu / pu isotope ratio determined by ams identified global fallout as the source of plutonium . The samples used for this work (t2f and t2 g in) were collected on the mountain pasture nafeld - alm at an altitude of 2530 m asl, at 47.018 n, 13.012 e on august 25th, 1999 . The chemical separation of the samples (separating am from pu) was carried out in summer 2006, the ams measurement in august 2006 . The discharge history of pu from sellafield is well documented . Though releases extended from 19521992, the peak discharge occurred between 1970 and 1980 (fig . 1). A marine sediment core was collected in 1993 at 54.416 n, 3.563 w by the research vessel gauss, federal maritime and hydrographic agency, germany . This core was investigated for a number of isotopes in and recently for pu isotopes and u with ams at vera . Generally, the chronology of the 48 cm long core is unclear, since mixing of the sediment is suggested by the oceanographic situation of the sampling site and hence expects an average integral ratio of the discharges in the core; despite this, clearly discriminated peaks in the pu, pu, and pu are observed in . For the present work, the ams sputter targets prepared for were re - measured for pu, pu, pu, pu and pu . The chemical sample preparation was performed in november 2009, while the ams re - measurement was done in december 2011 . For some of the samples, pu / pu and pu / pu data have already been given in, however the data in the present manuscript stems from the new measurement . The garigliano nuclear power plant was situated at sessa aurunca near the garigliano river in campania, italy . It consisted of a boiling water reactor with a thermal power of 506 mw, and was in operation from 1964 to 1978, and is presently in the decommissioning phase . Though mainly operated with 2.3% enriched uo2, mixed oxide fuel, consisting of uo2 and puo2, was also used . During operation and decommissioning, a core from the sediment inside the (now dry) drain channel (41.257 n; 13.832 e) and another one from the entry point of the channel into the river was collected in may 2006; pu was separated in autumn 2006, and the ams measurement was performed in august 2007 . Based on the pu depth profiles, in studies on the yield of heavy elements in thermonuclear explosions a general exponential trend with increasing mass was observed . This trend can be understood under the simplifying assumption of an identical cross section for all involved (n,) reactions; the chance to produce a mass n masses above the initial nucleus is then the probability of absorbing exactly n neutrons, which is described by a poison distribution . As long as the neutron absorption probability is small, furthermore, the higher binding energy of even - even nuclei leads to a higher abundance of isotopes with even masses; a factor of 1.58 was observed for the ivy mike device . Thus the initial ratio between mass 241 and 239 will be the same as that between mass 242 and 240:(1)241pu239pu0=242pu240puand thus we obtain the time t since irradiation(2)t = t1/2ln2ln(241pu239pu)(242pu240pu)with t1/2 = 14.4 yr the half - life of pu . Despite the present - day availability of improved nuclear data for a more detailed simulation, calculation of the actual isotope yields is complicated by the fact that a constant neutron flux throughout the uranium or plutonium used in the devices cannot be assumed . The resultant distribution will generally exhibit a higher yield of the heavier masses (more frequently produced in the high flux spots) than indicated by the lighter masses . Simulations are possible for simplified cases, especially for thermal neutrons, which corresponds roughly to the case of reactor production . Additionally, we assume that the duration of the irradiation is much shorter than the half life of pu . Both neutron capture and fission are taken into account, the corresponding cross sections were taken from the endf / b - vii.0 database . The system of differential equations was solved numerically with mathematica (wolfram research, champaign, il, usa) for different assumed burn - up levels . The methods to extract the plutonium from the environmental samples varied between the different projects; the (dry) sample mass was 1 g for sellafield, 212 g for garigliano, and 20 g for salzburg samples, respectively . Generally, the samples were leached, and plutonium was extracted with ion exchange . After co - precipitation with 1 to 3 mg iron, the sample was calcined to produce plutonium oxide embedded in an iron oxide matrix . The ams facility vera is especially equipped for the measurement of heavy ions . Uranium pilot beams are used for tuning the spectrometer, which is then scaled to the various pu isotopes for the measurement . The switching times between the different isotopes is typically 15 s, therefore fast variations of the ion source output are not completely averaged out and contribute to the uncertainty of the measured isotopic ratios . The high - energy beam analysers are designed to allow the transport of 5 + actinide ions at a terminal voltage of 3 mv . Oxygen is used as a stripper gas which results in a stripping yield of 5% for this charge state . For most previously published data a time - of - flight (tof) detector with 25% efficiency was used to further suppress potential background from u; but we have never observed such background, so measurements can also be carried out without tof, resulting in a theoretical detection efficiency of 4 10 . However, under typical sputter source conditions, for 10 pu atoms in the sputter target we obtain a count rate of 30 s (for puo2 only 1/10 of this count rate is observed). Since the sputter targets last for many hours, the available accelerator beam time usually limits the detection efficiency . We hope that we can improve on this by reducing the amount of iron matrix used, which requires new handling procedures . Counting statistics were no limitation for the sellafield marine core samples, since their concentration of radionuclides is exceptionally high; as already reported in, to avoid saturation of the detectors by the very high count rates we had to reduce the source output strongly, by running the cesium reservoir essentially at room temperature instead of the usual 150 c . Therefore, these sputter targets appeared almost untouched after the measurement . All data was normalized to a pulser running at a fixed frequency to correct for any detector dead time that arose as a result of high counting rates . Since all pu isotopes are measured in the same ionization chamber, we assume the same ion optical transmission . The ams device can in principle introduce large fractionation, since each isotope is essentially measured with a different tuning of the (relatively complex) mass spectrometer, and the tunings may differ in quality . Since we obtain our plutonium tunings by scaling from the u pilot beam the heavier plutonium masses are more likely to suffer from ion optical losses . The resultant uncertainty is difficult to assess, since no plutonium standard with isotopic ratios suitable for ams is available so far, which would allow to monitor and correct machine fractionation for every measurement . However, reference materials measured in the past suggest a typical precision better than 10% . To monitor whether the beam tuning degrades as a result of drift in the components, the u / u ratio of our in - house uranium standard vienna - kku is measured using three sputter targets per turn of the sample wheel . It should be noted that systematic trends in the ion optical transmission of the different pu isotopes will partly cancel out in the double - ratio used in eq . We expect little fractionation during chemical sample preparation, and blank material allows any laboratory contamination to be traced . The beam analysers will not distinguish between the in - growing am and the remaining pu . However, the time span between the separation of am and the ams measurement is still relevant . Am (most likely) has a different negative ion yield to pu, which introduces an uncertainty into the mass 241 determination . We are not aware of any published ionization yield for am, but has investigated other monoxide ions of actinides: the relative negative - ion formation probability of tho, uo, npo, and puo is approximately 1:3:5:7 . As an example, we assume that am does form negative monoxide ions even two times better than pu, and that the measurement is carried out one year after chemical separation . Since 5% of pu will have decayed into am, the observed count rate for mass 241 will appear 5% higher, which corresponds to a 1 yr lower age . As another extreme example, if we assume that am does not form any negative monoxide ions at all (which is definitively not the case, see), the sample will appear one year older - as it actually is . Since no information is available on the actual ion yield of am, which is moreover expected to vary with the matrix of the sputter target and with ion source conditions, we assume an additional uncertainty of 1 year for the age determination per year between separation and measurement . Fig . 2 and table 1 shows the results measured in this work, in comparison to the numbers given for the ivy mike 43 counts of pu were successfully measured on salzburg soil within 1.5 h. the resultant isotopic ratio for pu / pu is (5.7 1.0) 10 . A contribution of reactor material in our samples, in particular from the chernobyl accident, could cause a lower ratio . However, a significant contribution from chernobyl was excluded in . Additionally, while a pu / pu ratio of 2.7 10 is expected for chernobyl debris in 2011, our measured ratio of (1.43 0.06) 10 is more than one order of magnitude lower, and lies perfectly on the eye guide for global fallout (through masses 239 and 242 of salzburg soil). We think that our value better represents the northern hemisphere global fallout ratio than that taken from . A possible explanation for the higher ratio observed in the deep sea crust (va132, 918 n, 14603 w, 4830 m depth) would be influence from the various nuclear test sites in the south pacific . We think this assumption is supported by the pu / pu of 0.25 observed in the nodule sample (also taken from fig . 3 in), while the accepted northern hemisphere global fallout average is 0.185; this value is also observed in our salzburg soil samples . (2) for the measurements from literature and the simulations for thermal neutrons is shown in table 2 and fig . Since all ratios were calculated for the time of production, or corrected for pu decay, an age of 0 is expected in all cases . While the mike test appears 6 years older than expected, the simulated thermal neutron results appear too young, and the deviation grows with the assumed burn - up . The chernobyl data appears 5 years too young; a more accurate estimate is obtained if we assume production by thermal neutrons . In this case (2), but fit the simulation to the measured pu / pu ratio, which results in a total thermal neutron fluence of 0.0025 b, and in an estimated age deviating by less than 2 years from the known calendar date of the chernobyl explosion . This agreement is surprising, considering that the processes in the actual reactor deviated significantly from a simple thermal neutron irradiation . For the salzburg soil, an age of 52.5 1.5 yr is obtained, corresponding to the year 1954 . This age is 8 yr before the maximum of the nuclear weapons tests in 1963, and the deviation is similar to that for the ivy mike test . If this trend holds for all material produced in nuclear explosions, a correction could be applied reducing the deviation . The garigliano results suffer from a larger statistical uncertainty, due to lower pu concentration in the samples . The age is 42 8 yr, which corresponds to the year 1965, in agreement with expectations . Our new pu / pu data for the sellafield core agrees with previous measurements on the same sputter targets presented in, while the three pu / pu ratios given there have significantly lower precision . However, one sample (st60 k) shows a deviation of about 25% from the previously published data, which is much more than allowed by counting statistics (see table 1). We attribute this outlier to variations in the transmission of pu through the spectrometer, limiting the reproducibility of the present method . Since a deviation of 25%, corresponding to a dating error of about 5 years, is relevant for the present work, this emphasizes the need for an ams standard which would allow to trace and correct for such variations . Our sellafield dataset is well suited to illustrate the advantage of using the pu information for dating . (2) compared to an estimate of the initial pu / pu which is based only on pu / pu:(3)241pu239pu0=240pu239pu2 while the age shift due to the neglected even odd offset of this simpler method could probably be handled, the pu information establishes a monotonous age - depth curve for the sediment core, however the data covers only a short time span of seven years . The time range 1977 to 1984 falls well into the later part of the sellafield discharges; however, if the age is calibrated against a simulated thermal neutron irradiation, a fluence of 0.0015 b fits best, and leads on an estimated age correction of 5 years . The conventional approach towards source assessment of an unknown material is to measure elemental concentrations and isotopic ratios, and to compare them with a database of all viable sources . This works also for environmental plutonium isotopes, and probably would lead to the same source assignments as the method proposed in this work . However, we think that the calculation of predictive values like burn - up, irradiation date, or production mode (i.e. Reactor vs. explosion) makes the assessment more straightforward . The measurement of pu, pu, pu, pu and pu allows for more precise assessment of the history and origin of environmental plutonium . Additionally we have shown that the use of pu and pu allows dating of the time of irradiation accurate to within 6 years, while our data on the irish sea sediment core suggest that relative dating of material from the same source is possible with a precision of less than 2 years . The information contained in the other plutonium isotopes may allow further improvement in the precision . When a reactor origin can be assumed, possibly based on the absence of pu, and by calibrating the data against simulated thermal neutron data with the same pu / pu, an accuracy of 2 years is suggested by our data.
Since the introduction of tooth whitening by haywood and heymann in 1989, this trend is getting more popular . There are a few types of bleaching methods, but all of them share a common principle of the decomposition of peroxides from hydrogen peroxide or its compounds such as carbamide peroxide (cp) into unstable free radicals . These radicals further breakdown into large pigmented molecules either through oxidation or reduction reaction . The oxidation or reduction process changes the chemical structure of the interacting organic substances of the tooth, which results in the change in color . Furthermore, at 45% concentration of cp in different intracoronal tooth - bleaching procedures, maleknejad (2012) reported an increase in the diameter of dentinal tubules, and it also promoted alterations in the mineral content of the dentine . Types of bleaching methods include nonvital bleaching, in - office professional bleaching, and home bleaching . Nightguard home bleaching uses a relatively low level of whitening agent and is applied to the teeth via a custom - fabricated mouthguard and is worn at night for a duration of at least two weeks . A lot of research has been carried out to identify the effects of bleaching on the tooth surface and dental restorative materials . Jacob (2007) reported that bleaching with cp may alter the marginal leakage of resin composite restoration; however, amalgam restorations were not adversely affected in vitro . It was reported that the bleaching agent, regardless of the whitening products used, will reduce the microhardness of the enamel and promote an increase in surface roughness . Hence, it is widely used for anterior and also posterior restorations . Due to this some studies concluded that application of home bleaching agent gels may cause surface softening of composite resins, whereas others stated that there are no significant changes in surface microhardness found after the application of 15% cp on composite resin . Furthermore, a study was done to evaluate the effects of bleaching on toothbrush abrasion in three types of composites . It was concluded that nano - filled resin composites are to be used for restoration when bleaching treatment is required . There is a significant and positive correlation found between surface roughness and vital bacterial adhesion . It appears that bleaching agents may affect the adherence of certain cariogenic micro - organisms to the outer surfaces of composite resin restorations . Roughness has a major impact on aesthetic appearance, discoloration of restorations, plaque accumulation, secondary caries, and gingival irritation . The experimental composite tested in this study was a locally produced dental restoration nanocomposite (kelfil). This nanocomposite was produced by the school of dental sciences (ppsg), universiti sains malaysia (usm), kelantan, malaysia . The nanosilica fillers, synthesized locally by a sol - gel process, were in the size range of 1020 nm . The experimental nanocomposite (enc2) contained 35 wt% of nanosilica fillers . This material (enc2) the aim of this study was to study the effects of home bleaching on the hardness and surface morphology of a universal nanocomposite, an anterior nanocomposite, and a nanohybrid composite . Two commercially available resin - based composites, based on bisphenol a - glycidyl methacrylate / triethyleneglycol dimethacrylate / ethoxylated bisphenol - a - dimethacrylate (bis - gma)/ethoxylated bisphenol - a dimethacrylate (bis - ema)/triethylene glycol dimethacrylate (tegdma)] resin chemistry (filtek z350; 3 m espe, st . Paul, mn, usa and tph 3; dentsply caulk, milford, de, usa) and one experimental resin - based composite based on bis - gma / urethane dimethacrylate (udma)/tegdma resin chemistry (kelfil) were used in the present study [table 1]. All composites were packed into acrylic molds (4 mm diameter 2 mm thick) which were placed on a mylar strip . The excess composites were removed using another mylar strip on top of the mold and pressed flat with a glass slab . Eighteen samples of each composite material were prepared, with a total at 54 samples . All samples were light - cured for 20 seconds from each top and bottom surface using elipar freelight 2 (3 m espe st . All samples were then polished using sof - lex (3 m espe, st . Four different roughness of disks were used, which were coarse (55 m), medium (40 m), fine (24 m), and ultrafine (8 m), using a mandrel on a slow - speed handpiece, 10 seconds per disk without water . Conscious efforts were made to standardize strokes, downward forces, and number of strokes for each disk . All samples were stored in a distilled water bath at 37 c for 24 hours prior to the treatment procedure . Composites resin and bleaching agents tested the samples from each composite resin material were further divided into three groups; groups 1, 2 and 3 with n = 6 . Samples in group 1 as the control group were not bleached but stored in shaking water bath sw23 (julabo, seelbach, germany) for 14 days at 37 c of distilled water . Samples in group 2 were subjected to opalescence pf (ultradent product, south jordan, ut, usa) 10% cp for eight hours per day, for 14 days according to the suggested wear time of the manufacturer . Samples in group 3 were bleached with opalescence pf (ultradent product, south jordan, ut, usa) 20% cp for two hours per day for a total of 14 days . In groups 2 and 3, prior to each bleaching procedure, the samples were taken out from the distilled water bath, air - dried with an air jet spray for 60 seconds . The bleaching agent was applied on one surface of the sample using a microbrush (kerr corporation, california, usa) and left for the duration suggested by the manufacturer . After each bleaching procedure, the samples were washed with a water jet spray for 60 seconds before they were stored back in distilled water bath and ready for the next bleaching procedure the next day . All samples were subjected to surface roughness testing using atomic force microscopy (afm) (ambios technology, california, usa). Five different areas were selected randomly with a scan area of 40 40 m and resolution 512 512 pixels to obtain surface roughness values (ra). Then, three - dimensional (3d) images with 10 10 m sizes were acquired for each group of materials . After surface roughness testing, all samples were subjected to hardness testing using a vickers hardness tester, model vm50 (fie, maharashtra state, india). The samples were placed underneath the indenter and 1 kg load was applied for a dwell time of 15 seconds . Every sample was indented three times at three different points, and the mean readings were recorded . All statistical analysis was conducted at a significance level of p <0.05 using mann - whitney test and kruskal - wallis test . All statistical analysis was conducted at a significance level of p <0.05 using mann - whitney test and kruskal - wallis test . The results of the statistical analysis using kruskal - wallis test are presented in tables 2 and 3 . Table 4 reveals the result of mann - whitney test analysis of kelfil and table 5 is the result of mann - whitney test of tph 3 . Median roughness number (ra, nm) and interquartile range of the three tested composite resins after 14 days of bleaching with 10 and 20% cp median vickers hardness number and interquartile range of the three tested composite resins after 14 days of bleaching with 10 and 20% cp median vickers hardness number and interquartile range for kelfil after 14 days of bleaching with 10 and 20% cp median vickers hardness number and interquartile range for tph 3 after 14 days of bleaching with 10 and 20% cp table 2 shows the roughness number (ra) of the three types of composites that were bleached with 10 and 20% cp home bleaching agents . There was no significant change in roughness in all three composites after 14 days of bleaching with 10 and 20% cp compared to the control group . Table 3 illustrates the results of all the samples that were subjected to the vickers hardness test . Kelfil showed significant change in hardness with a p value of 0.001 and there was also a significant change in tph 3 with a p value of 0.021 . Table 4 shows that there was a significant change in kelfil after 14 days of bleaching . There was a significant increase in the surface hardness after bleaching with 10% cp home bleaching agent . However, there was a significant decrease after bleaching with 20% cp home bleaching agent . There was also a significant decrease in the surface hardness of kelfil after bleaching with 20% cp when compared to the group of samples bleached with 10% cp . Table 5 reveals a significant change in the surface hardness of tph 3 after being subjected to 14 days of bleaching . There was a significant decrease in the surface hardness in the groups bleached with 10 and 20% cp compared to the control group . However, there was no significant change when the group was bleached with 10% cp compared to the group bleached with 20% cp . In this study, filtek z350, a commercialized and widely used nanocomposite did not show any significant changes in surface hardness when compared to its control group, and also to groups of samples bleached with either 10 or 20% cp . However, there were significant changes in the vickers hardness number for both kelfil and tph 3 that had been subjected to 14 days of bleaching with either 10 or 20% cp . For kelfil, there was a significant increase in hardness after bleaching for 14 days with 10% cp but a significant decrease in hardness with 20% cp . After the curing process of a composite resin, a postpolymerization process continues to occur up to a certain period of time, which increases the hardness of the composite . In this study, the bleaching process was possibly being carried out at the same time as the postpolymerization process . The postpolymerization process in kelfil was observed with the 10% cp, and it was more prominent than the bleaching process; hence the increase in its surface hardness . . Nevertheless, with 20% cp, the bleaching process was more dominant than the postpolymerization process in all the three composites tested . The increase in hardness with 10% cp was observed in both filtek z350 and kelfil, and it maybe due to the similarities in their filler structure . In tph 3, there was a significant decrease when comparison was made between the control group and 10% cp group, and also between the control group and 20% cp . This significant decrease is consistent with the conclusion drawn from other studies . As a general rule, the higher the filler loading, the greater the physical properties of the composite resins . It has a lower filler loading of 29% by volume compared to filtek z350 with the filler loading of 78.5% by weight or 59.5% by volume which is suitable for anterior and also posterior composite resin restorations . The lower filler loading of kelfil may lead to loosely packed inorganic fillers, which in turn caused the significant decrease in the vickers hardness number . For tph 3, there was a significant decrease in vickers hardness number in samples that were subjected to 14 days of bleaching with both 10 and 20% cp . It was concluded that nanohybrid resins generally presented inferior properties compared to the nanofilled composite . Tph 3 has 58% volume filler loading, which is similar to filtek z350 . The possible explanation for the significant reduction in the surface hardness might be due to the presence of bis - gma monomer . Resin composites are reported to be highly susceptible to chemical softening due to the presence of bis - gma monomer if the chemicals have a solubility parameter ranging from 1.82 10 to 2.97 10 (j / m). In the case of tph 3, the content of bis - gma in its matrix might be higher than in filtek z350 . Another possible explanation is the degree to which the filler is bonded to the resin matrix . Although there was a significant decrease in the vickers hardness numbers of kelfil and tph 3, there were no significant changes in the roughness number in all specimens after 14 days of bleaching with either 10 or 20% cp opalescence pf home bleaching agents . This is consistent with the conclusion that no statistically significant differences were detected for any material . The cantilever sensor of afm senses any irregularities at the surface of the specimen; however, as all the composites tested have similar nano - sized fillers, approximately 20 nm, there was no significant difference between them . Furthermore, all samples were polished with the same polishing system, sof - lex disks which may contribute to a similar surface finish in the composite resins even after bleaching treatment . The 3d images of afm [figures 13] show that there was no plucking out of filler particles after the bleaching treatment . Afm topography 40 40 m of z350 after bleaching with 20% cp afm topography 40 40 m of kelfil after bleaching with 20% cp afm topography 40 40 m of tph 3 after bleaching with 20% cp generally, the surface roughness of all composites tested has a reading below 0.2 m or 200 nm . Ra above 0.2 m results in an increase in the accumulation of plaque and a higher risk for caries and periodontal inflammation . According to chung in 1994, when ra was lower than 1m, therefore, all composites surfaces evaluated after bleaching treatment have demonstrated a smooth surface, which from the clinical point of view, present no risk of accumulation of plaque . From this study, it can be inferred that following bleaching, the experimental anterior composite and nanohybrid composite may need to be replaced, due to the reduction in their surface hardness . However, this has to be further investigated with in vitro studies evaluating the effects of saliva, and controlled clinical trials are necessary to determine any clinical implication . Based on the result of this study, it can be concluded that 14 days of bleaching with 10 or 20% cp home bleaching agents did not cause any changes in surface roughness of the three tested composites . The afm evaluation of surface roughness showed that both bleaching agents yielded ra values below 200 nm for all materials tested, which poses no risk of the accumulation of plaque . The performance of kelfil after bleaching treatment is similar to that of the commercial nanocomposites . The afm evaluation of surface roughness observed in the 3d images promises to be an effective technique . Nanocomposite and nanohybrid composite resins bleached with 10 or 20% cp bleaching agents result in the same outcome . On the other hand,
Penile squamous cell carcinoma (pscc), the predominant histological type (> 95%) of penile cancer, is a relatively rare malignant tumor in western countries and japan . Pscc is a rare disease, making it difficult to establish a standard of care in any of the clinical stages, particularly in advanced disease . Multimodal treatments, including surgery, chemotherapy, and radiation therapy, should be considered for patients with advanced pscc . However, the optimal chemotherapeutic regimen is unknown, although cisplatin - containing chemotherapy is the mainstay of combination chemotherapy . Recent studies have suggested that taxanes in combination with cisplatin and fluorouracil (5-fu) have a significant effect on unresectable and recurrent penile cancer . Malignant wound, results in a decline in the quality of life because of the presence of bleeding, exudates, and/or strong odor . Mohs chemosurgery is a technique of chemical fixation of a cutaneous tumor and subsequent excision . Several studies have shown the efficacy of mohs paste for maintaining malignant wounds in advanced squamous cell carcinoma of the breast, skin, head, and neck [4, 5]. We report a case of a pscc with advanced lymph node metastasis treated with combination therapy consisting of taxane - based chemotherapy, irradiation, and mohs paste . An 80-year - old male presented to a community hospital with pain and redness in his left inguinal region . He had undergone penectomy and bilateral inguinal lymphadenectomy 1 year ago because of primary pscc (pt1pn0). A large solid mass with bleeding and a smelly exudate was observed in his left inguinal region . Macroscopically, the tumor mass of the left inguinal region was 7.0 cm in diameter with an ulcer, redness, and exudate (fig . A ct scan revealed a huge mass (5.9 5.8 cm) in the left inguinal region along with his left femoral vessels (fig . He was administered combination therapy consisting of a taxane - based chemotherapy, irradiation, and mohs chemosurgery . The chemotherapy regimen consisted of 60 mg / m docetaxel administered over 3 h on day 1; 750 mg / m 5-fu on days 15; and 70 mg / m cisplatin on day 4 . The patient also received 50-gy external - beam radiation therapy to the left inguinal region, initiated on the same day of chemotherapy . Mohs paste was applied every morning with a surrounding gauze to avoid attaching with normal skin (as previously reported). Mohs paste included 50 ml of zinc chloride - saturated aqueous solution, 10 g of zinc powder, and 15 ml of glycerin . Radiation therapy was temporarily deferred for 12 days beginning on day 9 because of neutropenia and general fatigue . The necrotic tissue fell off and the wound flattened on day 20 after starting chemotherapy . 2b), and a ct scan showed that the tumor had decreased by 70% in diameter at 1 month after the first course of chemotherapy (fig . 3b). Because of socio - economic reasons, the patient declined additional treatment . There was no progression or metastasis on the chest and abdominal ct for 8 months . However, the wound grew and became erosive again 10 months after discharge, and the patient died because of the progression of local recurrence 1 year after chemotherapy treatment . In our patient with inguinal recurrence of pscc, multimodal treatment, consisting of combination chemotherapy, irradiation, and mohs chemosurgery, was responsible for the tumor shrinkage and the successful local control of a malignant wound . Although this field is limited by a paucity of clinical trials or prospective data, the available single institutional retrospective reviews indicate that multiagent cisplatin - based chemotherapy regimens fight significantly against pscc . In 1991, dexeus et al . Reported a triple - drug chemotherapy regimen, which soon became a standard regimen for pscc with tolerable adverse effects . The regimen consisted of 20 mg / m cisplatin on days 26, 200 mg / m methotrexate on days 1 and 15, and 10 mg / m bleomycin on days 26 . They reported a response in 10 of 14 (71%) patients (with moderate side effects). In their follow - up studies, a lower rate of complete responses with severe toxicities was reported [8, 9]. With respect to scc of the head and neck, in which pscc is historically similar, a randomized trial revealed that the addition of docetaxel significantly improved progression - free, and overall survival in patients with unresectable scc compared to the standard regimen of cisplatin and 5-fu; therefore, docetaxel, cisplatin and 5-fu have become the current standard induction regimen for advanced scc of the head and neck . In pscc, pizzocaro et al . They reported a high activity of this regimen, with 5 of 6 treated patients showing a response . Based on these findings, we chose docetaxel for taxane - based combination therapy for the patient, although there are few reports demonstrating its efficacy against pscc . Further studies are needed to determine which taxane has a better response and less toxicity in patients with pscc . Radiation therapy has been used for many years in the treatment of pscc for the primary tumor, inguinal metastases and distal metastases . It may also play a role in the treatment of locally advanced penile cancer, particularly when inguinal adenopathy is initially unresectable . There are no randomized trials that have evaluated the impact of radiation on the prognosis and the local control of patients with inguinal metastasis originating from pscc . However, ravi et al . Revealed that palliative radiation therapy showed an amelioration of symptoms in 56% of patients with inguinal metastasis in growing pscc . In addition, the radiosensitizing effect of cisplatin, 5-fu, and docetaxel has been demonstrated in several types of cancers [13, 14, 15]. Therefore, it is plausible that radiation in combination with cisplatin, 5-fu, and the docetaxel regimen is a promising option for multimodal treatment of pscc . Malignant wounds from primary or metastatic carcinoma are generally incurable, and palliative methods to manage the wounds are needed . Frederic e. mohs developed and published a technique for the chemical fixation of a cutaneous tumor in 1941 . Mohs paste is effective for the hemostasis of bleeding, odors, and exudates, thereby contributing to the patient's quality of life . In japan, kakimoto et al . Reported on 5 patients with breast cancer who were successfully treated with mohs paste . Showed the efficacy of radical surgery followed by systematic therapy and mohs paste in patients with breast cancer . In our case, the tumor started to decrease in size after treatment and the necrotic tumor fell off by day 20, without any resection . To the best of our knowledge, this is the first report demonstrating the advantage of mohs paste for inguinal recurrence of pscc . Our result shows that mohs paste may be an effective and reliable option for multimodal treatment of inguinal recurrence . It is difficult to define which treatment modality was the most effective for the patient, because we used a combination of 3 different modalities . Although our result suggests that each treatment was synergistically effective, it is also important to delineate which modality is the most effective and then choose a suitable modality for each patient with pscc . Secondly, the socio - economic background of the patient did not allow him to continue systemic treatment and/or maintenance therapy . The study showed that paclitaxel was well tolerated, with a moderate activity against pscc . Therefore, the patient may have been able to avoid cancer progression for a much longer time if he had received additional maintenance chemotherapy . In conclusion, we report a case of successful local control of a recurrent inguinal mass of pscc treated with multimodal therapy . To the best of our knowledge, combination treatment with taxane - based chemotherapy, external beam radiation therapy, and mohs paste is a reliable option for the induction therapy for recurrent pscc . Further studies are needed to investigate maintenance treatment after effective induction therapy for recurrent pscc.
The majority of women living with hiv are in their reproductive years (ages 1549) [1, 2]. The dramatic decrease in the risk of mother - to - child hiv transmission (mtct) is leading to normality in the lives of couples affected by hiv, who want own children . In europe, the reduction in mtct to less than 1% is mainly due to highly active antiretroviral therapy (haart). Effective haart is resulting in suppressed viral load (vl); thus, a vaginal birth can be as safe as a planned caesarean section [3, 4]. Avoidance of breastfeeding and postnatal neonatal postexposure prophylaxis (pep) further supports the effective reduction in mtct [35]. The literature suggests that there is no increased rate of fetal malformations due to the hiv infection or haart [6, 7]. A pregnant woman with hiv infection large studies of noninvasive prenatal screening have already indicated that it will lead to a decrease of invasive prenatal screening procedures such as amniocentesis (ac) or chorionic villi biopsy (cvs). If invasive prenatal testing is necessary, it can be done, but in these circumstances, haart should be started prior to the procedure to suppress the vl below the limit of detection . In these cases current, evidence suggests that mtct is very unlikely; however, studies reporting on the risk of mtct in invasive prenatal testing are limited due to small study size . Haart is given during pregnancy for two reasons, first to women with an own indication for haart (they require treatment for their own health) and secondly to pregnant women starting therapy purely as a prophylactic treatment to reduce mtct . The aim of our study was to investigate if pregnant hiv - positive women get referred for special prenatal ultrasound screening services in our tertiary referral center, but also if and at what point the prenatal ultrasonography is performed . Pregnant hiv - positive women usually have a combined antenatal care in a tertiary referral center and with their own gynaecologists . As well as the prevalence of prenatal ultrasound screening, prenatal, and postnatal finding was recorded . We hypothesized that the fetal anomaly rate in women with hiv - infection is as low as in all other pregnancies (35%) [12, 13]. Hiv - positive pregnant women who presented in our tertiary referral center between january 1, 2002 and december 31, 2012 were included in this retrospective cohort study . Three categories were used: very preterm delivery (24 + 0 to 33 + 6 weeks of gestation), preterm delivery (34 + 0 to 36 + 6 weeks of gestation), and term delivery (37 weeks of gestation). All data regarding early prenatal screening (as, e.g., nuchal translucency measurements) and fetal anomaly scan at 20 weeks of gestation or at first presentation in our center were recorded . Only scans which were performed in our center were included, reflecting the fact that hiv - positive pregnant women are high risk pregnancies, and high - risk pregnancies are referred to a tertiary center or an equivalent specialized center for prenatal screening [1417]. An early anomaly scan was defined as a first trimester scan; in the study period, the fetal nuchal thickness was assessed; a formal nuchal translucency measurement was included if measured by appropriately qualified sonographers . A fetal anomaly scan was defined as a detailed scan in the second trimester (usually between 20 to 22 weeks of gestation). All the scans performed at a later gestation in our department prior to birth are recorded separately as late scans in the third trimester . Malformations were any fetal / neonatal disease, which required either surgery or special pediatric care including chromosomal anomalies . All cases with an ac, mtct, and any intrauterine or postnatal death were evaluated . Maternal information included age, ethnicity, gestational age at delivery, gravidity and parity, haart already before the pregnancy, vl (copies / ml), cd4 count (cells/l) prior to birth, and other risk factors such as coinfection with hcv . The last recorded vl prior to the delivery was used and classified in three risk groups . In the study, a vl below 50 is considered as negative / undetectable . The last cd4 count prior to birth was noted, and again three categories were used . The mode of delivery was classified as (1) planned caesarean section; (2) in cases of rupture of membranes and/or contraction it was recorded as elective caesarean section in labour; (3) emergency caesarean section; (4) caesarean section after planned vaginal birth; (5) vaginal birth; (6) unplanned vaginal birth and (7) instrumental vaginal delivery . In the unit the first planned vaginal birth was recorded in 2009 . Before that time, women were offered elective caesarean section at around 37 + 0 weeks of gestation [3, 8, 19]. With evidence for the safety of the vaginal birth with undetectable vl, the policy in the unit shifted towards planned vaginal birth, and if caesarean section was offered in these cases, the delivery was delayed until> 37 weeks of gestation according to the german - austrian guidelines [3, 4, 8]. The following neonatal data were included: apgar score, arterial cord ph (aph), cord base excess (be) and neonatal weight (stratified according to 10th, 1090th and> 90th percentile). A weight below the 10th percentile was considered to be intrauterine growth retardation (iugr). Information about scan findings was obtained from the record of the ultrasound department, and further information was collected from maternal case notes, pediatric notes, and discharge letters . Ethics approval for the retrospective study was obtained from the ethics committee at the j. w. goethe university, frankfurt (number 30/13). For categorical variables and nominal variables, frequency tables were used for descriptive statistical analysis . For ordinal and quantitative data, mean and these data were further analyzed using the wilcoxon - mann - whitney test, kruskal - wallis test, spearman - correlation, chi - test, and fisher's exact test as appropriate . In addition, multivariate logistic regression analysis was performed to identify factors associated with a woman having an early anomaly scan . Overall 330 pregnancies were recorded, with 322 singleton pregnancies (97.6%) and in eight twin gestations (2.4%). One twin pregnancy was conceived due to ivf with first diagnosis of the hiv - infection in the early second trimester . There were 122 preterm deliveries (36.5%) and 90 (26.9%) of these were between 34 and 36 + 6 weeks of gestation . Maternal and neonatal characteristics are presented in table 1, stratified by pregnancy duration in table 2 . Two thirds of women (66.4%) were of african ethnicity . In one quarter of women, more than three quarters 257 (77.4%) of the births were elective caesarean section . In 29 cases (8.7%), women delivered vaginally . The cd4 count (cells/l) prior to birth was in the majority of 175 (62.5%) 350, in 76 (27%) between 200 and 349, and in 30 (10.7%) <200 . The vl (copies / ml) in most women 168 (55.8%) was suppressed below 50 copies in 88 (29.2%) 50399 and in 45 (15%) 400 . One hundred and eight women (37.4%) were on no haart treatment at the beginning of the pregnancy . In 25 (8.9%), a positive anti - hcv test was recorded . Thirty newborns (9%) were classified as below the 10th percentile . In 100 of the 330 pregnancies (30.5%) the nuchal translucency was measured in 67 (20.3%) of the 330 cases (nt median 1.22 mm (range 0.63 mm)). A multivariate analysis for factors influencing a woman having an early anomaly scan (table 3) showed that african ethnicity and first diagnosis of hiv during the ongoing pregnancy were factors which significantly could be related to not having early prenatal ultrasound screening (figure 1). Invasive testing (ac) was done in three (0.9%) of 330 cases . Only one case was done at 25 weeks in our department, and we started haart and performed the ac after vl was fully suppressed . Two cases were done for advanced maternal age without control of vl and without specific precautions for example, haart, and both revealed a normal karyotype . In all of three cases, no mtct occurred . In the second trimester in 252 (74.5%) of 330, a detailed anomaly scan at 2022 weeks was done . In 18 (5.5%) patients, the scan was performed in the third trimester due to late presentation in our unit . In table 4, fetal and neonatal malformations as well as chromosomal anomalies are presented . In seven cases of 330 cases (2.1%), we diagnosed a fetal malformation . Postnatally, all of the seven cases were confirmed, and eight further malformations and two cases with trisomy 21 were detected . The chromosomal anomalies were not suspected . Both women, 33 and 39 years of age, had no early scan or biochemical screening but a scan in our unit (late in the second trimester with no anomalies seen). There were three cases with a skin tag, one nevus sebaceous of the occiput, and one case with a socalled sucking blister on the hand, all considered to be minor . Each of these cases had at least one scan in our department prior to the birth . However, the sucking blister and the nevus were leading to an upgrade in neonatal pep due to breaking down of protective skin barrier, and one newborn presented with a small omphalocele which was not seen prior to birth . . The overall fetal malformation rate (including the minor anomalies) was 4.5% . In table 5, the fetal and postnatal mortalities are recorded . In our cohort, we had six cases of intrauterine or postnatal loss and all were born by caesarean section . All of the three newborns were delivered by caesarean section, and all were preterm (33 + 6, 36 + 3 and 36 + 4 weeks of gestation). In all cases, the vl was detectable, all women were on haart, and one woman was coinfected with hcv . One woman had already a vertically infected child, and she had a poor compliance . There are conflicting results regarding the risks for hiv - positive mothers for possible adverse effects in their pregnancies [5, 6]. In our study, we confirm the low fetal malformation rate of 4.5% in women living with hiv . There are different national registers collecting data on haart and pregnancy outcome (e.g., apr: antiviral pregnancy registry; nshpc: national study of hiv in pregnancy and childhood (uk); ecs: european collaborative cohort; epf french perinatal cohort) [4, 2124]. These registers confirm the same malformation rate in women taking haart as in the general population (35%) [12, 13]. The postnatal anomalies were minor ones (skin tag, sucking blister) or missed due to minimal extend (omphalocele). The two cases with postnatal trisomy 21 were missed prenatally but were not seen in typical screening periods . There were no anomalies in the unscreened population . A change in treatment policies is evident over the 11 years of the study, reflected in the changes in delivery mode over time and the gestational age at delivery . A high preterm delivery rate is confirmed by other groups . In our population, 26.9% are late preterm deliveries (3436 + 6 weeks of gestation) and are mostly iatrogenic due to early caesarean section as in other studies and in the past . The updated national german - austrian guidelines now delay caesarean section to term in women with suppressed vl . The numbers of women with fully suppressed vl (vl <50 copies / ml) (p <0.001) and cd4 cells 350 (p> 0.20) prior to birth increased over the last years . The first is early screening which should take place between 11 + 0 and 14 + 0 weeks of gestation . This early screening was introduced by nicolaidis in 1992 as a combined method of screening (including ultrasound screening and two maternal biochemical markers: free human chorionic gonadotropin (free hcg) and pregnancy - associated plasma protein a (papp - a) [2628]. In germany this test is not covered by the national health system and there for is paid by the woman herself . Usually at that time an early anomaly ultrasound scan can be performed, which is covered by the health system . The second screening interval is the anomaly scan at 2022 weeks of gestation [17, 29]. The prevalence of first trimester screening of 97.5% in a low risk general population has been demonstrated . We demonstrate that prenatal screening is offered and available, but that the early screening interval is missed, as only 30.5% women get referred for early anomaly scan . Even so some women may have chosen not to undergo testing for ethical and cultural reasons . As a limitation of our data it could be that the screening which is done at the community - based care is missed, but as indicated usually, it warrants a referral to a highly qualified and specially trained team [16, 29]. In our study, population the majority of 188 (66.4%) women were of african origin, and in 79 (24.2%), the diagnosis hiv - infection occurred in the pregnancy, both factors were significantly related to having no early prenatal screening . Tariq et al . Are reporting on late booking for antenatal care in non - caucasian women compared with caucasian women regardless of time of diagnosis of hiv - infection . Three cases of mtct are low (0.9%) and confirmed by other groups (reporting mtct rates of 0.1%1.3%) [3, 4]. However, looking back in our data, the viral control has improved dramatically over the last 11 years; in 55.8% of all pregnancies, the vl is <50 . National health systems vary, and a complete first trimester screening (with inclusion of biochemical serum markers) has not been established on a national basis for high risk pregnancies in some european countries . In our cohort, two cases of trisomy 21 occurred, and the question remains open if these two cases could have been traced in a complete first trimester screening . In one pregnancy, an ac was performed due to suspected chromosomal anomaly, which revealed a normal karyotype . Due to the time required to initiate haart and to have a suppressed vl in hiv - positive pregnancies, invasive testing will be very likely to happen in the second trimester which will then raise the difficult ethical questions about late termination of pregnancy when an abnormal result is obtained [32, 33]. First trimester screening (including maternal markers as free hcg and papp - a) has been investigated in pregnant women living with hiv . Some groups feel that maternal markers could be less reliable than those in hiv - negative women [34, 35]. In our study data from 2002 to 2012, in the first years, the nuchal translucency was assessed but not formally measured . This could be due to the delay in having certified specially trained sonographers involved . In the future, the new methods of chromosome - selective sequencing of maternal plasma cell - free fetal dna (cfdna) in noninvasive prenatal testing (nipt) are valuable especially for our study group due to no risk of mtct . At present this interesting method is not widely available, and more data of this new method are needed.
Diabetes mellitus is a global health problem and an important cause of mortality and morbidity in many countries . The trend of increasing diabetes prevalence seems to prevail among developing countries . In brazil, diabetes affected 11.3 million people in 2011, and this number is likely to triple by 2030 . Estimates suggest that the diabetes rate in less developed countries will increase by 69% between 2010 and 2030 . Diabetes imposes a burden for society such as high socioeconomic costs that have an impact on productivity as well as life and health quality . This situation seems to be worse in developing countries, where the healthcare system often fails to meet demand . Studies have concluded that a western dietary pattern, sedentary lifestyle, and genetic factors play a central role in diabetes development . The brazilian ministry of health has followed the world health organization's recommendations and has taken some actions to monitor diabetes such as an annual telephone - based survey . Socioeconomic disparities might contribute to some degree of heterogeneity in measures of prevalence between regions . A study demonstrated that diabetes prevalence across the brazilian states ranged from 11% to 25%, with an overall rate of 16% in 2001 . Brasilia, the capital of brazil, is located in the central - west region of the country . The city has the highest human development index in brazil, but it has one of the highest levels of social inequality compared with other brazilian regions [11, 12]. These characteristics of brasilia warrant further investigation in many aspects, including the health status of its population . Thus, the goal of this study was to estimate the prevalence of diabetes and its associated risk factors in adults of brasilia, brazil . The present cross - sectional population - based study was conducted in brasilia, brazil, from february to may 2012 . The sample size was calculated based on an estimation of 16% of self - reported diabetes cases . Considering a 95% confidence interval (ci), precision of 2.25%, and a design effect of 1.8, we added 10% of the sample size to compensate for any eventual attrition, which resulted in a final sample of 2,019 individuals . Participants were selected by a two - stage probability sampling process by cluster and were stratified by sex and age . A total of 220 census tracts were randomly selected from 3,886 urban tracts with more than 200 inhabitants . Up to 10 households were selected from each census tract . In total, one adult per household was selected following the predefined quotas of sex and age to answer the interview . Trained professionals surveyed all of the participants in their homes using a semistructured questionnaire . To ensure reliability, 20% of the interviews were audited by telephone . To test the understanding and acceptability of the questionnaire, 150 pilot interviews were held prior to data collection . The dependent variable was self - reported diabetes . Independent variables included demographic characteristics (age group, sex, marital status, living arrangements, and household location), socioeconomic characteristics (level of education, occupation, and social class), chronic health conditions (self - reported hypertension, depression, respiratory diseases, cardiovascular diseases, and other chronic diseases), access to healthcare (health insurance, medical consultation, and hospitalization), and perceived health status (mobility, self - care, usual activities, pain / discomfort, and anxiety / depression). The stratification was based on the brazilian criterion of economic classification, which defines five classes, with a being the wealthiest group and e being the poorest . In all of the analyses, self - reported diabetes prevalence in the population was then calculated at a 95% ci . To identify factors related to diabetes prevalence, we calculated prevalence ratios (pr) using bivariate analysis and calculated the adjusted pr by a poisson regression model with robust variance . In this model, we preferred to use this more conservative model that included all of the variables to allow for better confounding adjustment . Other models that included only the most significant variables were tested and did not change the significance of the variables . Associations were considered to be statistically significant when p <0.05 . The stata software version 10.1 was used for all of the calculations . Approximately 60% of the participants were women, and 57% were aged between 35 and 60 years . Most of the participants belonged to economic class c, had completed high school, were married or cohabitating, lived with at least one more person in the household, and dwelled in a satellite town . Diabetes was self - reported by 10.1% (95% ci: 8.5%11.6%) of the adult population in brasilia . Table 1 depicts diabetes prevalence and prevalence ratios (pr) before and after adjustment by poisson regression . The age group of 3565 years, hypertension, respiratory disease, cardiovascular disease, and pain / discomfort were significantly associated with diabetes . Sex, marital status, living arrangements, social class, education level, employability, living location, health insurance, medical consultation, hospitalization, physical mobility, self - care, usual activities, and anxiety / depression revealed no significant association . Figure 2 illustrates differences in diabetes prevalence between all persons and the population with comorbidities . Diabetes prevalence in the age range 3065 years is higher among individuals with cardiovascular disease, followed by those with hypertension and those with respiratory diseases . This result suggests that the likelihood of diabetes increases with age and is greater in persons with comorbidities . An age of 35 years and over, presence of pain or discomfort, cardiovascular disease, hypertension, and respiratory disease were positively associated with diabetes in the adult population of brasilia . The main limitations of our study were the self - reported assessments of the primary outcome and independent variables . Self - reported diabetes might be a source of bias because individuals need to be aware of the diagnosis prior to answering, which could result in disease underestimation . However, performing a clinical test for diagnosing diabetes is not always possible in population - based studies . Thus, self - reported answers regarding diabetes have been a common practice according to the literature [17, 18]. Another shortcoming was the cross - sectional design of the study, which hampers a causal relationship between diabetes and the significantly associated factors identified herein . A previous population - based study developed in brazil in 2008 used telephone interviews to investigate self - reported diabetes prevalence and found low prevalence rates in brasilia . Another study found that brasilia was the region with the highest diabetes prevalence compared with other brazilian regions from 2002 to 2007 . Research identified a significant increase in self - reported diabetes in the brazilian population because it ascended from 3.3% in 1998 to 5.3% in 2008 . In south and central america, the estimated diabetes prevalence in 2013 was 8.0%; brazil demonstrated the highest prevalence, followed by colombia and argentina . The variability of diabetes prevalence may be due to a poorer diet and a lack of physical activity, or it could be related to better access to diagnostic testing . As expected, our results demonstrated that the likelihood of having diabetes increases with age . From a healthcare policy perspective, diabetes prevention and management programs should target young people and not only the elderly population . Diabetes prevalence was higher among individuals with cardiovascular disease, hypertension, and respiratory disease compared with the general population . There is convincing evidence of the association between diabetes and hypertension, which increases the risk of a cardiovascular event . A 2003 study conducted in so luis, a city located in one of the poorest areas of brazil, observed a positive association between diabetes and hypertension . A cross - sectional study conducted between 2004 and 2005 in so jose do rio preto, a city in the brazilian southeast region, revealed that the diabetes prevalence was almost threefold higher in a population of hypertensive individuals compared with the general population . A cohort study performed in women between 1988 and 1996 throughout 11 states in the united states found that chronic obstructive pulmonary disease was a diabetes risk factor . A retrospective cohort study conducted in northern california reported that individuals with diabetes are at a greater risk of developing asthma, chronic obstructive pulmonary disease, fibrosis, and pneumonia . In contrast, a systematic review of 10 studies suggests that growing up in a socioeconomically disadvantaged environment may contribute to diabetes in later life . An australian study also described a positive association between socioeconomic variables and diabetes in adults aged 45 years and over . The perceived health dimensions physical mobility, self - care, usual activities, and anxiety / depression were not associated with diabetes in our sample . In 2012, a literature review found that diabetes was considered a potential risk factor for the poor performance of daily life activities among individuals aged 50 years and over . A study conducted with older adult other than depression, this finding might depict an association between diabetes and activities of daily living, which may be developed at older ages . Diabetes is a common health condition in adults living in brasilia and is positively associated with older age, cardiovascular disease, hypertension, respiratory disease, and presence of pain or discomfort.
Natural killer t (nkt) cells are innate - like lymphocytes typified by coexpression of receptors characteristic of natural killer and conventional t cells . As such, murine nkt cells generally bear ly49 receptors, nkg2 family of receptors, cd94, and nk1.1 (the latter only being expressed in specific strains, including the commonly used c57bl/6). Human nkt cells often express similar surface molecules including cd56, cd161, cd94, nkg2d, and nkg2a . Both human and mouse nkt cells display a variety of stimulatory and inhibitory t cell - associated receptors and ligands (e.g., cd28 and cd154), whose expression depends on the activation status of the cell . Finally, both human and murine nkt populations include cd4 and cd4cd8 (double negative; dn) subpopulations; while cd8 nkt cells are found in humans, they are rare in mice . The t cell receptors (tcrs) expressed by nkt cells recognize the conserved and nonpolymorphic mhc class i - like molecule, cd1d . Unlike classical mhc class i - like molecules, the expression of cd1d is largely restricted to cells of bone marrow origin including antigen presenting cells (apcs) such as dendritic cells (dcs), macrophages, and b cells . Furthermore, the cd1d molecule (via heterodimerization with 2-microglobulin) specializes in displaying lipid moieties rather than protein polypeptides . Importantly, intact expression of cd1d is critical for the development of nkt cell populations, as cd1d/ mice are devoid of these cells . Nkt cells are further subclassified into type i or ii lineages, depending on the composition of their tcr and the cd1d - presented glycolipid antigens to which they respond . Type i or invariant nkt (inkt) cells express canonical tcr chains comprised of specific gene segments (v14-j18 in mice and v24-j18 in humans) that preferentially pair with specific tcr chains (v8, v7, or v2 in mice and v11 in humans). These invariant tcr pairings confer reactivity to cd1d and a restricted array of presented glycolipid antigens . The dependence of inkt cells on the v14-j18-comprised tcr is demonstrated by v14 tcr transgenic mice, in which a higher frequency and number of inkt cells are observed, and also j18/ mice, in which no mature inkt cells develop . Despite the conserved use of the invariant tcr, inkt cell populations are phenotypically (e.g., presence or absence of cd4 expression) and functionally (e.g., preferential production of certain cytokines, such as il-17) diverse . The prototypical (and first discovered) inkt cell stimulatory glycolipid, alpha - galactosylceramide (-galcer), was identified during a screening for compounds from marine sponges (agelas species) with antitumor activity . Since this initial discovery, a number of naturally occurring and synthetic lipid antigens have been described to bind cd1d and activate inkt cells . These cells are now typically identified using cd1d tetramers loaded with -galcer or its synthetic analogs (e.g., pbs-57;). In contrast, type ii or variant nkt (vnkt) cells bear a more diverse array of tcr and chains and have been shown to recognize sulfatide moieties presented by cd1d . More recently, type ii nkt cells have also begun to be better characterized through development of cd1d tetramers loaded with sulfatide [9, 10], but these cells are still less well characterized than their invariant brethren . Given that far more is known regarding the antitumor activity of inkt cells, we will predominantly focus our attention on these cells . Inkt cells develop in the thymus, by originating from cd4cd8 double positive (dp) thymocytes . Positive selection of inkt cells is mediated by homotypic interactions of dp cells and recognition of glycolipid antigen - cd1d complexes [1114]; however, the nature of the self - antigens involved in this process remains somewhat elusive . Like conventional t cells, maturation of inkt cells at the dp stage and beyond depends on the ability to construct a functional tcr and intact signaling . As such, inkt cells are profoundly diminished or absent in mice lacking expression of rag, cd3, lck, zap-70, slp-76, itk, lat, or vav [1521]. Transcriptionally, development of inkt cells at the dp stage is regulated by the transcription factor rort, which prolongs the survival of dp thymocytes by upregulating bcl - xl, to allow sufficient time for distal tcr gene segment rearrangements to occur [22, 23]. More recent studies have shown that heb, the e protein family of basic helix - loop - helix transcription factors, regulates inkt cell development by regulating rort and bcl - xl mrna . Finally, the absence of the transcription factor runx1 also blocks inkt cell development at the earliest detectable inkt cell - committed subset . Inkt cell development at the dp stage also critically depends on the signals generated by engagement of the signaling lymphocyte activation molecule (slam) family of surface receptors, which are expressed on developing inkt cells, as well as conventional dp thymocytes . Slam family receptor signaling is transduced by the adaptor molecule sap (slam - associated protein), which in turn binds to the tyrosine kinase fyn, and results in propagation of a phosphorylation signal [25, 26]. Accordingly, inkt cells fail to develop in mice and humans bearing mutations in the gene that encodes for sap [2729], in mice lacking fyn or expressing a mutant version of sap that cannot bind fyn [23, 30], in mice in which both ly108 and slam signaling are simultaneously disrupted, and in those lacking the transcription factor cmyb (which is necessary for appropriate expression of sap and certain slam family members). Taken together, these studies establish the importance of the slam - sap - fyn signaling axis in inkt cell development . Following positive selection, inkt cells undergo distinct stages of maturation that are characterized by the sequential acquisition of cd24, cd44, and nk1.1: cd24cd44nk1.1 (stage 0), cd24cd44nk1.1 (stage 1), cd24cd44nk1.1 (stage 2), and finally cd24cd44nk1.1 (stage 3). As these cells progress through these developmental stages, they begin to upregulate nk cell markers (e.g., nkg2d and ly49 receptors), cd69, and cd122 and acquire distinct effector functions (e.g., production of il-4, ifn-, perforin, and granzymes). One of the key regulators of inkt cell development and acquisition of an effector / memory phenotype and functions is the broad complex tramtrack bric - a - brac - zinc finger transcription factor plzf, whose expression is highest in stage 0 and 1 populations [36, 37]. Plzf - deficient animals exhibit a severe reduction in inkt cell number and plzf - deficient inkt cells fail to cosecrete th1 and th2 cytokines upon stimulation [36, 37]. Recently, it was demonstrated that the lethal-7 microrna posttranscriptionally regulate plzf expression and inkt cell effector functions . The transcription factor t - bet is indispensable for the final maturation stages of inkt cells [39, 40] and absence of this transcription factor results in reduced inkt cell numbers due to developmental blockade at stage 2 . T - bet - deficient inkt cells fail to proliferate in response to il-15 as they lack surface expression of cd122, a component of the il-15 receptor . In addition, t - bet - deficient inkt cells fail to produce ifn- in response to tcr stimulation and exhibit defective cytolytic activity [39, 40] as t - bet directly regulates the activation of genes associated with mature inkt cell functions, such as perforin, cd178, and ifn- . As inkt cells progress to stage 1, a proportion of cells downregulate cd4, giving rise to dn inkt cells . Generation of the cd4 inkt cell lineage and production of th2-type cytokines is critically regulated by the transcription factor gata-3 . Similar to plzf - deficient inkt cells, gata-3 deficient inkt cells fail to produce th1 or th2 cytokines in response to -galcer . Recent studies have identified a unique subpopulation of nk1.1cd4 inkt cells that are transcriptionally regulated by rort and capable of producing large quantities of il-17 upon stimulation . As such, inkt cells are also sometimes classified into nkt1, nkt2, and nkt17 based on their cytokine production profiles and respective expression of t - bet, gata-3, and rort [43, 44]. Finally, mechanistic target of rapamycin (mtor) signaling has also been shown to be important for inkt cell lineage diversification and acquisition of effector functions [4548], and loss of mtor2 may result in loss of nkt17 cells . Taken together, these recent studies provide new insights into the transcriptional regulation of inkt cell maturation and functional differentiation . The importance of inkt cells in mediating protection against tumors is highlighted by several findings . First, a number of independent studies have shown a decrease in the number of inkt cells in the peripheral blood of patients with a variety of cancers and even precancerous myelodysplastic syndromes [4951]. Moreover, the inkt cells that persist appear to have decreased proliferative and functional responses [5254]. Interestingly, an increased frequency of peripheral blood inkt cells in cancer patients portends a more favorable response to therapy [55, 56]. While these observations identify an association between inkt cell numbers and/or function and development of malignancy, they do not provide a direct causal link this link has been established in a number of mouse studies in which the biology of the host and initiation of tumors can be more systematically manipulated via gene knockouts, antibody depletion strategies, and adoptive transfer of various lymphocyte populations into cancer - predisposed or tumor - challenged hosts . In mice that are prone to development of tumors due to loss of one allele of a tumor suppressor (p53+/), absence of inkt cells (by virtue of genetic knockout of the j18 gene segment or cd1d) results in earlier and more frequent development of tumors and thus shorter survival, when compared to inkt - sufficient littermates . Similarly, treatment of cd1d/ and j18/ mice with a carcinogen resulted in increased incidence and earlier onset of tumors in comparison to treated wild type mice . Conversely, administration of -galcer to mice controlled the growth and metastasis of adoptively transferred [59, 60] or carcinogen - induced [61, 62] or spontaneous tumors . Moreover, adoptive transfer of inkt cells into j18/ inkt cell - deficient mice prevented the growth of subcutaneous sarcomas . Finally, adoptive transfer of small numbers of purified inkt cells into lymphocyte - deficient nod - scid - il2r/ (nsg) mice was sufficient to protect mice from challenge with a cd1d tumor . These findings collectively argue that inkt cells play a central and nonredundant role in the response to tumors . Further studies would shed light on the mechanisms by which inkt cells exert these antitumor effects . Engagement of the invariant tcr by cd1d / glycolipid antigen complexes results in inkt cell activation, an event that is typified by rapid and robust production of a variety of cytokines and chemokines, including but not limited to il-2, il-4, il-10, il-13 il-17, ifn-, tnf, tgf, gm - csf, rantes, eotaxin, mip-1, and mip-1 [65, 66]. The nature and magnitude of the inkt cell cytokine response depend on the glycolipid antigen; for example, -galcer - mediated inkt cell activation elicits a strong ifn--dominated cytokine response, while och (a synthetic analog of -galcer with a truncated lipid chain) elicits a response with significantly higher level of il-4 production . The rapidity of this cytokine response is attributed to the semiactivated state of inkt cells and the presence of preformed cytosolic mrna for a variety of cytokines . Indeed, administration of -galcer to inkt cell - sufficient, but not inkt cell - deficient, mice results in polyclonal activation of conventional t, b, and nk cells within 3 - 4 hours and also eventually leads to the mobilization of macrophages and neutrophils . Intriguingly, it was previously believed that mammalian species are incapable of producing glycolipids (such as -galcer), in which the sugar moiety is attached via an o - linkage to the ceramide backbone in an alpha - anomeric configuration . Despite the absence of -glucosyl or -galactosyl transferases in mammals, recent findings indicate that a small percentage of the glycolipids that are constitutively presented by mammalian cd1d are indeed -anomeric . Whether the percentage of cd1d - presented -anomeric glycolipids is altered in tumor tissues nonetheless, following encounter with cd1d / antigen complexes displayed by apcs, inkt cells not only produce cytokines but also upregulate surface expression of cd154 (see figure 1(a)). Ligation of apc - expressed cd40 is especially important for mediating subsequent maturation and functional activation of dcs, subsequent upregulation of cd80 and cd86, and amplified production of ifn- [72, 73]. In addition, the ligation of the chemokine receptor cxcr6 on inkt cells by cxcl16 expressed on apcs also provides costimulatory signals resulting in robust -galcer - induced inkt cell activation . Importantly, matured dcs are potent producers of il-12, which induces sustained ifn- production by inkt cells [7577]. The importance of inkt cells in il-12-mediated tumor rejection was effectively demonstrated by the defective clearance of a variety of tumors in j18/ mice . Mature dcs also support the priming and activation of cd8 t cells, culminating in optimal effector and memory cell formation [72, 78]. Finally, the sustained release of ifn- by inkt cells leads to activation and proliferation of nk cells and nk cell secretion of ifn-. The combination of cytokines (e.g., il-2, il-12, and ifn-) as a result of inkt cell activation also leads to upregulation of death - inducing ligands (e.g., cd178 or cd253) on nk cells and cd8 t cells [79, 80]. These sequential activation events are believed to be critical for the -galcer - induced inkt cell - mediated antitumor effects [76, 81, 82]. As such, inkt cells not only bridge the activation of innate and adaptive immunity, but also indirectly potentiate the antitumor activity of other cytotoxic effector lymphocytes . Tumor establishment and growth are believed to be intricately modulated by a myriad of soluble and contact - derived signals obtained from the tumor microenvironment (tme), which consists of the tumor cells themselves, tumor - infiltrating lymphocytes (tils), and stromal cells that communicate in a dynamic and bidirectional manner . In addition to their indirect modulation of other effector lymphocyte populations, inkt cells may also regulate tumor growth via their effects on the tme (see figure 1(b)). Indeed following intravenous administration, inkt cells were shown to represent a significant percentage of the tils in patients with head and neck carcinomas [83, 84]. Importantly, higher frequency of tumor - infiltrating inkt cells correlated with overall and disease - free survival as an independent prognostic factor in primary colorectal cancer patients and with tumor regression in head and neck carcinomas . Conversely, in patients with primary hepatocellular or metastatic cancer, cd4 inkt cells that produced high levels of th2-type cytokines and had low cytolytic activity were enriched within the tumor and appeared to inhibit the expansion of antigen - specific cd8 t cells, suggesting that these particular inkt cells may contribute to generate an immunosuppressive microenvironment . In experimental studies, cotransfer of human monocytes and inkt cells to tumor - bearing nod - scid mice suppressed tumor growth when compared with mice that received monocytes alone . Importantly, inkt cells can target tumor supportive cells such as tumor - associated macrophages (tams), a highly plastic monocyte - derived subset of inflammatory cells that can exert immunosuppressive functions, and promote tumor proliferation and matrix turnover [88, 89]. Indeed tams are known to produce il-6, a cytokine that appears to promote the proliferation of many solid tumors, including neuroblastomas and breast and prostate carcinomas . Found that macrophage density correlated positively with microvessel counts and negatively with patient relapse - free survival . Since tams cross - present neuroblastomaderived endogenous cd1d ligand(s), they can be specifically recognized and killed by inkt cells in an il-15-dependent process . Other potential inkt cell tme targets include myeloid - derived suppressor cells (mdscs). Mdscs have been found to accumulate in the blood, lymph nodes, and bone marrow and at tumor sites in most patients and experimental animals with cancer and inhibit both adaptive and innate immunity . The absence of inkt cells in mice during influenza virus infection resulted in the expansion of mdscs, high viral titer, and increased mortality . The adoptive transfer of inkt cells abolished the suppressive activity of mdscs and restored virus - specific immune responses, resulting in reduced viral titers and increased rates of host survival . Thus, certain populations of inkt cells may help alter the tme via their effects on tams and mdscs, to help create a tumor - suppressive or immune - permissive milieu . In addition to their indirect control of tumor growth, inkt cells can mediate direct killing of tumor targets (see figure 1(c)). Inkt cells alone, or in combination with nk cells, have been shown to kill a variety of tumor targets in vitro [6, 93, 94]. While this mechanism of killing appears to be dependent on the presence of stimulatory glycolipids and cd1d [95, 96], inkt cell cytotoxicity also appears to be triggered via ligation of nkg2d by target - expressed stress ligands . It remains to be seen whether mult1, the newly identified shed form of high affinity nkg2d ligand that triggers nk - mediated tumor rejection in mice, also activates inkt cells . Consistent with their direct cytotoxic capacity, inkt cells express perforin and granzymes, as well as cd178 [34, 96, 99, 100]. In our hands, blockade of cd1d - mediated lipid antigen presentation, disruption of t cell receptor (tcr) signaling, or loss of perforin expression was found to significantly reduce inkt cell killing in vitro . Moreover, we demonstrated that inkt cells alone were sufficient for control of the growth of a t cell lymphoma in vivo that preferentially relies on perforin and the adaptor protein sap [64, 69]. Mechanistically, inkt cells rely on sap for formation of stable conjugates with the tumor targets as well as proper orientation of the lytic machinery at the immunological synapse . Despite the majority of studies implicating inkt cells as having an antitumor role, a limited number of studies also implicate inkt cells as suppressing antitumor responses, but these paradoxic responses may be related to the level of tumor cd1d expression [102, 103]. Alternatively, these differences may stem from the fact that contrary to the use of c57bl/6 mice in the previously discussed studies these last two studies were performed in balb / c mice, in which there is a predominance of il-4-producing th2 phenotype inkt cells . Interestingly, the antitumor responses of inkt cells may be regulated by the activity of type ii nkt cells . Demonstrated that type ii variant nkt (vnkt) cells were sufficient for the downregulation of tumor immunosurveillance and relapse growth of a model fibrosarcoma in an antigen - dependent manner, while a second study found that activation of vnkt cells with sulfatide antigen could suppress the activation of inkt cells . Conversely, type ii vnkt cells were, in at least one study, suggested to promote the antitumor activity of cpg oligodeoxynucleotides . This suppression appears to be mediated through a contact- and il-10-dependent mechanism [109, 110]. Indeed, induction of treg cells suppressed the protective effect of adoptive transfer of inkt cells into j18/ mice . Consistent with these findings, depletion of treg cells or short - term elimination of their suppressive activity results in enhanced inkt cell - mediated antitumor responses and increased nk and cd8 t cell activation and ifn- production . Interestingly, the ability of treg cells to suppress inkt cell proliferation depends on the degree of invariant tcr agonism, such that responses to weak (e.g., och), but not strong (e.g., -galcer), agonists were effectively suppressed . When viewed collectively, these findings suggest that inkt cells possess inherent capacity for direct cytotoxicity but their antigenic exposure may modulate whether their antitumor effects can be suppressed by treg and vnkt cells . Given the preponderance of evidence suggesting that the activation of inkt cells provides protection against the growth and metastasis of a variety of tumors, safety of -galcer administration was examined in a phase i trial . While administration of -galcer was well tolerated at a range of doses, no clinical responses were observed in patients with advanced solid tumors . On the heels of this study, nieda et al . Showed that treatment of metastatic cancer patients with -galcer - pulsed immature monocyte - derived dcs resulted in dramatic increases in serum ifn- and il-12 and activation of nk and t cells in the majority of subjects . Importantly, this phase i trial also documented reduction in tumor biomarkers and tumor necrosis in several patients . These findings were extended in a study of a small number of patients, in which the -galcer - pulsed dcs were matured prior to adoptive transfer . This study demonstrated a> 100-fold increase in blood inkt cell numbers in all patients, and this increase was long - lived (> 6 months). A number of subsequent clinical trials, all with limited number of patients with advanced head and neck or non - small cell lung cancers, have since employed similar strategies of adoptive transfer of -galcer - pulsed apcs [115118]. Collectively, these studies demonstrate increases in blood ifn- levels and inkt cells in some but not all patients, stabilization of disease in a few of the subjects, and absence of severe treatment - related toxicities . In a different approach, chemotherapy - refractory 5 lymphoma patients were treated with autologous peripheral blood mononuclear cells (pbmcs) stimulated with anti - cd3, il-2, and ifn-. This ex vivo stimulation resulted in enrichment of nkt cells (to ~20% on average), and this cell fraction was shown to possess the highest cytotoxic capacity in vitro . Of the nine patients who received adoptive transfer of these cells, two showed partial responses and two others had stabilization of disease . Two subsequent studies by motohashi et al . Evaluated the adoptive transfer of ex vivo expanded inkt cell - enriched cells to patients with advanced cancer . In the first, 6 patients with advanced non - small cell lung cancer were treated with either a low or a high dose of ex vivo expanded inkt cells . Of the 3 patients treated with the high dose, all had an increase in the frequency of ifn--producing pbmcs and 2 showed expansion of inkt cells . Although no clinical responses were observed in this study, a follow - up trial of 17 patients with advanced head and neck cancers treated with a high dose of inkt cell - enriched autologous pbmcs showed a significant increase in ifn--producing pbmcs in 10 of 17 patients . Importantly, while none of these patients displayed tumor regression, 5 had disease stabilization and the mean survival time for the subjects with higher frequencies of ifn--producing pbmcs was tripled above those with low percentages of ifn--producing pbmcs (29.3 versus 9.7 months). Administered both in vitro expanded inkt cells and -galcer - pulsed apcs to patients with advanced head and neck squamous cell carcinomas . Treatment increased the frequencies of inkt cells and ifn--producing pbmcs, and a partial clinical response or disease stabilization was observed in 7 of 8 patients . Although the responses in these studies have not been profound, it must be noted that these inkt cell - based immunotherapies have all been conducted on patients with advanced malignancies often those in whom standard chemotherapy, irradiation, and/or surgical excision treatments had failed . Future studies of inkt cell - based immunotherapy may be able to take advantage of two recent technologies . As mentioned previously, many malignancies are associated with a decrease in the numbers and proliferative capacity of peripheral blood inkt cells . In order to circumvent the difficulty of being able to expand these infrequent and potentially defective cells from patients, watarai et al . Generated induced pluripotent stem (ips) cells from mature inkt cells and then expanded large numbers of inkt cells from these established ips cells . Ips - nkt cells generated in this fashion were demonstrated to be able to activate and expand antigen - specific cd8 t cell responses to limit the growth of leukemia in mice without inducing graft versus host disease (gvhd). Described inkt cells engineered to express cars bearing specificity for gd2, a highly expressed moiety on neuroblastoma cells . In their studies, they showed that inkt cells expanded from the pbmcs of healthy human donors and transduced with retroviral car constructs could protect humanized nsg mice against metastatic neuroblastoma without inducing gvhd . Whether these two technologies could be combined to generate functional car - bearing ips - nkt cells remains to be seen . Inkt cells are innate - like effector lymphocytes that not only are directly cytotoxic, but also possess the unique ability to nucleate the antitumor responses of other effector lymphocytes and alter the cellular and angiogenic makeup of the tumor microenvironment . As such, the promise of an effective inkt cell - based immunotherapy can only be realized by devising and evaluating strategies that simultaneously maximize each of these antitumor effector mechanisms . The challenge for the future will thus be to identify these strategies and apply them to tumors against which inkt cells wield the most optimal responses.
As a service to our authors and readers, this journal provides supporting information supplied by the authors . Such materials are peer reviewed and may be reorganized for online delivery, but are not copyedited or typeset . Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.
Clinicians rely on patients recall of their symptoms to adjust therapies and monitor responses, but the accuracy of patients recall, particularly between clinic visits, is poor.1 patients recall recent symptoms more clearly than earlier, more remote symptoms, and more severe symptoms more clearly than mild symptoms.2 recognition of day - to - day variability in symptoms is also important for revealing important trends (improvement or worsening), but measuring such variability is difficult without close tracking of symptoms . Daily symptom variation is important because it may obscure the ability to detect signal (of improvement) from the noise (of random daily variation). Taken together, these challenges highlight the problems of symptom recall as the basis for making treatment recommendations, and either altering or escalating therapeutic interventions.1,3 inflammatory bowel disease (ibd) causes chronic inflammation of the gastrointestinal tract, often resulting in severe symptoms . Despite optimal treatment, over 40 percent of patients continue to have symptoms that reduce their quality of life (qol) such as persistent fatigue,4,5 abdominal bloating, or fecal urgency.6 careful attention to a patient s symptomatic response to therapies using paper symptom logs is often inadequate or too cumbersome to provide sufficient evidence to guide treatment.7 improved methods are needed to ease symptom data collection and to facilitate conducting individual (n - of-1) therapeutic trials to improve symptoms and qol . Currently, 78 percent of teenage patients carry mobile devices capable of easily collecting and transmitting symptom data on a daily basis.8 there is growing interest among patients (e.g., the quantified self community)5,9,10 and physicians1115 in leveraging mobile technologies for improving patient outcomes . According to a recent study by the pew research center, over 40 percent of americans who have a chronic health condition track their symptoms or health indicators such as headaches, blood sugar, blood pressure, etc.5 but there is little evidence of the impact that frequent monitoring of self - reported data has on patient qol or other health outcomes.16 while some limited progress has been made in integrating formalized patient - reported outcome (pro) measures into clinical practice, these early efforts provide little evidence to guide the effective integration of pros into real - world patient care setting.16,17 efforts at implementation are made more difficult by technical limitations (e.g., many care centers lack electronic tools to collect pros, and completing repeated online surveys are not sustainable for most patients over time). Most published studies utilizing pros in real - world patient care settings have demonstrated that the pros have little or no impact on patient outcomes.16 this may be due to a lack of validated tools to monitor patient symptoms or qol reliably and prospectively in real time.16 standardized qol indicators for general assessment have not been studied widely for assessing patients in clinical practice . The patient - reported outcomes measurement information system (promis) survey instruments may be appropriate for this purpose, but promis surveys have been validated only in population studies.18,19 promis tools have not been studied in individual patients or over short periods, and have not been broadly incorporated into routine patient care . As part of an ongoing study exploring innovations in health care, we evaluated a prototype tool to determine if continuous symptom tracking aids in improving patients and physicians understanding of symptom patterns and if tracking can help patients and physicians work together to better target treatments.20,21 we also attempted to determine if promis measures can be used in individual patients to track qol over short periods, as opposed to populations of patients for which they were developed . We report, here, important learning from a small number of patients who completed prototype testing of this tool . This study was conducted as part of the ongoing work of the collaborative chronic care network (c3n) project that is developing innovations to facilitate more continuous, collaborative relationships between patients and families with chronic illness and their physicians . The tools described here emerged from a structured design process involving patients, families, clinicians, and innovators that entails testing novel concepts in small - scale prototypes and larger pilot studies prior to deployment.21 this manuscript describes the prototype testing of the novel concept . We took advantage of the collaborative network of patients, physicians, and researchers who participate in the improvecarenow network to engage participants.21 we recruited adolescent patients with ibd who were engaged in their care and were interested in further improving their symptoms and qol . Each patient (along with a parent if under 18 years old) met with his or her pediatric gastroenterologist and a researcher at the time of study enrollment to discuss the patient s medical history and to plan the details of the patient s individual study . The researcher and patient agreed upon a means of assessing symptoms by selecting from a list of preassembled measures . They were allowed to create their own symptom measure if none was available to fit their needs . We used a daily, automated text - message service to prompt patients to submit symptom scores via text message . We did not limit the number of questions that could be asked per day, but suggested each patient select no more than three to four . At study enrollment, the questions were delivered each day at this prespecified time, and did not vary from day to day or with responses to prior questions . We also used the promis survey instruments to measure fatigue and pain interference across all patients.22,23 these short surveys have been extensively validated in a wide range of chronic medical conditions and children.18,19,24 the promis surveys query information on specific outcomes for the preceding seven days . The results were converted to t - scores, where a score of 50 represents the mean score of the general pediatric calibration sample (standard deviation 10).25 t - scores greater than 50 represent worse fatigue or pain interference . We deployed the appropriate promis survey weekly via a web - based interface to each patient . We compared individual patients promis survey results with their average symptom scores from the same period . We used individual - level, statistical process control methods for time - sequence data to monitor the patient s daily symptom data and weekly promis survey data as they were collected over time.26 we established each patient s individual baseline pattern of symptom variation, or common cause variation . Symptom data points that fell outside three sigma control limits were noted as special cause variation, or variation outside stable baseline variability.26,27 these data, along with calculated control limits, were graphed on a microsoft excel spreadsheet for visual representation of data for patient and physician review . For prototype testing, we used a heavily manual process including creating microsoft excel graphs by hand and sharing data with patients and providers via email that was possible for only a small number of patients . The purpose of this type of manual prototype was to inform more robust pilot testing facilitated by automated tools being developed by the research team . Patients were given access to their own data and were provided with graphical displays of their data at each meeting . Physicians and patients jointly reviewed the data on a regular basis in prearranged consultations (in person or by phone, according to each family and physician s preferences). These meetings were opportunities for patients and physicians to learn from patterns of data and to make treatment decisions if they felt this was appropriate . This study and the consent procedures were approved by the institutional review boards of both the university of michigan health system (hum00051651) and cincinnati children s hospital medical center (20103380). All adult participants provided written informed consent to participate in this study . Written informed consent was obtained from parents of minors and written assent from minors themselves for participation in this study . Patients tracked symptoms in order to monitor for changes in disease status, to generate hypotheses as to what makes their symptoms better or worse, or as part of a specific n - of-1 study . We present three patients who tracked their symptoms, prior to implementation of n - of-1 testing, and for whom symptom patterns led to important revelations . A 19 year old with longstanding indeterminate colitis underwent proctocolectomy with ileal pouch anal anastomosis at the age of 10 years . She had frequent nocturnal stools that woke her up to six times per night and caused fatigue contributing to her poor qol . She had been treated every eight weeks with long - term infliximab therapy by intravenous infusion . In collaboration with her physician, she decided to track her total number of daily stools and the number of stools waking her from sleep each night . She historically awoke to have a bowel movement from three to six times per night on average . During her baseline observation period, there was a span of nine consecutive nights with fewer episodes of nocturnal stools (fig . 1). However, she failed to recognize this nine - night interval of improved nighttime waking for stools despite the shift in symptoms that qualified as special cause variation.26 this shift occurred immediately following an infusion of infliximab, which led her treating physician to conclude that infliximab may have played a role in her transiently improved stool pattern . However, this postinfusion improvement was not reproduced after her subsequent four infliximab infusions, during which time her daily symptoms were continuing to be recorded . She did experience reproducible improvement in her stools with repeated courses of antibiotics that were prescribed for non - ibd related symptoms by her primary care physician (previously illustrated in kaplan et al.20). During those times when she experienced decreased nighttime bowel movements, she also felt less fatigued (fig . 2). Upon reviewing these data she confirmed that she felt less fatigued and more energetic . The patient and her physician both felt that the graphical representation of her symptom data aided in their visualization and understanding of the treatment effects . Both the patient s mother and her physician were interested in trying different probiotics to determine if one would be more effective than others for managing her fecal urgency . Over the course of repeated periods on and off different probiotics,20 her fecal urgency gradually improved (fig . 3, green dashed ovals), though there was no discernable difference during periods with or without the use of probiotics.20 the reduction in fecal urgency over time was reflected in improved promis fatigue score (fig . After three months of monitoring, the patient developed an exacerbation of the disease (fig . After he was diagnosed with crohn s disease, his running pace decreased and had not returned to his pre - illness baseline, and he was interested in assessing his running potential after starting treatment with biological therapy . At his enrollment in this study, his baseline promis fatigue t - score was 35.7, indicating that he was less fatigued than the average healthy individual . He received his first infliximab infusion on may 25, 2011, with induction regimen completed by july 6, 2011, (usually consisting of three infusions spread over six weeks). His number of daily bowel movements decreased within four days of completing the induction regimen, and attained a steady state along with a special cause (fig . His running pace also improved significantly from a mean 8.4 to 7.6 minutes / mile, indicated by a special cause shift as well . A 19 year old with longstanding indeterminate colitis underwent proctocolectomy with ileal pouch anal anastomosis at the age of 10 years . She had frequent nocturnal stools that woke her up to six times per night and caused fatigue contributing to her poor qol . She had been treated every eight weeks with long - term infliximab therapy by intravenous infusion . In collaboration with her physician, she decided to track her total number of daily stools and the number of stools waking her from sleep each night . She historically awoke to have a bowel movement from three to six times per night on average . During her baseline observation period, there was a span of nine consecutive nights with fewer episodes of nocturnal stools (fig . 1). However, she failed to recognize this nine - night interval of improved nighttime waking for stools despite the shift in symptoms that qualified as special cause variation.26 this shift occurred immediately following an infusion of infliximab, which led her treating physician to conclude that infliximab may have played a role in her transiently improved stool pattern . However, this postinfusion improvement was not reproduced after her subsequent four infliximab infusions, during which time her daily symptoms were continuing to be recorded . She did experience reproducible improvement in her stools with repeated courses of antibiotics that were prescribed for non - ibd related symptoms by her primary care physician (previously illustrated in kaplan et al.20). During those times when she experienced decreased nighttime bowel movements, she also felt less fatigued (fig . 2). Upon reviewing these data she confirmed that she felt less fatigued and more energetic . The patient and her physician both felt that the graphical representation of her symptom data aided in their visualization and understanding of the treatment effects . Both the patient s mother and her physician were interested in trying different probiotics to determine if one would be more effective than others for managing her fecal urgency . Over the course of repeated periods on and off different probiotics,20 her fecal urgency gradually improved (fig . 3, green dashed ovals), though there was no discernable difference during periods with or without the use of probiotics.20 the reduction in fecal urgency over time was reflected in improved promis fatigue score (fig . After three months of monitoring, the patient developed an exacerbation of the disease (fig . After he was diagnosed with crohn s disease, his running pace decreased and had not returned to his pre - illness baseline, and he was interested in assessing his running potential after starting treatment with biological therapy . At his enrollment in this study, his baseline promis fatigue t - score was 35.7, indicating that he was less fatigued than the average healthy individual . He received his first infliximab infusion on may 25, 2011, with induction regimen completed by july 6, 2011, (usually consisting of three infusions spread over six weeks). His number of daily bowel movements decreased within four days of completing the induction regimen, and attained a steady state along with a special cause (fig . His running pace also improved significantly from a mean 8.4 to 7.6 minutes / mile, indicated by a special cause shift as well . We found that monitoring daily symptom data detects the daily variation in patients symptoms . Establishing a patient s background pattern of symptom variation can serve as a baseline from which to make subsequent comparisons when making changes in diet or medications . Significant (special cause) deviations in symptoms from the baseline pattern are also associated with variation in promis instrument assessments . Our data from these patients suggest that the promis measures of fatigue and pain, in particular, are responsive to changes in patient symptoms over short periods and on the individual level . These findings lend face validity to daily remote monitoring of patient symptoms, and pave the way for broader use of the promis instruments for use in patient - centered improvement research . Using standard promis instruments, in addition to more customized patient symptoms, may enable greater learning across patients because measures are well validated and standardized in pediatric populations . We also found that knowledge about daily symptom variation and promis assessments of key outcomes may be clinically relevant . Improving patients and physicians understanding of symptom patterns for the purpose of tailoring treatments to the patients needs is essential for understanding response to attempted therapies . It is also essential to measure symptoms that are important to patients in order to help patients achieve good qol even if other traditional measures show minimal symptoms . This work is important and expands upon the very few previous studies of the impact of daily monitoring on outcomes for chronic diseases.16 the most widely documented evidence is for asthma in which daily monitoring is associated with improved asthma control.28 there is also evidence that daily monitoring via mobile phone of adults with heart failure results in improved qol, improved self - care,12 and in one study decreased mortality.29 similarly, in insulin - dependent diabetes mellitus, daily monitoring leads to improved glucose control, more symptom - free days, and decreased perceived disease - related anxiety.13 despite limited evidence of benefit from mobile symptom tracking in some studies, the majority of studies do not demonstrate a measurable improvement in patient outcomes.16 one significant issue that has not previously been addressed is the day - to - day variability of patient symptoms . As we ve seen in our study, there is substantial daily variability in symptoms, which may obscure the detection of improvements, when they occur . The work reported here builds on previous studies by developing tools that enable daily symptom tracking to better understand and detect changes in patient symptoms . Understanding the nuances of symptom variation at baseline, and in response to therapeutic challenges is critical for targeting therapies for improvement and for determining the individual efficacy of therapeutic changes . Comparing daily symptom data with weekly promis surveys provides internal consistency of the pro measures collected for each patient . Together, the collection of these pro data from patients facilitates real - time feedback to patients and their physicians about the effectiveness of therapies . These methods offer substantial improvements over routine clinical practice and over prior mobile health (mhealth) studies lacking such feedback . These were highly engaged patients who volunteered for this study (as is the case in many studies). It is possible that daily or weekly symptom measurement may not be feasible in less - engaged patients . However since 75 percent of young people have mobile phones and 88 percent of them send and respond to numerous text messages daily (typical 14 to 17 year olds send and receive 100 text messages per day),30 it remains to be determined how important engagement with care is to these assessments . There is also some concern that daily monitoring may lead to increased attention to symptoms and worsening of perceived physical health.31 however others have demonstrated that patients with chronic constipation who monitored daily symptoms had no worse outcomes than those monitored weekly in a prospective randomized clinical trial.32 others found improved outcomes with daily monitoring for irritable bowel syndrome, asthma, and heart failure.12,13,33 it will be important to determine if certain patients may benefit more from these methods and if others should avoid these methods to minimize attention to symptoms . Note that a patient with longstanding daily symptoms may have a significant improvement in symptoms without being aware of such changes (as was seen with patient 1). Conversely, perceived improvement may not actually represent real improvement, but may be misattributed background variation which, when graphically evaluated, represents common cause variation (as seen with patient 2). This was seen with the initial improvement in nocturnal stools following infliximab infusion (patient 1), which was not reproduced after subsequent infusions . It is tempting to infer a causal relationship between two events because of their close proximity in timing, even if the association is coincidental . The accuracy of the interpretation of associations has direct implications for how most physicians and patients routinely test therapies by ad hoc trial and error . It is only with careful monitoring of outcomes, and with replication (through experimentation), that we can distinguish between causal relationships or associations.34,35 this study also highlights the importance of tracking the symptoms that patients feel are most important to them . The example of patient 3, whose gastrointestinal symptoms and fatigue were not severe, illustrates this additional benefit of our individualized, patient - centered approach . By closely monitoring the symptom that was most important to the patient (his running pace), he and his physician were able to determine when a significant improvement occurred in the outcome that was of greatest importance to the patient . Our findings in this early study highlight the difficulty in assessing treatment efficacy based on casual assessment of symptom relief for an individual patient . These findings are of great importance to improving the physician s ability to adequately understand their patient s disease activity and the symptoms contributing to qol for the purpose of tailoring treatments to the needs of the patient . This applies when new therapies are initiated and also during changing, modifying, weaning off, or withdrawing therapies (as could be the case with steroid weaning for ibd treatment). These new methods offer the potential to support more collaborative decision - making and to improve clinicians and patients abilities to identify the right treatments both through informal learning and planned experimentation with individual patient therapeutic (n - of-1) trials . We must have a clear understanding of the effects of an intervention (medical, dietary, behavioral, etc .) On the patient s symptoms . Only with a clear understanding of symptom variation, and an ability to discern a meaningful change in symptom pattern from the background variation, can we fully understand the effects of therapies on symptoms and know with certainty whether or not a therapy improves symptoms and qol . The new methods described here are the building blocks for transforming the doctor - patient relationship to become more collaborative and to provide improved evidence for understanding individual patient treatment efficacy for better treatment individualization . Based on the success of daily tracking using customized and standardized measures (promis measures), and the utility of graphical display of symptoms using statistical process control, we are developing more advanced technology (web platform and mobile app) to enable tracking and real - time visualization of this data for the patient and provider . We are also examining ways to better support patients and providers in using pro data to enable formal n - of-1 studies of pediatric patients with ibd . N - of-1 studies are well established as a method to improve patient symptoms . However, until now these methods have been limited in their scope of use . We are currently expanding these methods that we have described here to develop the means of conducting n - of-1 studies concurrently with larger numbers of patients who may have different complaints and different treatments . These methods have the potential to benefit patients broadly and to improve patient - physician engagement and communication across a wide range of clinical settings . Clinicians and patients should consider the use of mobile daily symptom data collection to improve care and outcomes . Daily symptom tracking can facilitate improved informal learning as well as rigorous n - of-1 experimental studies to objectively assess individual treatment efficacy and individualize patient care.34,35 prototype studies have provided proof of concept so that these methods can be further expanded to enable more robust learning designed to improve symptoms and qol for pediatric patients with ibd and others with differing chronic health conditions.
The vast majority of cancers in the oral cavity and in the head and neck are squamous cell carcinomas (sccs). It is the sixth most common cancer worldwide, and its incidence is rising in industrialized nations [1, 2]. Similarly, other benign lesions of the oral cavity such as lichen planus may have a prevalence of 0.52% in the general population and may have a risk of malignant transformation of 1% . Many benign oral mucosal lesions are not cancerous which presents a clinical dilemma to the physician . Furthermore, precancerous lesions such as leukoplakia may exhibit mild structural alterations in the mucosa that can be difficult to distinguish from normal healthy tissue . Currently, obtaining histopathology via biopsy is the gold standard of diagnosis; however, this procedure can pose significant morbidity to the patient such as the risk of bleeding, wound infection, and potentially impairment of speech and swallowing if multiple biopsies are performed . Moreover, it becomes a clinical challenge to monitor patients for progression of diffuse dysplasia or leukoplakia, and many of them may require multiple biopsies over many years . The discomfort of biopsy and compromisation of tissue integrity can lead to problems with future biopsy interpretation or in the case of laryngeal biopsy, considerable problems in individuals with high vocal demands . Subsequently, any technique that can yield histopathological information without injuring tissue has obvious advantages over biopsy . Detailed examinations of the texture, color, contour, and extent of mucosal lesions have been performed utilizing many instruments such as the hopkins' rod - lens scopes, flexible endoscopes, direct laryngoscopes, and advances in microlaryngoscopic visualization techniques . However, these methods are limited by their inability to provide histopathological data during the clinical examination . As a result, over the last decade, technological advances in optical imaging detection techniques have emerged with a variety of methods employed to facilitate detailed examination and provision of histopathological information of mucosal lesions . Examples of such novel optical techniques include: aminolevulinic acid - induced fluorecence, autofluorescence, confocal endomicroscopy, and contact endoscopy . Aminolevulinic acid - induced fluorescence is a technique whereby neoplastic cells undergo preferential fluorescence after aminolevulinic acid (ala) has been applied to the mucosa surface . In the presence of ala, once this dye - like substance has been applied, mucosa containing neoplastic cells will fluoresce orange red and normal mucosa will retain the normal green fluorescence . Coupled with autofluorescence, several authors have noted that these techniques can diagnose laryngeal carcinoma and dysplasia with good accuracy . Autofluorescence was first described in identification of neoplastic cells of the larynx by harris et al . . Certain molecules then transform into photonic energy, which is emitted as long - wave scattered light which can be detected . The autofluorescence imaging method detects the fluorecence given off by the different concentrations of fluorophores seen in normal and neoplastic mucosa . Thus, autofluorescence videoendoscopy for photodiagnosis of head and neck squamous cell carcinomas has been described as being quite accurate with good sensitivity and specificity in several studies [616]. Unlike ala and autofluorescence where histological detail is not appreciated, other optical techniques such as narrow - band imaging endoscopy (nbie) nbie uses filtered light with wavelengths preferentially corresponding to peaks of absorption of hemoglobin to enhance superficial neoplasms based on their neoangiogenic pattern . These light wavelengths penetrate superficial mucosal and deep submucosal layers to enhance capillary and submucosal vasculature . The obtained image is further enhanced by using high - definition television (hdtv). Carcinomas can then be identified based on the changes in the microvascular pattern of the mucosal lesion . Several studies have shown good sensitivity, specificity, negative and positive predictive value, and accuracy in detection of squamous cell carcinoma of the oral cavity, oropharynx, larynx, and esophagus [1720]. However, instead of solely relying on neoangiogenic patterns for diagnosis of carcinoma, further histological detail can be obtained with the use of confocal endoscopy which is an in vivo optical imaging method whereby mucosal lesions can undergo significant magnification to allow examination of cellular histology . This technique also allows reconstruction of three - dimensional structures based on the acquired images . Utilization of various stains to help highlight cellular structures has been tried by some authors to distinguish normal from invasive carcinoma cells . The utility of this new technology is highlighted in its capability to distinguish between benign or low - grade mucosal dysplasia thereby potentially reducing unnecessary biopsies . Contact endoscopy is another novel noninvasive optical diagnostic imaging method that allows in vivo and in situ examination of the cellular architecture of the superficial layers of the mucosal epithelium . Magnified images are obtained using hopkins' rod - lens endoscope placed on the surface of the dye stained mucosal tissue . This technique allows assessment of precancerous and cancerous lesions in vivo and has significant potential in the histopathologic diagnosis of many suspicious head and neck mucosal lesions without tissue biopsy . Ce was originally described and used by hamou in 1979 as a technique for visualization of cervical and uterine epithelial cells for screening and diagnosis of cervical and uterine pathology . The first reported use of ce in otolaryngology head and neck surgery was by andrea et al . As a diagnostic tool in the evaluation of various pathologies in the larynx in the 1990s [4, 2330]. They were able to visualize and diagnose laryngeal mucosal pathology from the magnification of vocal fold epithelium and microvasculature during microlaryngoscopy after staining the vocal cords with methylene blue dye . Diameters, of these scopes come as either 4 mm or 5.5 mm and lengths of 23 cm and 18 cm . Straight forward (o) and forward - oblique telescopes (30) are also available, and all are capable of 1x, 60x, and 150x magnification . These endoscopes require a high intensity xenon light source, and images can be digitally captured for real - time photographic and video documentation, figures 1 and 2 . The most basic technique of ce involves staining of the superficial cells of the mucosa with a contrast dye, 1% methylene blue (mb) after which the magnifying endoscope (karl storz 8715 aa, tuttlingen, germany) 0 is then placed in contact against the mucosal surface, and the documented magnified cytological images (at 60x or 150x) are then recorded, figure 3 . Both a cytopathologist and an otolaryngologist can then assess these images, comparable to histology, figure 4 . Contact endoscopy and its efficacy in head and neck oncology, advantages, limitations, and future potential diagnostic utility will be briefly reviewed in this article . The literature search was conducted using the following key terms: contact endoscopy, contact microlaryngoscopy, aminolevulinic acid induced fluorecence, autofluorescence, confocal endomicroscopy, oral mucosa, oral cavity, larynx, oropharynx, hypopharynx, head and neck carcinoma, leukoplakia, and lichen planus . Relevant search terms and combinations using boolean operators were performed, and relevant article selection was limited to the prospective, human and english studies without restriction to year of publication . Examined 42 consecutive patients at a tertiary care center with suspicious lesions of larynx, pharynx, and esophagus under general anesthesia . The results obtained by the cytopathologist and otolaryngologist were based on images generated from the ce . They found that the more experienced the examiner, the higher the sensitivity of ce was in the diagnostic differentiation of benign versus malignant mucosal lesions . They included 142 patients undergoing microlaryngoscopy at their institution with various laryngeal diseases all underwent ce and subsequent biopsy for histopathological diagnosis . All malignant lesions identified by ce was confirmed by histopathology, but ce did not identify malignancy in 10 patients diagnosed histopathologically thus giving ce a sensitivity of 79.6%, specificity of 100%, and accuracy of 93% . Their results were based on computer - assisted analysis of all ce images based certain nuclear morphometric parameters to determine benign from malignant lesions . Thus, based on their computer - assisted analysis of ce images, their sensitivity was 91% and specificity 81% . All patients were examined with contact rhinoscopes under local anesthesia and biopsy of the area under examination was done . In all 5 cases of malignancy, ce and histological diagnosis directly correlated with each other . Most significantly, for the prediction of persistent and recurrent disease, sensitivity and specificity for ce was 100% with an accuracy of 92.1% . Pilot study examined 83 patients using both autofluorescence and contact endoscopy during microlaryngoscopy . For contact endoscopy, the calculated sensitivity was 94.7, specificity of 95.5 and an accuracy of 94% . In summary, authors of the above prospective trials have obtained the following results: a sensitivity of 79100%, a specificity of 81100%, and an accuracy of 8894% . Overall, it appears that sensitivity, specificity, and accuracy of ce are similar across the trials . Since the development of contact endoscopy, this technology has been used successfully by several authors in analyzing and diagnosing various pathologies of the larynx, oral cavity, oropharynx, and nasopharynx via real - time examination of mucosal cytological detail [4, 2631, 3336]. Despite its introduction into otolaryngology, ce has yet to find a place in routine clinical practice despite its potential advantages . From the above clinical trials, ce appears to have good sensitivity, specificity, and accuracy as a noninvasive method for distinguishing between benign and malignant mucosal lesions in the head and neck . However, some authors state that it may be difficult for ce to detect mild (grade i) mucosal dysplasia because most of the cellular anomalies occur in at the level of the basal epithelium, and this technique can only examine cellular architecture found at the superficial epithelial layers [4, 21]. Despite this limitation, most significantly, ce accurate ability to diagnose and tease out the histological differences between squamous metaplasia, atypia, and carcinoma even in the presence of irradiated mucosa was highlighted by the study performed by pak et al . . At present, most authors seem to agree that it has significant potential as a noninvasive detection method that could play a role as a future substitute for histological examination . Most significantly, it offers a noninvasive, rapid, and repeatable in vivo assessment of the cytological architecture while avoiding the need for an invasive biopsy and its associated risks . Ce provides immediate results, with the possibility of examining multiple mucosal areas in a short time . Ce can also assess a wider surface mucosal area, providing more information than a selected histological section taken by biopsy . It also avoids tissue damage and alteration of cellular architecture which may occur in the biopsy and histological preparation . This noninvasive technique also helps to direct the site of biopsy by identifying areas with cellular atypia and thus avoiding the need for multiple biopsies . Subsequently, this results in a dramatic improvement of the diagnostic yield of the biopsy . Other potential roles of ce include the rapid diagnosis of benign and malignant mucosal lesions in an outpatient or operating room setting, surveillance, guided biopsies, and intraoperative evaluation of tumor resection margins . Despite its advantages most notably, ce can only evaluate the most superficial cell layer of the mucosal epithelium . This is most likely due to a number of factors including (i) poor penetration of methylene blue which only stains a few superficial layers, (ii) short focal distance of the scope (i.e., ce can only assess to a depth of 80 um at 60x magnification and 30 um at 150x magnification), and (iii) optical artifact at high magnification due to glare from light reflected from cells not in focus . Subsequently, assessment of submucosal lesions or lesions occupying deeper cell layers becomes more difficult [4, 27, 28, 30, 32, 33]. The lack of depth of penetration prevents the evaluation of important histological information especially when vertical extent of dysplasia is crucial in distinguishing the different grades of dysplasia from carcinoma in situ and invasive carcinoma . As a result, these factors could affect the sensitivity of ce, thus accounting for some of the false negative diagnostic results noted by authors . The potential impact of ce missing a malignant lesion needs to be taken into consideration if this technology is to one day substitute histopathology . Future investigation into better penetrating dyes, advances in digital optics, and image enhancements will eventually allow better vertical staining and increased resolution of the deeper cell layers which would translate ce in becoming a much more sensitive and accurate diagnostic tool . A pilot study conducted at our institution also investigated some of the limitations and potential advantages of ce in the evaluation of head and neck mucosal lesions . From our preliminary experience, technical difficulties with line of sight, access difficulties to mucosal surfaces, scope positioning, and problems with consistent image quality due to artifact were consistent with those found by previous authors [30, 33]. Our pilot study also demonstrated that although ce is a simple, rapid, repeatable, noninvasive examination performed with standard equipment, there is a learning curve associated with its use . However, once one is accustomed with this detection system, ce can be performed almost as quickly as an outpatient flexible fiberoptic nasopharyngoscopic examination . In conclusion, in vivo assessment of head and neck mucosal pathology may be applied to (i) early detection of premalignant and malignant lesions, (ii) serial follow - up examinations of suspicious lesions such as leukoplakia and lichen planus, and (iii) assessment of resection margins . Despite its limitations, ce represents a promising optical technology that may afford reliable, accurate, and noninvasive in vivo assessment of cytological pathology . Prospective investigation with ce we hypothesize that future study will demonstrate improved sensitivity, specificity, and accuracy of contact endoscopy in the diagnosis of head and neck tumors.
The first historical study of quality assessment in the medical field was reported by ernest amory codman, md, of massachusetts general hospital in 1920 . To support his end results theory, he made public the results of the review of his own hospital in a privately published book, a study in hospital efficiency, in which he emphasized the importance of patient follow - up and quality assessment . He helped to found the hospital standardization program, which eventually became the joint commission on accreditation of healthcare organizations in 1987, and the joint commission in 2007, with the motto helping health care organizations help patients. From the 1970s through the 1980s, the rapid increase in medical lawsuits and the medical malpractice insurance crisis promoted risk management of medical practices in the united states . In 1989, the society for thoracic surgeons (sts) started to establish national databases as an initiative to improve quality and patient safety among cardiothoracic surgeons and to respond to strong public opinion about the importance of accountability . In 1997, an initiative was begun to improve data quality and auditing, and staff were hired to support these efforts . In the sts congenital heart surgery database data specification, (http://www.sts.org/sites/default/files/documents/congenitaldataspecsv3_22.pdf), the patient national identification (social security number) is listed, but this field should be collected in compliance with state / local privacy laws . The sts national database complies with the health insurance portability and accountability act, and the federal government protects the sts national database . In 1998, the sts contracted with the duke clinical research institute (dcri) for data warehousing and data analysis . In 1999, the institute of medicine published a report titled to err is human: building a safe health system, which stated that 44,000 to 98,000 persons die in hospitals as a result of medical errors that could have been prevented . Today, the management of the national database is one of the most important tasks of the sts . The database contains three components: adult cardiac surgery, general thoracic surgery and congenital heart surgery (fig . The sts was the first professional organization to seek approval for its measures from the national quality forum (nqf), a multi - stakeholder health policy organization head - quartered in washington, dc . In this manner, the sts has gained a positive reputation with the government and with health policy organizations . In addition, in 2010, the sts started to publicly report isolated coronary artery bypass grafting (cabg) composite star ratings not only on its own website but also on a consumer report website (www.consumerreportshealth.org). Later, public reporting of aortic valve surgery (avr) and cabg+avr began, and this year, will be extended to congenital heart surgery . The nqf has been releasing quality indicators in medical fields under the rubric of nqf - endorsed standards (http://www.quality-forum.org/home.aspx). Participation in the sts national database, operative mortality stratified by the five sts - eacts (european association for cardiothoracic surgery) mortality categories, and risk adjustment in congenital heart surgery (rachs-1) pediatric heart surgery mortality . The sts states on its website that sts believes the public has a right to know the quality of the surgical outcomes, and considers public reporting an ethical responsibility of the specialty . In turn, in 1998, at the 7th annual meeting of the asian society for cardiovascular and thoracic surgery in singapore, the need for an asian cardiovascular surgery database was discussed . First, a database ad hoc committee was formed by the japanese society for cardiovascular surgery (jscvs) and the japanese association for thoracic surgery (jats) (table 1). Moreover, quality improvement of cardiovascular surgery has been discussed by the members of the board of jscvs and jats since early 2000 . In pursuit of this goal, three committees were organized by the jscvs and jats among its academic groups: 1) a board certification committee, 2) a center aggregation committee, and 3) a nurse practitioner and physician assistant committee . In 2000, before this movement, the japan cardiovascular surgery database (jcvsd) was established with close ties to the jscvs and jats . The jcvsd and jscvs invited the founder of the sts national database to discuss starting the construction of the database . The jcvsd established input items comparable to those of the sts national database . In the congenital heart surgery database, the common terminologies and the definitions of congenital heart diseases published in the annals of thoracic surgery were adopted, and 193 input items were established in the japan congenital cardiovascular surgery database (jccvsd). Thus, the congenital heart surgery databases in the united states, europe, and japan were integrated by using common language in these databases . As a result, although the results were not reported, for example, the discharge mortality in the jccvsd was 0.2%, 0.7%, 3.6%, 7%, and 17.6% for rachs-1 categories 1, 2, 3, 4, and 5/6, respectively, during 2008 to 2010 (fig . This result is comparable to that reported from the sts congenital heart surgery database . Unlike the sts national database, the jcvsd employed web - based data collection . Data on adult cardiac surgery (japan adult cardiovascular surgery database [jacvsd]) were collected beginning in 2001 by five participating units and data on congenital heart surgery (jccvsd) were collected beginning in 2008 by seven units . Jcvsd required informed consent from each patient according to the opt - in rule to comply with the private information protection law . For web - data transmission, high level secure socket layer was adopted for coding of the individual patient s information . The jacvsd and jccvsd grew to become national databases by the end of 2013 (fig . The most recent annual number of submitted procedures are 49,507 in jacvsd and 10,835 in jccvsd . Twenty frequently cited papers dealing with topics such as risk models of isolated coronary bypass surgery, thoracic aortic surgery, and valve surgery have been published in indexed international journals . The performance of the congenital heart surgery risk model as measured by the c - index is over 0.8 . On the basis of these risk models,, adult cardiac surgeons can estimate the 30-day mortality rate, in - hospital mortality rate, and major complication rate after inputting the patient s covariates before the surgical procedure . Japanscore contributes to obtaining adequate informed consent from the patients and the families, leading to increased satisfaction . In addition, benchmark reports have been released as support tools for quality improvement of participating institutions . In japan, many adult cardiac surgeons learn about the risks faced by their patients, as well as their own performance as a surgeon, through the risk - adjusted mortality and benchmark report . To ensure fairness and transparency in evidence - based medicine (ebm), the jcvsd organized a data access and usage working group . This working group meets twice a year, and requests 100% of their data during at least for the immediate 2-years . After the working group accepts an application, the department of health quality assessment (hqa) of the university of tokyo analyzes the newly submitted data . The role of the hqa is similar to that of the dcri for the sts national database . The members of the working group include two to three adult cardiac surgeons; visits to 70 sites have been carried out so far . Recently the hqa reported the details and outcomes of the site visits to the jccvsd . In 2011, the jcvsd started to collect a participation fee of 10,000 yen per year for each section in the jacvsd and the jccvsd . The total number of sections was 658 (541 in the jacvsd, 117 in the jccvsd) by the end of april 2014 . This participation fee is much lower than that required by the sts national database; however, it is an important financial resource, especially for site visits . In 2011, the japanese board of cardiovascular surgery (jbcvs) decided to adopt the data of the jacvsd and the jccvsd for board certification . In 2013, there were 162 new applicants and 1,003 renewals . The jbcvs held its first web - based and paperless review in september 2013 . Compared with the previously employed review method that relied on the submission of operation records, the web - based and paperless review method had higher quality, lower cost, and required less time in 2010, the jcvsd served as the basis for the establishment of the national clinical database (ncd) in japan, which includes clinician - initiated databases reflecting all surgical fields . Data collection with the same security level of jcvsd as mentioned above to protect the individual patient s information . Through the central institutional review board in the university of tokyo, an opt - out rule was adopted, and informed consent became unnecessary . The ncd is governed by a committee whose members are representatives of medical associations related to surgery, such as the japan surgical society (jss), jscvs, jats, the japanese association for chest surgery, the japanese society of gastroenterological surgery, the japanese society of pediatric surgeons, the japanese society of vascular surgery, the japanese society of endocrine surgery, the japanese society for mammary cancer, and the japanese thyroid association . The ncd establishes the surgical board certification system for the jss, which requires 13 input items at the first level in the hierarchy of specialties . Six board certification systems, including the jbcvs and the databases of nine academic associations, are set at the second level as subspecialties . The main server was transferred from the hqa to the university hospital medical information network (umin) with a mirror - image backup . The hqa focused on data analysis and site visits, whereas the umin is responsible for data warehousing . The ncd uses cutting - edge statistical techniques to detect any trace of data inconsistency . The participating associations have supported the ncd financially and the database has grown rapidly; the total number of participating hospitals is 4105, and the number of cumulative procedures was 4,138,000 at the end of april 2014 . The participating associations will release, or have released, their own risk models [1618], and papers have been published based on data from the ncd . The administrative database, diagnosis procedure combination (dpc)/par - diem payment system (pdps) was introduced in japan by the japanese ministry of health, labour and welfare (mhlw, a government agency) in april 2003 to comprehensively assess fixed daily payments and to control medical expenditure in the acute setting based on the quality assessment . The number of participating hospitals by the end of april 2014 was 1,585, including all advanced - treatment hospitals, that is, university hospitals . In japan, total health care expenditures have been increasing by 1 trillion yen annually, and health care expenditures make up 9.5% of the gross domestic product, which puts japan in the 16th position of the 34 member countries of the organization for economic cooperation and development . On the other hand, the population aging rate in japan is over 24%, which is the highest rate in the world . Changes in population makeup and the growing proportion of elderly persons are the underlying issues relating to rising health care expenditures, and successive cabinet office members and the mhlw have set policy directions to address this national issue . Quality improvement, quality assessment, and the pay - for - performance system provide methods to control medical expenditures . The quality and outcomes framework (qof), a system for the performance management and payment of genaral practitioners (gps) in the national health service in england, wales, scotland and northern ireland was introduced as part of the new general medical services contract in april 2004 . In contrast, in the united states, the agency of healthcare research and quality (ahrq) has defined never events or errors of medical care for which medicare, the government healthcare insurance for aged and disabled persons, does not pay . In the c. walton lillehei lecture of the 49th sts annual meeting, the director of the ahrq emphasized that the federal government will pay for the quality, not for the volume . In japan, the ncd and dpc / pdps could play complementary functions for quality assessment through adequate risk adjustment and the complete enumeration of procedures in various surgical fields . In the future, balancing professional autonomy and administrative leadership might be a recurrent issue for quality assessment and quality improvement in japan . Recently, the japanese association of cardiovascular intervention and therapeutics proposed to the ncd a comparative study between percutaneous coronary intervention and cabg that would use well - tested statistical methods such as propensity score matching . Thus, the participation of units from nonsurgical fields, such as medical therapy, intervention, radiation therapy, and chemotherapy, will facilitate risk stratification of each treatment modality, and will contribute to the search for the best management of diseases and patients . A longitudinal follow - up database is needed for the design of such studies, and it is under construction . Recently, the pharmaceutical and medical device agency (pmda), a consultative organization of the mhlw, suggested to enroll in the jacvsd and perform follow - ups on the use of artificial valves for trans - aortic valve implantation . The pmda recognized the completeness and reliability of the data of the jacvsd, and from a cost - performance point of view, the pmda decided to outsource the post - market surveillance of newly covered medical devices in the cardiovascular surgical field . This demonstrates how the national database could contribute to the post - marketing surveillance of drugs and medical devices, and could help control randomized trials and multicenter studies . The ncd will start to collect fees from participating hospitals according to the total number of enrolled surgical procedures . Clerical assistants have been widely employed throughout the country, which has gradually lightened the data input workload of young surgeons . Governmental support and some government funds clinicians are responsible for patient safety and quality improvement, and the database will aid in achieving these goals . As reinertsen stated, to truly improve quality, the system must, 1) eliminate unnecessary variation (standardize processes), and 2) achieve and document continuous improvement (in care processes and outcomes). In recent years, the importance of certainty, not excellence of operations, and that of the concept of structure, process and outcome have been emphasized, and multiple approaches, for instance, postgraduate education systems, reporting systems of malpractice to prevent recurrence, introduction of information technology, introduction of simulators, ebm, and other techniques, have been used for patient safety . Since it is methodologically based on the jcvsd, the ncd represents an interface between medical databases and board certification systems, which is its point of difference from the sts national database . In 2014, a new organization for medical board certification was established in japan that, beginning in 2017, will certify all medical boards in close collaboration with medical associations . This new organization will adopt the standards of the jcvsd and the ncd for evaluating the clinical practices of applicants . For the assessment of medical outcomes and quality, the jcvsd and the ncd will continue to be the sole reliable data source for surgical fields in japan, where medical system reform will be implemented quickly and based on professional autonomy . The national database is fundamental for quality improvement, patient safety, and the adequate control of medical expenditures in the country.
Migraine is a disabling neurological disorder considered by the world health organization as the 19th leading cause of all years lived with disability among both males and females of all ages, and as the 12th leading cause of years lived with disability among females of all ages . Apart from pain, the disability caused by migraine is aggravated by accompanying symptomatology, such as gastro - intestinal symptoms, the most common being nausea and vomiting, to such an extent that their presence is one of the diagnostic criteria for migraine . A telephone interview survey of 500 self - reported migraine sufferers found that nausea occurred in more than 90% of all migraineurs; nearly one - third of these had nausea during every attack . Vomiting occurred in almost 70% of all migraineurs; nearly one - third of these vomited in the majority of attacks . Indeed, 30.5% of those who had nausea, reported that it interfered with their ability to take their oral migraine medication . The american study ii stated that 73% of the migraineurs studied reported to have suffered from nausea during attacks and that 29% had vomited . One of the most interesting approaches to nausea adopted by traditional chinese medicine and, in particular, by acupuncture is the stimulation of the acupoint pc6 neiguan . Indeed, there is documented evidence as to the efficacy of stimulating this point to alleviate chemotherapy - induced nausea and vomiting (cinv), postoperative nausea and vomiting (ponv) and motion sickness, both with acupuncture and acupressure . However, to the best of our knowledge, there are no studies in indexed medical literature as to the efficacy of treating pc6 acupoint for gastrointestinal symptoms in migraine attacks and particularly for nausea . Therefore, our preliminary study aimed at verifying if pressure applied to the point pc6 was effective on the presence of nausea during migraine attacks . A total of 40 female patients were enrolled into this study, after having given their informed consent, and all were suffering from migraine without aura, diagnosed according to the criteria established by the international classification of headache disorders (ichd - ii). The patients were examined at the women s headache center, department of gynaecology and obstetrics of turin university . Inclusion criteria were: at least two migraine attacks per month for a 1-year period before enrollment; no more than 15 days of pain per month . None of the patients were on prophylactic therapy, but were allowed to continue taking their usual symptomatic treatment . The patients medical history had to include the presence of nausea as accompanying symptomatology of their migraine, documented by a diary noting at least 1 month of attacks with nausea, prior to the inclusion in the study . Subjects taking antiemetics to control their nausea, whether as a single product or present as a compound in a combination product for the control of migraine, were excluded from the study . The patients enrolled were asked to fill in a dedicated diary recording the details of the length and intensity of the migraine attacks along with the accompanying symptomatology, paying particular attention to the presence of nausea . A device known as the sea - band the sea - bands are elastic wristbands with a 1 cm protruding round plastic button; these devices apply continual pressure to the pc6 acupuncture point with the aim of decreasing, or completely eliminating nausea (fig . 1). The pc6 point, also called neiguan, is located on the anterior surface of the forearm, 3 fingers widths up from the first wrist crease and between the tendons of the flexor carpi radialis and palmaris longus . The sea - bands were applied bilaterally on both wrists on the neiguan point, starting from the onset of the migraine attack and left in place for no less than 4 h, or for the whole attack period.fig . 1the localization of the point pc6 neiguan and the correct positioning of the sea - bands the localization of the point pc6 neiguan and the correct positioning of the sea - bands the patients were asked to document a total of six migraine attacks: three without the use of the sea - band wristbands (phase c, control) and three with the application of the sea - bands (phase sb). The sequence of the treatment given for the attacks (with, or without sea - bands) was chosen at random according to a scheme provided by the computer and was applied to each single patient . The section of the diary provided that covered the symptom of nausea was detailed to include information as to the time of symptom onset and symptom resolution, the intensity of nausea at the onset (t0), at 30 (t1), 60 (t2), 120 (t3) and 240 (t4) minutes evaluated on a scale from 0 to 10, where 0 indicated no nausea and 10 the maximum sensation of nausea . Diary analysis was carried out by an impartial operator who did not know in which attacks the sea - bands were used or not . In this preliminary study, the average values of nausea in phases c and sb were calculated at different times throughout the study and a statistical evaluation of the differences between the values obtained in t0, t1, t2, t3 and t4 in the two groups studied was performed using a non - parametric friedman test for repeated measures . Moreover, a non - parametric wilcoxon test for paired data was always performed for each level of the variable time to evaluate the difference between phase c and phase sb . This test also took into consideration the average intensity of the three attacks in each of the two phases . All values given in the following text are reported as arithmetic means (sem). All analyses were performed using the statistical package for the social sciences (spss) software program . Only 32 patients (mean age 39.65 years, range 1961) completed the study . Four patients were lost to follow - up, three handed over a diary with incomplete, unreliable data and one patient did not suffer from any migraine attacks in the 3-month observation period . The friedman test for repeated measures showed a highly statistically significant reduction in the intensity of nausea in the sb group (p <0.001) during treatment (at t1, t2, t3 and t4). The wilcoxon test for paired data showed that the nausea intensities were significantly higher in phase c than in phase sb (fig . 2): after 30 min (t1 c 5.55 0.36 vs. t1 sb 4.6 0.40, p = 0.006), 60 min (t2 c 4.93 0.33 vs. t2 sb 3.11 0.40 p <0.001), 120 min (t3 c 3.48 0.35 vs. t3 sb 1.89 0.31 p <0.001) and 240 min (t4 c 2.05 0.28 vs. t4 sb 0.93 0.23 p <0.001). There was no difference between groups at t0 (t0 c 5.96 0.38 vs. t0 sb 6.36 0.35; p = 0.276).fig . 2average values of nausea score before treatment (t0), after 30 min (t1), after 60 min (t2), after 120 min (t3), after 240 min (t4), in phase sb (black columns) and in phase c (white columns). Non - parametric wilcoxon test for paired data at t0, t1, t2, t3 and t4: at t0 p = 0.276, n.s . ; at t1 * p = 0.006; at t2, t3 and t4 * * p <0.001 average values of nausea score before treatment (t0), after 30 min (t1), after 60 min (t2), after 120 min (t3), after 240 min (t4), in phase sb (black columns) and in phase c (white columns). Non - parametric wilcoxon test for paired data at t0, t1, t2, t3 and t4: at t0 p = 0.276, n.s . ; at t1 * p = 0.006; at t2, t3 and t4 * * p <0.001 the number of patients who reported having had at least a 50% reduction in the nausea score was: 0/32 at 30 min in phase c and 7/32 in phase sb (chi square test: p = 0.16 rr 0.43; ci 95% 0.320.58); 1/32 at 60 min in phase c and 15/32 in phase sb (chi square test: p <0.001 rr 0.37, ci 95% 0.250.56); 11/32 at 120 min in phase c and 23/32 in phase sb (chi square test: p = 0.003 rr 0.44, ci 95% 0.240.80); 21/32 at 240 min in phase c and 27/32 in phase sb (chi square test: p = 0.083 rr 0.55, ci 95% 0.251.1). Moreover, when the consistency of the treatment (response in at least two out of three treated attacks) is taken into consideration, it was reached: in 9 patients (28%) at 60 min; in 13 (40%) at 120 min and in 19 (59%) at 240 min . Noteworthy, the nausea was significantly reduced by acupressure in 3/3 attacks: in 5/32 patients (15%) at 60 min; in 10/32 (31%) at 120 min and in 17/32 (53%) at 240 min . Some studies have reported that nausea was present in 73 to more than 90% of the subjects studied and that almost one - third of these experienced nausea during every attack . Moreover, 30.5% of the subjects who reported nausea indicated that its severity even interfered with their ability to take their oral migraine medication [3, 4]. Traditional chinese medicine and especially acupuncture, stimulates some points that can be considered extremely valid from the point of view of nausea and/or vomiting control . In particular, the treatment of the acupoint pc6 neiguan may be applied to this aim, even with the application of acupressure alone, as has been validated by various studies . International literature reports numerous studies on the efficacy of stimulating the acupuncture point pc6 and its capacity to reduce nausea under various clinical conditions . A cochrane review on ponv concluded by stating that, compared with sham treatment, acupoint stimulation significantly reduces nausea (rr 0.71, 95% ci 0.610.83) and the need for rescue antiemetics (rr 0.69, 95% ci 0.570.83). From a cochrane review on cinv, it emerged that acupressure is effective for both mean and worst acute nausea severity, and, therefore, acupressure is able to offer a no - cost, convenient, self - administered intervention for chemotherapy patients to reduce acute nausea . On the basis of the data obtained in this study, the application of acupressure for the control of nausea during a migraine attack seems to be justified . Indeed, the application of the sea - bands on the acupoint pc6 neiguan was observed to be effective in the control of nausea . The average nausea scores drop in the sb phase from 6.36 0.35 in t0, to 4.60 0.39 in t1, to 3.11 0.40 in t2, to 1.88 0.31 in t3 and to 0.92 0.22 in t4 . At each time step taken into consideration after the application of the sea - bands, there was a statistically significant improvement over the non - treated phases . Moreover, there was a high percentage of responders to the treatment: i.e. 46.8% at 60 min; 71.8% at 120 min; 84.3% at 240 min with a consistent response over time . Even when the fact that our study is both preliminary and open is taken into consideration, the results obtained seem to be encouraging and advocate the continuous application of pc6 acupressure in all migraine attacks with the accompanying symptom of nausea . Further controlled studies are, of course, required to validate the findings of this study.
My experience in state government has been that after natural or manmade point - source disasters, a governor reflexively turns to the office of emergency management, the department of public safety (or homeland security), or the national guard for advice and counsel . The staffs of these agencies, however, have neither the expertise necessary to guide the response to an epidemic nor an established, ongoing communications and surveillance system with hospitals, laboratories, and medical providers . It is essential, therefore, to establish a formal process that allows public health and medical experts to assist elected officials in analyzing and interpreting information about the outbreak and in coordinating the public health response to the outbreak . Guidance for fiscal year 2002 supplemental funds for public health preparedness and response for bioterrorism issued by the centers for disease control and prevention (cdc) requires that a state establish an advisory committee that includes representatives from health departments, first responders, hospitals, and voluntary organizations such as the red cross (11). In colorado this advisory committee includes not only the nine groups listed in the cdc announcement but also the presidents of the state board of health, state medical society, and state hospital association; the state veterinarian; a wildlife disease specialist; a medical examiner; a specialist in posttraumatic stress management; a pharmacist member of the board of pharmacy; the attorney general; the chief public information officer for the state health department; and, as an ex - officio member, the chief of the colorado national guard (3). These persons were named to the committee because they possess useful expertise or connections to the community . The statute authorizing the formation of the committee provided legal immunity to members for their advice (4), and the members pledged that they would attend the committee meetings during a bioterrorist attack rather than report to their regular jobs . By meeting regularly the committee members learn about each other s skills, experience, and roles and develop a working relationship that, by itself, can be extremely valuable during a crisis . One notable absence in the composition of the advisory committee is representation from federal agencies, such as cdc, the federal emergency management agency, the environmental protection agency, and the federal bureau of investigation . Although these agencies cannot, as a practical matter, attend meetings in every state and large municipality, during a crisis they will have an integral role, and disputes are more likely if the leaders are meeting for the first time in a highly stressful situation . For example, local - state - federal disagreements occurred in the management of the pneumonic plague epidemic in los angeles in 1924, the last instance of person - to - person transmission of plague in the united states, as well as during the anthrax outbreak in 2001 (12,13). Some existing state regulations, which in normal times are intended to ensure quality medical care, could hinder community efforts during a bioterrorist attack . For example, consideration should be given to modifying, for a limited period through executive orders, the regulations that control the prescription and dispensing of medicine, licensing of physicians and nurses, and transfer of patients between hospitals . Providing antibiotics or vaccinations in mass clinics and obtaining the services of retired or out - of - state physicians and nurses may be necessary . In colorado, executive orders that address these concerns have been drafted by the governor s technical advisory committee . The orders would permit a) health - care providers other than pharmacists and physicians, such as nurses and emergency management technicians, to dispense medications, b) medicines to be distributed without an identified patient s name on the packet or bottle, c) practice of medicine and nursing by professionals who are not currently licensed in colorado, provided the practice is restricted to caring for epidemic - associated illnesses and the persons are working under the supervision of a licensed practitioner (who is given legal immunity for the supervisee s work), and d) persons seeking medical care at one facility to be redirected to another facility without initial assessment or stabilization attempt if the initial hospital is unable to care for any more persons or if a specific facility (established or temporary) has been directed to receive epidemic patients, e.g., those with smallpox . These draft orders must still be tailored to the actual emergency and signed by the governor, but the background legal work can be completed ahead of time . Two additional features of the colorado bioterrorism statute exist; these features were designed to encourage volunteers and remove legal barriers to cooperation among institutions and agencies . First, the statutory definition of civil defense worker was modified to include a physician, health care provider, public health worker, or emergency medical service provider who is ordered by the governor to provide specific medical or public health services during and related to an emergency epidemic and who complies with this order without pay or other consideration (7). With this amendment, civil defense workers may receive compensation for injury, including illness caused by bioterrorism, which is suffered as a result of civil defense service . Second, the statute provides that persons and entities [including hospitals] that in good faith comply completely with board of health rules regarding the emergency epidemic and executive orders shall be immune from civil or criminal liability for any action taken to comply with the executive order or rule and that the state shall provide compensation for property if the property was commandeered or otherwise used in coping with an emergency epidemic (4). To ensure that a sufficient number of health - care providers, laboratory technicians, public health epidemiologists, and administrative support workers show up for work during a bioterrorist attack, appropriate personal protection (e.g., respiratory protection, vaccination, or chemoprophylaxis) for the worker and, probably, for household members of the worker are essential . When performing nonstandard work, the worker may also need legal protection, as discussed above . Plans for a bioterrorist attack should include these factors and be written by the employer who knows how the agency operates and is staffed because people work for an agency, hospital, or institution, not a region . Nonetheless, coordination of resources should develop mutual aid agreements with neighboring jurisdictions and integrate single institution or agency plans into community, regional, or statewide plans . In the 2000 colorado bioterrorism statute, the state board of health was given the new authority to promulgate rules requiring each state and local health department, general or critical access hospital, and managed - care organization to write a plan for responding to bioterrorism (7). While hospitals and health departments may have previously written plans for managing mass casualties resulting from aircraft, bus, or train crashes or natural disasters, such plans need to be modified to include consideration of the special circumstances of bioterrorism (e.g., chemoprophylaxis and personal protective equipment for workers, infection control, and handling of laboratory specimens). Because pandemic influenza may pose challenges to the medical and public health systems similar to those of bioterrorism, a single plan for both types of epidemics should be drafted . During typical outbreaks of communicable diseases, clear and timely communication by the state health department with multiple local health departments and hospitals can be a challenge . In a bioterrorist attack, the communications challenge will likely be greater because many more persons and agencies will be involved . The telephone system may not have sufficient capacity for the increased demand or it may be damaged and disorganized, as happened during the response to the attacks on the world trade centers in new york city in september 2001 (14). Furthermore, a large, sometimes overwhelming, number of inquiries made by members of the public to the public health agency usually occur during public health crises, and therefore, administrative plans for a bioterrorist event should include consideration of this workload . Rather than relying on hospital personnel, public health agencies may find it advantageous to station their own personnel with mobile telephone or radio communications equipment in individual hospitals to assure that public health agencies get the information they need as rapidly as possible . Accomplishing this may require an executive order of the governor that commandeers two - way radios . In colorado, board of health regulations require the state and local health departments to include assignment of employees to hospitals in the agency s emergency plan (8). Disease reporting requires specification of what to report in what manner and timeframe to which parties . A first legal step in this process is to require immediate reporting of any suspected or confirmed illness, syndrome, or outbreak caused by any potential bioterrorist agent . For example, colorado regulations were modified in 1999 so that cases of plague, which had been required to be reported within 24 hours of diagnosis by telephone, fax, or through a web - based system, were to be reported immediately only by telephone to an on - call person if the physician or hospital suspected the case was related to a bioterrorist event (9). Disease surveillance systems are critical not only for the initial detection of an outbreak but also for monitoring the extent and spread of the outbreak and for determining when it is over . Managing a large outbreak would require gathering information from contact tracing and source - of - exposure investigations as well as information about the availability of critical medicine, medical equipment, and the handling of corpses . These information needs are much different than those needed for early detection of an attack . Therefore, legal authority for surveillance should be modified as necessary to ensure collection of all information that could be needed by the public health agency to fulfill its duties throughout the epidemic . This legal authority may include requirements for groups that do not commonly report information, such as pharmacists, to provide it . Administrative public health orders restricting personal behavior of persons with certain diseases, such as tuberculosis, are relatively common in this country (15). Such orders are usually hand - delivered to a specific person(s), and the restrictions are removed after a specified period, such as after one incubation period or when an ill person is no longer infectious . Another type of public health order might involve work restriction, e.g., health - care providers who cannot demonstrate evidence of immunity to a vaccine - preventable disease are not permitted to work during an outbreak of such disease . Few, if any states, however, have experience issuing and enforcing large - scale quarantine orders that last more than 12 days . Orders restricting large numbers of contacts of cases of plague to home were issued in florence, italy, in 1630 and described in the 1999 book, galileo s daughter (16). The enforcement of orders restricting the movement of residents of an entire town in which there was an outbreak of viral hemorrhagic fever was depicted in the 1996 movie, outbreak . The images of severe disease and enforced quarantine are similar in the book and movie and are plausible and disturbing to lay audiences . A more recent, well - documented example of a large - scale movement restriction was the british epidemic of foot - and - mouth disease of 2001, which affected many farms and businesses and led to the quarantine and slaughter of 4 million sheep, cattle, and pigs for disease control purposes (17). In all three examples, a decentralized quarantine was imposed . In general, the advantage of a decentralized strategy (e.g., persons are restricted to home) is that it may reduce the risk for transmission of disease because fewer persons congregate . Alternatively, the centralized strategy (e.g., restricted persons are taken to a sports arena, auditorium, theater, school, or hospital) is seemingly easier for the government to care for restricted persons and to enforce the order but could allow contagious and noncontagious persons to come into contact with each other . Another example of large - scale quarantine occurred in los angeles in 1924 during the last epidemic of pneumonic plague in this country (12). Three days after the first 15 cases in this outbreak became known to public health officials, eight city blocks that housed approximately 2,500 mexicans were placed in quarantine . Public health nurses were sent to the area to make house - to - house inspections to identify new cases, and all patients with suspected cases in the area were examined by physicians at the patient s home and then sent to the county hospital . All persons who lived at addresses where cases had occurred were quarantined in the county general hospital, and a spanish - speaking priest and social workers were placed in the area to reassure and calm the residents . The quarantine actions taken in this outbreak were a combination of centralized and decentralized strategies . As has been discussed by barbera et al . (18), numerous concerns regarding large - scale quarantine exist . All states currently have in place varying degrees of legal authority enabling isolation, quarantine, or travel restrictions if needed to maintain the welfare and safety of the public . Drafting restrictive orders in advance is less helpful than with the other types of orders discussed above because restrictive orders require more tailoring to the specific circumstances and parameters of an outbreak . Factors such as duration and location of restriction are dependent on what the bioterrorist agent is, how it is transmitted, how widely the agent has been disseminated, whether exposed persons can be personally identified, and what resources are available to care for restricted persons . Not drafting such orders in advance, however, means that they may be written during the turmoil of multiple agencies trying to control an outbreak . Authorities should never hesitate to revise the orders on the basis of on updated information . At the end of the operation topoff exercise, for instance, when the governor had issued a travel restriction order for all of metropolitan denver and cdc had quarantined the entire state of colorado, such orders created many unforeseen problems, including how to enforce the orders, maintain essential community services, and distribute foods and prescription medicines . Accurate and substantive information given to the public by credible public health and medical experts can do much to allay the fears of the public and encourage their cooperation and participation in constructive, organized community response efforts (19,20). I have discussed a number of ideas about legal and administrative preparations for a bioterrorist attack, but more work can be done, including development of strategies addressing issues related to mental health, disposal of corpses, performing forensic autopsies, signing death certificates, and managing potential animal vectors of disease . I have not discussed the sharing of medical and epidemiologic information between public health agencies and law enforcement agencies, such as the federal bureau of investigation . Under normal circumstances, public health officials typically argue that release of disease surveillance information to the criminal justice system will discourage persons with reportable conditions from disclosing to public health officials where they have been and with whom they have had contact . However, a bioterrorist attack is not a routine event, and i recommend that state and local public health agencies review the laws and regulations governing the confidentiality of disease surveillance records and develop a legal and administrative protocol for sharing pertinent and relevant information with law enforcement agencies during a bioterrorist attack (21). Finally, i have not discussed the protection of civil liberties and due process for persons affected by executive orders of the governor and public health officials . This is an important and difficult issue, especially when well persons are quarantined solely on the basis of their having visited, worked, or resided in a particular location at a particular time, as opposed to having had face - to - face contact with a known contagious person . Public health officials and attorneys general should review existing safeguards for the protection of civil liberties and determine whether modifications need to be made for the special circumstances created by a bioterrorist attack.
The name of mucosa - associated lymphoid tissue (malt) lymphoma was first established in 1983 by isaacson and du . From the beginning, it was adopted well and is still used in an unchanged form . Marginal zone lymphoma of malt is, apart from diffuse large b - cell lymphoma, the most frequent type of lymphoma that occurs in the stomach . What is important is that it can develop in almost every organ and tissue, for instance lungs, breast, thyroid gland, bladder, skin, or orbital adnexa . It is an indolent type, but clinical outcomes and response to treatment vary among patients . Malt lymphoma arises from the extranodal sites reach in b - lymphocytes, which appears in response to chronic antigenic stimulation caused by infection (helicobacter pylori) or autoimmune process (hashimoto disease). This disorder is the best example of how infectious pathogens and genetic abnormalities lead to malignant transformation . Gastric malt lymphoma pathogenesis is a complex process including many gene alternations that result in cancer appearance . Better understanding of the background of the disease is crucial for discovering new prognostic factors, helpful in deciding when more aggressive treatment should be employed . The incidence of malignant lymphomas is at the rate of 3%-4% of all malignancy worldwide and has been increasing during the last 50 years . Lately, some stabilization in the number of diagnosis was observed, but only in developed countries . Malignant lymphomas are observed to be more frequent in north america, australia, and europe than in asia and africa . Malt lymphomas determine almost 7% of all non - hodgkin's lymphoma, and at least 40% is primarily located in stomach . It is confirmed that gastric malt lymphoma occurs in younger patients than the rest of malignant lymphomas . The malt lymphoma is mainly a disease of older adults, with a median age of 60 years ., there is much higher proportions of malt lymphomas, which can be caused by more frequent prevalence of helicobacter pylori in this region of the world . Although 90% of population worldwide have confirmed bacteria colonization, only 2% will develop malignant lymphoma . It was confirmed by weber et al . That almost 90% of patients with gastric malt lymphoma are infected with helicobacter pylori . This curved bacillus, previously called campylobacter pyloridis, is a gram negative pathogen found in the stomach . Although over 80% of people are asymptomatic, chronic infection can lead to gastritis, gastric and duodenal ulcer, gastric adenocarcinoma, and malt lymphoma [5, 6]. Nowadays, it is widely accepted that helicobacter pylori gastritis is crucial in an evolution of malt lymphoma localized in stomach . It was confirmed by several studies that chronic gastric inflammation causes constant antigenic stimulation, which leads to clonal expansion of b - cell lymphocytes [7, 8]. In the gastric mucosal cells, there are elevated levels of some cytokines, including proliferation - inducing ligand (april), which belongs to the tumour necrosis factor (tnf) family . April is produced by macrophages present in the gastric malt infiltrate, located close to the neoplastic cells . April may also induce b - cells transformation and the progression to the diffuse large b - cell lymphoma (figure 1). They come either from t cells or directly by the antigenic autostimulation of lymphoma cells . Gastric inflammation causes the appearance of a large number of macrophages, which, under a helicobacter pylori infection, release large amounts of april . Importantly, a number of april - producing macrophages significantly decrease in complete remission after eradication therapy . Other pathogens, are also suspected to play an important role in malt lymphoma pathogenesis . There are bacteria such as campylobacter jejuni, borrelia burgdorferi, and chlamydia psittaci and viruses like hepatitis c virus (hcv) that are potentially responsible for oncogenesis . These pathogens were found in histological material, but so far no strong evidences were established . Patients with autoimmune disease have for sure higher risk of developing malt lymphoma . Autoreactive b cells infiltrate the healthy organs and create lymphoid infiltrate similar to normal malt tissue with huge amount of reactive clonal b lymphocytes . This situation is observed in salivary gland in patients with diagnosis of sjgren syndrome and in the thyroid gland in hashimoto disease . Sjgren syndrome is associated with 44 times increased risk of lymphoma, whereas hashimoto's thyroiditis causes 70 times increased risk of thyroid lymphoma . Some of them are proven to be strongly associated with the disease, but some are still not confirmed . It is believed, that on the background of chronic inflammation not only reactive b - cells are stimulated but also activated neutrophils which can lead to production of oxygen species . As a result, this genotoxins provoke dna damages, which are responsible for mutations and transformations of genetic material . It originates from a fusion of two proteins: apoptosis inhibitor 2 (api2) and paracaspase malt lymphoma - translocation gene 1 (malt1). What is more important is that while t(11;18)(q21;q21) is detected, no other chromosome abnormality can be found . Unfortunately, positive cases do not response to helicobacter pylori eradication, but, in contrast, they do not transform to more aggressive diffusive large b - cell lymphoma . It is known that complete remission can be seen in at least 20% of patients with t(11;18)(q21;q21). The incidence of positivity for this translocation malt lymphoma is at approximately 20% in europe [16, 17] but is not as common in the united states where only 5% are positive . Moreover, it is usually connected with an advanced stage of disease and poor outcomes . Bcl-10 gene is relocated from chromosome 1 to 14, which in consequence triggers overexpression of bcl-10 protein also known as ciper, carmen, or me10 . In healthy organisms, higher expression is observed in lymph nodes, spleen, and testis . So far, it is believed that bcl-10 protein expression is responsible for proliferative effects [19, 20]. The t(14;18)(q32;q21)(igh - bcl-2) is commonly present in follicular lymphoma, in about 20% of diffuse large b - cell lymphoma and sometimes in chronic lymphocytic leukemia . Although this aberration is extremely rare in other types of lymphomas, it can be found in some cases of gastric malt lymphoma . Bcl-2 is an antiapoptotic protein, which helps in survival and expansion of clonal b cells . So far, the role of t(14;18) in gastric malt lymphoma is not fully understood . Overexpression of bcl-2 is found not only in translocation positive patients but also in the negative ones . It is believed that similar to other types of lymphomas, t(14;18)(igh - bcl-2) must coexist with other genetic abnormalities in order to develop neoplasm . T(3;14)(p14;q32)(igh - foxp1) is a newly described abnormality present in patients with malt lymphoma . The first study showed that positivity for this translocation is approximately 10% of all malt lymphoma patients . The most recent studies described the presence of t(3;14)(p14;q32) in diffuse large b - cell lymphoma, outside the lymph nodes especially [23, 24]. Only one study, so far, confirmed the existence of this translocation in gastric malt lymphoma which involved bad clinical outcomes . In pathogenesis of malt lymphoma, the above described translocation promotes oncogenesis by similar well - known mechanism . The majority of them involve the same pathway, which leads to antigen receptor - mediated activation of nfb . This is a crucial transcript factor which plays a key role in malt lymphogenesis [26, 27]. It regulates processes connected with b - cell development, growth, and survival by production of cytokines and growth factors, for example, tnf- family (baff). Latest studies have shown that b - cell activation in malt lymphoma can be strictly connected with tnf family . It can be also responsible for activation of cell apoptosis [28, 29]. It is observed that in patients with higher baff levels in serum, the prognosis and survival are much worse . Based on recent knowledge about genetic abnormalities in gastric malt lymphoma, there is a model of multistep pathogenesis . On the background of chronic inflammation and antigenic stimulation occurs genetic instability . As a result, many possible translocation and unbalanced aberrations are observed . Gastric malt lymphoma can be long - time asymptomatic or associated with dyspepsia, abdominal pain, vomiting, diarrhea, obstruction, and nausea . Sometimes bleeding from gastrointestinal tract or even perforation may occur while extensive lesions are present . As a result, symptoms of anemia like paleness, weakness, or easy fatigue can be observed . B symptoms (weight loss, unexplained fever, and night sweats) in gastric malt lymphoma are very rare, but the most common of the above is weight loss . A prompt diagnosis is crucial, but, unfortunately, it is usually made by incidence . Patients with early stage of disease have usually low tumor growth and minimal possibility to spread . In contrast, patients with advanced stage of disease can undergo transformation to more aggressive lymphoma and may become resistant to treatment . Not only symptoms but also endoscopic picture can be inconclusive . Difficulties often arise to differentiate between chronic gastritis or ulcer from an early - stage lymphoma . In order to confirm the diagnosis, there always must be made pcr or fish analysis for t(11;18), which is important to separate groups that will not respond to standard treatment . Characteristic for gastric malt lymphoma are lymphoepithelial lesions (lel) with the presence of mainly two types of cells: neoplastic centrocyte - like or small lymphoid . There is no specific immunohistochemical profile typical for gastric malt lymphoma diagnosis . In 50% of patients, there is coexpression of cd43/bcl2 . Neoplastic cells are positive for cd-20 and negative for cd-10, cd-23, and cyclin d1 . If it is negative in histochemistry, rapid urea breath test or fecal antigen test have to be made . Another analysis to prove absence of helicobacter pylori infection is serological test for caga antibodies and helicobacter pylori - igg antibodies . Sometimes, there is possibility to detect other helicobacter species, for example, heilmannii or felis . Before taking any decision on how aggressive the treatment should be, it is extremely important to perform a complete staging of the disease . What is more important is that risk factors and individual parameters, which can affect later therapy, are crucial . Medical history must include information about the age, time of the first symptoms, the family history, and medical condition . The most important factor that we rely on during choosing method of treatment is clinical stage of the patient . During physical examination, it is important to remember about waldeyer's ring, which is mandatory in every gastric lymphoma patient . Staging in gastric malt lymphoma is similar to that in other types of lymphomas . According to recent european society for medical oncology (esmo) recommendations, it should include morphology with basic biochemical studies . If the blood cell count is lower, it can be caused by infiltration of bone marrow . Biochemical tests can detect liver or kidney problems, which can be important before the beginning of a chemotherapy . Lactate dehydrogenase (ldh) and 2-mikroglobulin are prognostic factors and will be abnormally high in patients with fast - growing tumor . Every newly diagnosed patient should be examined in case of certain viral infections that can affect treatment, such as hepatitis b and c or human immunodeficiency virus (hiv). In every case, computed tomography (ct) scans of neck, chest, abdomen, and pelvis, which are crucial to evaluate enlarged lymph nodes, should be performed . Core needle biopsy of bone marrow is made to diagnose possible infiltration of neoplastic cells . It was confirmed that 15% of gastric malt lymphoma patients have lymphoma cells in bone marrow . Positron emission tomography (pet) has still not confirmed clinical necessity, but it can be extremely helpful in controversial cases . Moreover, during staging procedures of gastric malt lymphoma, gastroduodenal endoscopy must be made . Biopsies are taken from different sites of gastrointestinal tract (e.g., stomach, duodenum, and gastroesophageal junction) and every location that looks suspicious . Most often, ann arbour staging is employed, which describe the extend of all types of non - hodgkin lymphoma in adults . Thus, staging of gastric lymphoma based upon the ann arbor system includes stage i e, which is disease limited to the stomach without nodal spread . Stage ii e1 is tumor in the stomach with spread to adjacent contiguous lymph nodes . Stage ii e2 is tumor in the stomach with spread to lymph nodes that are noncontiguous with the primary tumor . Moreover, if the spleen is affected, we add s. if the person has any of the b symptoms, we add letter b, and if is asymptomatic, we assign a (table 1). Prognostic factors in gastric malt lymphoma are similar to the value for non - hodgkin b - cells lymphoma . Factors that determine poor outcome are age, high level of ldh in serum, higher ecog performance status, stages iii and iv in ann - arbour scale, white blood count, and more than one extranodal site . It was observed that patients with nodal invasion has difficulty with complete remission after eradication treatment . For instance patients, with t(11;18)(q21;q21) especially, are resistant to the first line therapy, and remission rate was lower than that in patients of api2-malt1 negative (78% versus 22.2%; p = 0.0001). Only one study so far proved that the presence of t(3;14)(p14;q32) is connected with poor clinical outcomes of patients with gastric malt lymphoma . While helicobacter pylori plays a main role in the pathogenesis of malt lymphoma, it is also crucial in approach to the treatment . According to current international guidelines, first line treatment for localized helicobacter the treatment may be used with every highly effective antibiotics against helicobacter pylori, taking into consideration the locally expected antibiotic resistance . If there is no response to the therapy above, second line triple or quadruple therapy is used . It was reported that after two lines of treatment, 99.8% of patients were cured from gastritis . In a large study of 1408 patients, remission after eradication treatment in early stage unfortunately, in 5%10% of gastric malt lymphoma patients, we cannot confirm helicobacter pylori infection . Moreover, more than 30% patients are resistant to first line treatment, and 30% of them have t(11;18)(q21;q21). Treatment for this patients should be chosen individually depending on the clinical stage of disease . For those who have stable disease without any symptoms, radiotherapy, chemotherapy, and/or surgery can be considered after unsuccessful eradication treatment . Further recommended surgery is considered to be a standard therapy in therapy of patients with gastric malt lymphomas, but, recently, the value of this therapy has been not confirmed . Even if the lymphoma is localized at early stage, the gastrectomy should be rather extensive due to the nature of the disease . Moreover, it is a major surgery and can be associated with serious complications and worsen a quality of life . German multicenter study group (gmsg) presented no difference between survival in patients treated with gastrectomy compared to eradication (overall survival rate 82% to 84%). What is more important is that there were observed 50% long - term complications were observed after surgery . In few studies the use of a modest dose of involved fields was performed on resistant - to - eradication therapy patients with early - stage disease . The dose was 2535 gy to the stomach and perigastric nodes for the period of 4 weeks [42, 43]. Compared to surgery, no serious long - term complications and toxicity were observed . Only nausea and anorexia were present during the time of radiotherapy . For a long time it was believed that gastric malt lymphoma is just a localized disease and that surgery and radiotherapy are the best treatment strategy . Now, when it is well known that it is disseminated disorder chemotherapy, it became more important . Still, there are no standard recommendations for relapse or progressive patients after therapy and for those with late stage of the disease from the beginning . It was observed that chemotherapy alone is more effective than surgery apart from some cases with gastric obstruction . Complete remission (cr) after oral monochemotherapy with cyclophosphamide was 83% in a study by nakamura and coworkers . Unfortunately, patients with positive translocation t(11; 18) are resistant to second line therapy with oral monochemotherapy with alkylating agents . Nucleoside analogs are confirmed to be effective in treatment of different kinds of indolent lymphomas . A polychemotherapy with fludarabine and mitoxantrone (fm) has a very good effect on patients with gastric malt lymphoma in both first and second line treatment . The complete remission after 4 cycles achieved 84% of investigated and all of them reacted to the treatment . After 2-cda, there were observed complications such as toxicities of 3 and 4 grade of who, mainly leukopenia, infections, and secondary neoplastic disease . Nowadays, immunotherapy became an extremely important part of treatment of non - hodgkin lymphomas . It is a chimeric mouse / human monoclonal antibody specified to cd20 antigen expressed on the surface of b lymphocytes . Now it is widely used alone or in combination with chemotherapeutic drugs in many types of b - cell non - hodgkin lymphomas . Rituximab binds to cd20 antigen and activates the lysis of b cells by mediating cytotoxicity of complement dependent (cdc) and cell - mediated cytotoxicity antibody dependent (adcc). The role of this drug is still not clear in gastric malt lymphoma . In 2003, there was a first - phase study by conconi et al . The cr was observed in 29% and overall response rate (orr) was 64% . The toxicity of this treatment was moderate or even mild, but the relapse rate was 36% . An important fact is that patients with translocation t(11;18) are responsive to rituximab treatment [58, 59]. What is more important is that in a study by the international extranodal lymphoma study group (ielsg), it was confirmed that chlorambucil in combination with rituximab was more effective than chlorambucil alone . The conclusion is that rituximab may a benefit in individual patients, but for the majority it is not sufficient when used alone . The efficacy of the combination of rituximab with chlorambucil was evaluated in a randomized study (comparator was chlorambucil alone) by the international extranodal lymphoma study group (ielsg) in gastric malt lymphomas that had failed antibiotics and in nongastric malt lymphomas . The preliminary report showed that the 5-year event - free survival was significantly better for patients treated with chlorambucil plus rituximab . There were also studies by raderer et al . With cycles generally used in more aggressive lymphomas . Twenty - six patients were administrated rituximab plus cyclophosphamide, doxorubicin or mitoxantrone, vincristine, and prednisone . Lately, bortezomib, the first therapeutic proteasome inhibitor, was examined by kiesewetter et al . In 2012 with cr in 33% and pr in 27.8% . The results on phase ii studies with chemotherapy and immunotherapy are shown in table 2 . Outcomes in gastric malt lymphoma patients with progressive, disseminated disease are very comparable with outcomes in follicular lymphoma . Although gastric malt lymphoma has a very favorable outcome, it is still important to have a proper followup . It is possible that the disease will return even after 5 years of complete remission . The relapse can be due to reinfection of helicobcater pylori . In a study by zullo et al . The followup is obligatory in patients with gastric malt lymphoma to identify early phase of the recurrence of the disease . To confirm a complete remission, although, there are no specified recommendations for a followup, the biopsy of gastric sites should be made every 6 months in first two years, and later once a year for the next five years . Systemic followup consist of blood tests and minimal adequate radiological and ultrasound and should be made at least once a year in the first 5 years . The transformation in more aggressive lymphoma is low at the level of 0.05%, but there is a higher risk of occurrence of secondary neoplasm and gastric cancer . These studies confirm that patients with gastric malt lymphoma need a long - term followup not only to detect early recurrence but also to find secondary disease . Recently, enormous progress has been made in better understanding of pathogenesis of gastric malt lymphoma . It has a great influence on the development of new and more effective treatment strategy . Still, not enough clinical trials are performed due to rare expression and high effectiveness of first line treatment of gastric malt lymphoma . What is more important is that early diagnosis of gastric malt lymphoma is extremely important . While the symptoms are unspecific or not, always during the endoscopic exam the complete histological biopsies must be taken to make diagnosis correctly . The less advanced the stage of the disease, the bigger the chances to achieve complete remission
Airprom: airway disease, predicting outcomes through patient specific computational modeling (fp7); bioshare - eu: biobank standardization and harmonization for research excellence in the european union (fp7); ict: information communication technology; medall: mechanisms of the development of allergy (fp7); naepp - epr3: national asthma education and prevention program, expert report 3; ncd: non - communicable disease; p4, predictive, preventive, personalized and participatory; u - biopred: unbiased biomarkers in prediction of respiratory disease outcomes (fp7); un: united nations; who: world health organization . The authors declare that they have no competing interests in relation to the content of this article . May 2008: 61st world health assembly . Who recommended a worldwide priority policy on ncd prevention and control (2008 to 2013), including cardiovascular disease, cancer, chronic respiratory diseases and diabetes, not least because they often have common environmental risk factors . May 2010: united nations (un) general assembly unanimously adopted resolution a / res/64/265:' tackling ncds constitutes one of the major challenges for sustainable development in the 21st century' . December 2010: the council of the european union adopted conclusions based on innovative and global approaches for ncds in public health and healthcare systems to further develop population - based and patient - centered policies . 2010: us center for disease control and prevention (cdc) says that' an essential strategy for keeping older adults healthy is preventing ncds and reducing associated complications' . The classical definition of prevention includes: primary prevention: to avoid the development of disease . Secondary prevention: recognize a disease before it results in morbidity (or co - morbidity). Tertiary prevention: to reduce the negative impact of established disease by restoring function and reducing disease - related complications . Expanding on the traditional model of prevention, gordon proposed a three - tiered preventative intervention classification system on the basis of the population for whom the measure is advisable based on a cost - benefit analysis: universal prevention addresses the entire population (for example, national, local community, school, and district) and aims to prevent or delay risk factor exposure . All individuals, without screening, are provided with information and skills necessary to prevent the problem . The subgroups may be distinguished by characteristics such as age, gender, family history, or economic status . Indicated prevention involves a screening process . According to these definitions, health promotion should be used for primary universal and selective prevention strategies, whereas p4 medicine (predictive, preventive, personalized and participatory) should be used for primary, secondary and tertiary indicated prevention strategies . To prevent the occurrence of ncds by implementing effective action at societal and individual levels: to detect and diagnose disease at an early stage, when it can be controlled effectively . To stratify patients into groups, enabling the selection of optimal therapy . To reduce adverse drug reactions through the predictive or early assessment of individual drug responses and assessing genes leading to ineffective drug metabolism . To improve the selection of new biochemical targets for drug discovery . To reduce the time, cost, and failure rate of clinical trials for new therapies . To shift the emphasis in medicine from reaction to prevention and from disease to wellness . Part of the conceptual work presented has received support from the european commission fp7 projects airprom (grant agreement fp7 270194), bioshare - eu (grant agreement fp7 261433), medall (grant agreement fp7 264357), synergy - copd (grant agreement) and u - biopred (grant agreement imi 115010). Jb, jma, ac - t, fk, mlk, sp, cp and ca were supported by medall; pjs, i m a and kfc were supported by u - biopred; jr and aa were supported by synergy - copd; cb was supported by airprom; ac - t was supported by bioshare - eu . The positions, proposals and ideas expressed in this paper have been discussed by several authors (ca, zc, lh, ab, jb, ac, sa, dc, dn) during the inaugural event of the european institute for systems biology and medicine of the systemoscope international consortium at the biovision world life sciences forum in lyon on 28 march 2011.
The adventitia is more than just the outermost layer of the artery; it is now known to play a critical role in vascular remodeling and other important processes of the artery [13]. Recent attention to the role of the adventitia in vascular remodeling has increased reporting of common carotid artery interadventitial diameter (iad), a noninvasive measure of vascular geometry and health . The results from one of these studies, an ancillary of the study of women's health across the nation (swan) called swan heart, suggest that declining endogenous estrogen that accompanies the menopausal transition has a direct effect on the peripheral vasculature . The study found lower levels of estradiol were significantly associated with larger common carotid artery iad even after adjustment for cardiovascular risk factors . Other studies have shown that larger iad is associated with increasing age [57], cardiovascular risk factors [46, 813], prevalent cardiovascular disease [12, 14, 15], and incident cardiovascular events . Thus, the increase in iad observed with declining endogenous estrogen suggests that lower levels of endogenous estrogens are associated with a less healthy vasculature . The strong association between iad and endogenous estrogen suggests that a similar association may exist with the use of exogenous estrogen . The purpose of this study was to determine whether postmenopausal current ht users had significantly different iad than those who were former users of ht in the women on the move through activity and nutrition (woman) randomized trial . We also wanted to determine if there were differences between other measures of vascular health . This study evaluates cross - sectional associations using measurements from the baseline visit of the clinical trial (clinical trials registry number: nct 00023543). The woman trial tested the ability of nonpharmacological lifestyle intervention to modify cardiovascular risk factors in postmenopausal women . The study recruited 508 eligible african american and caucasian women from allegheny county, pa, between april 2002 and october 2003 through direct mailings . Eligible women were postmenopausal, between 52 and 62 years of age, able to walk, currently using ht, and willing to participate in either intervention group regardless of assignment and had a waist circumference 80 cm, a body mass index (bmi) between 25.0 and 39.9 kg / m, blood pressure <160/95 mmhg, and low density lipoprotein (ldl) cholesterol between 100 and 160 mg / dl . Women were ineligible if they were taking medication for cholesterol, diagnosed with or on medication for diabetes, diagnosed with a psychotic disorder, or suffering from depression . The results of the women's health initiative estrogen / progestin arm were published in the middle of recruitment; as a result the eligibility criterion of current use of ht was modified to current or recent history of hormone use . Recent history of hormone use was defined as prior use of at least 2 years within 6 months of randomization . The decision to remain on ht was determined by the participant and her physician . At baseline, 40% of the women had discontinued use of ht (these women will be referred to as former ht users) and 60% remained on ht (these women will be referred to as current ht users). For those who discontinued use of ht the median time off therapy was 7 months prior to study randomization . Common carotid artery intima media thickness (imt), iad, lumen diameter (ld), and plaque were assessed by b - mode ultrasound using a toshiba ssa-270a duplex scanner (toshiba american medical systems, tustin, ca, usa) with a 5 mhz - linear array transducer . Right and left carotid images were taken of the near and far walls of the distal common carotid artery 1 cm proximal to the carotid bulb . Imt was defined as the distance from the lumen - intimal interface to the medial - adventitial interface (figure 1). Iad was defined as the distance from the adventitial - medial interface on the near wall to the medial - adventitial interface on the far wall (figure 1). Ld was defined as the distance from the intima - lumen interface of the near wall to the lumen - intima interface of the far wall (figure 1). Using a semiautomated edge detection software, the interfaces were traced electronically over the distal cca and a computer generated measurement was obtained for each pixel in the area of interest; these measurements were averaged to determine imt, iad, and ld used for this analysis . A reproducibility study, conducted in 20 women who were similar to the women in the current study, provided an intraclass correlation of 0.98 for imt and 0.99 for iad . The reproducibility study took place at the same lab and used the same equipment and readers as the current study . The presence of plaque was determined for each of the 5 segments of the left and right carotid artery (distal and proximal cca, carotid bulb, and proximal internal and external carotid artery). Plaque was defined as a distinct area protruding into the vessel lumen at least 50% thicker than the adjacent imt . The first of two prerandomization screening visits included a 12-hour fasting blood draw, physical measures of height, weight, waist circumference, blood pressure, the long distance corridor walk, medical, physical activity, and weight history . Conventional enzymatic methods were used to obtain total cholesterol, high density lipoprotein (hdl) cholesterol, and triglyceride concentrations from the blood samples . Low density lipoprotein (ldl) cholesterol was estimated using the friedewald equation . Medical history included history of drug, vitamin / mineral supplement, and alcohol use . Common carotid artery iad, ld, imt and plaque were measured at the second screening visit . Seventeen women had incomplete data for the calculation of iad or imt and were excluded leaving 491 women for analysis . Means and standard deviations are presented for normally distributed variables and medians and 25th and 75th percentiles are provided for nonparametric variables; dichotomous variables are presented as percents . Differences between the current ht users and the former ht users were determined using chi - square analyses for categorical variables and t - tests and wilcoxon - rank sum tests for continuous variables . Simple linear regression was used to assess univariate associations between iad and ld with ht and the following cardiovascular risk factors: age, race, systolic blood pressure, diastolic blood pressure, pulse pressure, bmi, weight, height, waist circumference, total cholesterol, ldl and hdl cholesterol, triglycerides, glucose, insulin, smoking status, and antihypertensive medication use . When collinearity between covariates was suspected (r> 0.4), the variable most strongly correlated to iad or ld was selected for the analysis . The following variables were collinear: glucose and insulin; systolic blood pressure and pulse pressure; bmi, weight, and waist circumference; weight and height; total cholesterol and ldl . Based on spearman correlation results glucose, pulse pressure, weight, and ldl were chosen for the multivariable models . Multivariable linear regression was used to test for the following predetermined covariates: age, race, pulse pressure, and smoking status . In addition, any statistically significant variable in the univariate analysis and any variable that differed by ht use status were also tested . Total cholesterol, hdl, ldl, glucose, and insulin differed by ht use status; addition of these variables did not alter the regression model so they are not presented in the results . The median (25th, 75th percentiles) age of the women was 57 (55, 60) years, median bmi was 30 (28, 34) kg / m; 11% were african american and 6% were current smokers . There were 197 former ht users and 294 current ht users at the time of the baseline carotid ultrasound scan . Former ht users were older and had a higher percent of african americans (table 1). Overall former ht users had a significantly worse cardiovascular disease risk profile than current ht users: higher total cholesterol, higher ldl cholesterol, higher glucose and insulin, and lower hdl cholesterol (table 1). There were, however, no differences by ht status in blood pressure, measures of general or central obesity, and smoking status (table 1). The mean iad was 6.94 mm for former ht users and 6.79 mm for current ht users (p = 0.001, table 2). Ld was also significantly larger in the former ht users than in the current ht users (5.44 mm versus 5.31 mm, p = 0.002, table 2). However, imt and presence of plaque were not different between the two groups (table 2). Simple linear regression showed that in addition to former ht use, larger iad was significantly associated with greater systolic blood pressure, pulse pressure, bmi, weight, height, waist circumference (all p <0.0001), glucose, insulin (both p = 0.001), age, caucasian race, current nonsmoking status and use of antihypertensive medications (p <0.05) (table 3). The most parsimonious model in the multivariate analysis revealed that higher pulse pressure, higher weight and former ht use were the key factors independently associated with larger iad . The model was also run forcing age, race, and smoking status (table 4). In this model, hormone therapy, pulse pressure, and weight remained significantly associated with iad (all p <0.01, table 4). The model was also run controlling for antihypertensive medication use, but this variable fell out of the multivariable model when pulse pressure was added . Current ht use was associated with a 0.14 mm smaller iad (table 4). African american women had a 0.05 mm smaller iad than the caucasian women, although this was not significant (table 4). Current cigarette smokers had 0.18 mm smaller iad than current nonsmokers, with borderline significance (table 4). Former ht use, greater bmi, weight, height, waist circumference, insulin (all p <0.01), glucose, and pulse pressure (both p <0.05) were associated with larger ld in univariate linear regression (table 3). The variables that yielded the best multivariate model to explain larger ld were former ht use, higher pulse pressure, and higher weight . When age, race and smoking were forced in the model only ht use and weight remained significantly associated with ld (table 4); the same results were seen when antihypertensive medication was added to the model (data not shown). Current ht use was associated with a 0.13 mm smaller ld (table 4). Each kg of weight was associated with 0.009 mm larger ld (table 4). Postmenopausal current ht users had statistically significant smaller iad than the former ht users; this relationship remained significant after adjustment for known cardiovascular risk factors . The current ht users also had statistically significant smaller ld than the former ht users . In contrast, imt and plaque were not statistically different between current ht users and former ht users . It also demonstrates the value of measuring iad and ld in this type of study . The adventitia, the most outer layer of the artery, is composed of supportive connective tissue, fibroblasts, collagen, and elastin fibers . Estrogen is known to preserve arterial structure by slowing elastin and connective tissue degradation, and by slowing age- and estrogen - related increases in collagen which lead to increased vascular stiffening . A small diameter reflects a healthy vasculature that is able to maintain an optimal balance of shear and tensile stress [1921]. This can make the artery vulnerable to injury and atherosclerotic development [1, 11]. The results of the current study, specifically the association of current exogenous estrogen use with smaller iad, is in line with the swan heart study that showed an association between higher levels of endogenous estrogen and smaller iad . The current study observed a 0.15 mm difference in iad between the current and former ht users . A longitudinal study observed 0.03 mm increase in iad each year for women (with similar mean age and mean height as the women in the current study). So the difference in iad observed in the current study translates to the change in iad expected over 5 years (0.03 mm / year 5 years = 0.15 mm). These findings agree with the results of a cross - sectional study that found smaller ld among non - oral (percutaneous gel or transdermal patch) ht users compared to ht non - users . Together, the findings from iad and ld may suggest the positive effect of estrogen on the vasculature through maintenance of vascular structure and function . Both the current study and the swan heart study found that larger diameter was associated with older age, higher systolic blood pressure, higher glucose, and higher insulin: all risk factors for cvd . Additional supporting evidence that enlarged diameter is an indicator of poor vascular health come from several studies showing enlarged iad is associated with cardiovascular disease risk factors [46, 813], increased imt [5, 12], plaque [5, 12, 23], and prevalent [12, 14, 15] and incident cvd . Polak et al . Recently published an article that identified a positive relationship between iad and left ventricular mass, an indicator of left ventricular hypertrophy . Each 1 gram difference in left ventricular mass was associated with 0.006 mm larger iad in a multiethnic population of women after adjustment for height, weight, and imt . Arterial diameter differences in current ht users and former ht users were observed in this study but differences in imt were not . Consistent with our findings, a cross - sectional study of an american cohort from the atherosclerosis risk in communities (aric) study, found no significant difference in imt by ht use . The women in the aric study were of similar age to the women in this study and also had an undefined ht regimen that was predetermined by the woman and her physician prior to the study . Selection bias may be present in both studies since women who chose to go on ht or women who chose to continue ht may have been different from the women who did not chose ht . A longitudinal study of oral therapy with one year of follow - up did not find a difference in imt progression in ht users and non - users . Three studies evaluated differences in imt by ht and age [22, 26, 27]. The women were dichotomized into younger versus older (using 55 or 60 as the age cut - point). Significant differences in imt were observed only in the older women who had longer use of ht than the younger women [22, 26, 27]. This may suggest that the effects of estrogen on imt are evident after long - term use . Other explanations are that the differences observed are attributed to differing vascular effects of oral ht compared to transdermal ht, and fewer years on ht in the negative studies than the positive studies . One study that compared oral and non - oral therapy found transdermal ht had greater statistically significant effects on imt than oral ht . The aric study and this study participants used oral ht, were relatively younger and had fewer years on ht compared to the positive studies . A limitation of this study is that the ht regimen was varied since the dose, hormone composition (estrogen only or estrogen plus progestin), and form were chosen prior to the study by the participant and her health care provider . A standard dose and regimen of ht would be easier to evaluate and compare this study to previous studies . This would likely be especially true for the imt results that were not significant in this study . Another limitation is that the adherence to ht and the level of estrogen or estradiol in the current users and former users was not assessed in this study . Although the women reported ht use we do not know their adherence rates or the level of estrogen metabolites present during the ultrasound measurements . In the future, assessment of estradiol levels should be included to improve our understanding of carotid diameter associations and dose - related effects . Strengths of this study are that it fills a gap in the literature, the methods used are valid and reliable, the lab that performed the ultrasound measures has high quality control, and it is one of the first to evaluate iad and ht . This study demonstrates the importance of iad and ld as more sensitive indicators of vascular health than imt . High resolution b - mode ultrasound is a valid and reliable detector of structural atherosclerotic changes of the arterial walls . The ultrasound measures in this study were performed with excellent reproducibility (class intra correlations of 0.98 for imt and 0.99 for iad) and continuous quality control to ensure reliable and valid data . In conclusion, these data suggest that current ht use is associated with vascular geometry in the postmenopausal women independent of cardiovascular risk factors . It also demonstrates the importance of measuring iad and ld in postmenopausal women with differing ht use . These measures should be included in addition to imt to provide a more complete story of vascular response and health.
Prostate cancer (pca) is the most common solid organ malignancy in american men with global statistics mirroring those found in the united states . Screening with prostate specific antigen (psa) has resulted in a significant stage migration such that the majority of new cases of pca are now detected while the disease is still clinically localized . These patients can choose from several treatment options and must weigh the potential morbidity of each treatment modality on their quality of life . In the vast majority of cases, urologists are the primary physicians that diagnose these patients with pca and are typically involved in the initial work up, discussion of all treatment options, and counseling of patients . Urologists are not only intimately involved with the treatment of the primary disease but also the consequential treatment - related complications while overseeing the long - term followup of these patients . Several centers have been recently established where urologists partner with radiation oncologists acquire ownership interest in intensity - modulated radiation therapy (imrt) equipment and provide integrated prostate cancer care . Although not yet validated in the literature, this may allow for improved quality of care and decreased cost . Unfortunately, these efforts have been much maligned in both the media and radiation oncology literature as conduits to increased revenue for the urologists with only debatable patient benefit [3, 4]. These reports have not yet been supported by data . After acquiring financial interest in an integrated prostate cancer center, we sought to evaluate whether our investment in imrt resulted in an increased utilization of radiation therapy in our patients with newly diagnosed prostate cancer . In september of 2008, we acquired financial interest in an integrated pca center offering imrt . Following institutional board approval, we identified all patients who were diagnosed with pca in the 12 months before and after the center became operational . Newly diagnosed cases of pca were identified by searching our electronic medical record using both prostate biopsy cpt codes (transrectal ultrasound - guided [trus] needle biopsy of the prostate, 55700/76942/76872) and the icd-9 prostate cancer diagnostic code (prostate cancer, 185.0). All men were diagnosed with pca after pathologic review of biopsy needle cores obtained after trus . Indications for biopsy included elevated psa, abnormal digital rectal exam, abnormal pca 3 test, and/or strong family history of pca . Prostate biopsies were performed utilizing a routine sextant pattern with at least 12 cores obtained . In cases where clinically indicated, the medical records of these patients were retrospectively reviewed and the data pertaining to the patients' demographics, cancer parameters, and initial pca treatment modality were extracted . Patients were assigned to two discrete groups based on the date of their pca diagnosis as it related to the date of the first availability of imrt at our integrated prostate center . All consecutive patients diagnosed with pca on a biopsy within 12 months prior to availability of imrt constituted the pre - investment group and all consecutive patients diagnosed with prostate cancer on a biopsy in the 12 months following initiation of imrt services constituted the post - investment group . The primary treatments received were designated as active surveillance (as), brachytherapy (bt), radiation therapy (xrt), radical prostatectomy (rp), and androgen deprivation therapy (adt). Treatment data were available for all patients and were stratified by the patient's age and gleason score . The age of 70 years old served as a cut - off point as in our clinical practice most of these patients are deemed suboptimal surgical candidates because of increased risk of postoperative urinary incontinence and erectile dysfunction . Our integrated pca center was established in collaboration with an academic radiation oncology department from a national cancer institute (nci) designated cancer center with a nationwide reputation for clinical and academic excellence . The radiation oncologists that treat our patients are not employed by the cancer center, but serve as full time academic faculty and have no financial interest in imrt . Radiation oncology residents have the opportunity to participate in all aspects of planning and delivery of radiation therapy . The center also employs a nurse - practitioner charged exclusively with the coordination and support of on - going clinical research programs . Patients who were referred to us for second opinion with biopsy - proven pca as well as men referred to us specifically for robotic - assisted laparoscopic radical prostatectomy were excluded to eliminate the potential biases resulting from the treatment recommendations rendered by an outside urologist . The overwhelming majority of these patients choose surgical therapy and their inclusion would skew the results toward lower utilization rates of xrt . Unpaired t - tests, chi - squared tests, and fisher's exact tests were implemented as appropriate . A total of 344 patients were diagnosed with pca on trus biopsy over the designated 24-month time period . Of the total patient population, 198 men were diagnosed with pca in the 12 months preceding availability of imrt, while 146 men constituted the post - investment group . Patient and cancer characteristics were similar between the two groups (table 1 and figure 1). The percentage of patients with gleason 7 pca was higher in the post - investment group but did not reach statistical significance, p = 0.073 (figure 1). Overall, the use of radiation therapy for those patients with newly diagnosed pca following investment in imrt (20.55% versus 20.71%, p = 1.00) was similar between the two patient populations (table 2 and figure 2). The number of patients treated with rp (67.81% versus 71.72%, p = 0.729), as (9.59% versus 4.55%, p = 0.177), adt (1.37% versus 2.02%, p = 0.999), and bt (0.68% versus 1.01%, p = 0.999) were not significantly different between post- and pre - investment groups . While overall treatment trends afford succinct analysis, clinical decisions regarding treatment of pca are often driven by a multitude of patient - specific factors . As such, the data was analyzed stratifying both gleason score and age (70 years old serving as a cutoff point). Treatments stratified by patient age are shown in table 3 and figure 3 . Despite the increased incidence of gleason score 7 disease in the post - investment group, there was no significant difference between the groups in all treatment patterns in men less than 70 years of age (table 3). An increase was found in the use of xrt in men older than 70 years of age in the pre - investment group (45.45%) as compared to men following acquisition of imrt (55.32%), but this did not reach statistical significance (p = 0.355). Analyzed by age and gleason score simultaneously, there was no difference between the treatment groups in patients younger than 70 regardless of the gleason score, table 4(a) and figure 4(a). For men 70 years or older with gleason 6 disease, there was a trend toward increased use of as (34.78% versus 15.79%) and decreased use of rp (21.74% versus 31.58%), but did not reach statistical significance (table 4(b)). There was no difference (43.48% versus 42.11%) in the use of xrt for men over 70 with gleason 6 disease . For patients with gleason 7 disease, there was a statistically significant increase in the utilization of xrt (pre - investment 41.38% versus post - investment 68.42%, p = 0.035) and decrease in the use of rp in the post - investment group (15.79% versus 55.17%, p = 0.006) seen in table 4(b) and figure 4(b). There was no difference between treatment groups in men over 70 with gleason 8 disease . External beam radiation therapy is widely used and is an effective treatment option for localized pca . There is now level i evidence that high - dose radiation therapy decreases the risk of biochemical failure in men with clinically localized prostate cancer as compared to conventional dose conformal radiation [57]. This improvement, however, comes at a cost of increased gastrointestinal (gi) and genitourinary (gu) toxicity . Observed that 2% of men receiving high - dose radiation experienced acute urinary or rectal morbidity of radiation therapy oncology group (rtog) grade 3 or greater . Intensity modulated radiation therapy allows for delivery of radiation with greater conformality to the target volume compared with traditional 3 d technique . Several randomized trials have shown that imrt reduced gi and gu toxicity compared with 3 d conformal radiation [810]. Were able to deliver 81 gy with less than 2% of grade 2 rectal morbidity and no grade 4 or greater rectal complications in patients with clinically localized pca . Furthermore, imrt has been shown to reduce the acute and late gi toxicity of patients treated with high - dose radiation therapy and adjuvant androgen deprivation as compared to 3 d conformal radiotherapy . This reduction in gu and gi morbidity has made imrt extremely popular in the delivery of high - dose external beam radiation for patients with clinically localized pca in the united states . Since 2004 several large urology groups in partnership with radiation and medical oncologists have established centers of integrated prostate cancer care . The integrated care model is patient - centered and disease specific, where the equipment and the staff are dedicated to the treatment of pca and no other disease entity . Although yet to be validated, this model may potentially result in better recognition and management of treatment - related complications, improved access to care, and increased experience with each treatment modality and thus better clinical outcomes . Recently, these centers have become the targets of intense criticism [3, 4]. The detractor's claim that integrated pca care centers lead to self - referral by financially motivated urologists and radiation oncologists and result in over - utilization of imrt contributing to the increased cost of health care . They further claim that these centers have a negative impact on residency training in radiation oncology by shifting patients away from the academic radiation oncology training programs . Unfortunately, these claims are not substantiated by data, but rather rely on indirect analysis of medicare claims and a 12% negative impact report from a single 3-point questionnaire survey of 81 radiation oncology training programs [3, 4]. To our knowledge, this is a first study conducted to directly evaluate whether financial interest in imrt as part of the integrated prostate cancer care model changed treatment recommendations for newly diagnosed patients with prostate cancer . We compared the distribution of treatments choices of all consecutive patients diagnosed with prostate cancer on a biopsy in our practice during a 12-month period prior to acquiring financial interest in imrt to a 12-month period following that acquisition . Our analysis revealed that overall there was a small, but statistically insignificant decrease in the use of radiation therapy and radical prostatectomy and a small increase in the use of active surveillance following investment in imrt . The increased use of active surveillance is likely due to the emergence of data from several large trials supporting the safety and efficacy of this approach in appropriately selected patients [1113]. Once the data were stratified by gleason score and patient age, a statistically significant increase in the use of radiation was found in men over 70 with gleason 7 disease (41.3% versus 68.3%). However, because of the overall low number of patients in this subgroup, this increase was due to a single patient difference between the groups (12 versus 13). These findings are not surprising as we believe that several important attributes of our integrated prostate cancer program provide for many patient benefits without the recently theorized, yet unsubstantiated risks of overutilization of imrt . As previously described, our center was established in collaboration with an academic radiation oncology department from an nci designated cancer center with a nationwide reputation for clinical and academic excellence . The radiation oncologists that treat our patients are not employed by the integrated prostate cancer center, but rather serve as full - time academic faculty and have no financial interest in imrt . Furthermore, the final determination on whether a patient is an appropriate candidate for primary or adjuvant radiation therapy is made entirely by the treating radiation oncologist . Radiation oncology residents have the opportunity to participate in all aspects of planning and delivery of imrt, thus deriving an educational benefit from this partnership . The center employs a nurse - practitioner charged exclusively with coordination and support of the clinical research program . Additionally, we are privileged to have cme accreditation by our state medical society and conduct regularly scheduled multidisciplinary morbidity and mortality conferences and discussions of challenging cases . Finally, we maintain a very high volume surgical program that ranks second in the number of radical prostatectomies performed annually in the greater philadelphia, pa, usa . First, this study is underpowered due to a fairly low number of patients and therefore the results of our statistical analysis must withstand the test of a larger trial for our conclusions to be validated . Second, even though we attempted to minimize limitations of the retrospective study design by including all consecutive patients diagnosed with prostate cancer within the 24-month period, the selection bias inherent in retrospective study design was not completely eliminated . Third, we did not include patients undergoing adjuvant or salvage radiation therapy in the trial and therefore did not ascertain the effect of financial interest in imrt on utilization of radiation in these patients . Finally, our findings may not be applicable to other integrated prostate cancer centers because of the unique structure of our specific program . Financial interest in imrt does not result in an increased utilization of radiation therapy in the treatment of newly diagnosed patients with clinically localized prostate cancer in our integrated prostate cancer center.
Neurofibromatosis type-1 (nf-1) is a multisystem, autosomal dominant disorder of peripheral nerves affecting nearly 1/3000 individuals worldwide.1 it was first described by a german pathologist, friedrich daniel von recklinghausen . Inherited or spontaneous mutation of the neurofibromin gene located on chromosome 17 is responsible for this diverse disorder . Common skeletal manifestations of nf-1 include spinal deformities, congenital tibial dysplasia (congenital bowing and pseudarthrosis), sphenoidal dysplasia and cystic lesions in bones . Pathological fracture of the acetabulum with anterior dislocation of hip secondary to osseous involvement of the acetabulum, femoral head, and pubic rami has never been documented in a case of nf-1 . A 16-year - old boy presented with the complaints of pain in the left hip associated with the inability to bear weight following a trivial fall . On examination, the affected limb was 1.5 cm short, abducted and externally rotated . On general examination, patient had 8 caf au lait spots over the body, bilateral axillary freckles and multiple palpable neurofibromas in the subcutaneous tissues of forearm, thighs and back [figure 1]. Patient met three out of seven criteria described for the diagnosis of nf-1 [table 1].2 plain radiograph and computed tomography scan of pelvis revealed an ill - defined lytic lesion causing pathological fracture - dislocation of the left hip [figure 2]. Magnetic resonance imaging (mri) showed additional soft tissue involvement and joint effusion [figure 3]. Ultrasound guided fine - needle aspiration cytology showed scanty cellularity with round to oval cells having minimal pleomorphism; hyperchromatic nucleus and moderate cytoplasm with spindle cells and osteoblasts . Clinical photograph showing skin lesions - caf au lait spots (black arrows) and axillary freckling (white arrow) criteria for diagnosis of nf-1 (at least 2 or more features) preoperative radiograph anteroposterior view (a) and computed tomography scan (b) of pelvis showing an ill - defined lytic lesion destroying anterior column of acetabulum, pubic rami and part of the femoral head coronal (a) and axial (b) sections of magnetic resonance imaging of pelvis showing expansile lytic lesion of acetabulum and pubic rami with soft tissue involvement and joint effusion a wide local excision followed by arthrodesis of the joint was planned . Considering the extent of bony and soft tissue involvement, we used a modification of the ilioinguinal and iliofemoral approach to have a wide exposure . We used the conventional ilioinguinal incision and combined it with femoral part of the iliofemoral incision [figure 4a]. On exposing the pelvis, anatomy was distorted . The deformed femur head and acetabulum with deficient pubic rami there was extensive soft tissue involvement adjacent to the acetabulum and lower part of the ilium . The entire acetabulum with 2 - 3 cm clear margin of the ilium was resected along with the abnormal soft tissue . Iliofemoral arthrodesis was done using a 14 hole stainless steel dynamic compression plate [figures 4c d, and 5]. Intraoperative photographs showing (a) skin incision (b) deformed femoral head (c) 14 hole dcp (d) iliofemoral arthrodesis postoperative radiograph after tumor resection and iliofemoral arthrodesis histopathology revealed dense collagenous tissue cores with spindle cells having blunt nuclei with minimal atypia and no mitosis or necrosis [figure 6a]. On immunohistochemistry, cells were s-100 positive and of neural origin [figure 6b]. Partial weight bearing was allowed at 6 weeks and full weight bearing at 10 weeks . At 1-year followup, the patient was comfortable, pain free, able to ambulate unassisted, stand on one limb, sit and climb stairs without any difficulty [figure 7]. (a) histopathological photomicrograph showing dense collagenous tissue cores and spindle cells with blunt nuclei . (b) immunohistochemistry showing neural marker s-100 positivity clinical photographs showing functional outcome at 1-year followup type 1 neurofibromatosis or von recklinghausen disease, is a multisystem disorder that primarily affects the cell growth of neural tissue and characterized by involvement of skin, peripheral nerves, subcutaneous tissue, eyes, and skeletal system . Although involvement of the musculoskeletal system is common, there have been only a few cases of subluxation / dislocation of hip in patients with nf [table 3].34567891011121314 on reviewing the literature, the etiology of hip instability leading to pathological subluxation / dislocation in patients with nf-1 can be classified as local and remote . Most of the cases are secondary to local (intra and peri - articular) neurofibromas, which can result in mass effect, bony erosions (ilium, acetabulum, and femoral neck), acetabular dysplasia, narrowing of the femoral neck, coxa valga, increased femoral neck offset, capsuloligamentous laxity, and synovial membrane proliferation.3458101113 remote causes of hip instability include intra spinal neurofibromas / schwannomas leading to motor deficit (hip abductor weakness) or sensory deficit (charcot's neuropathic arthropathy), limb length discrepancies secondary to hemi - hypertrophy of lower limb and abnormal biomechanical alteration in the spinopelvic alignment due to scoliosis.6791011 endo et al . Described anterior subluxation of hip secondary to decreased femoral head coverage resulting from decreased lumbosacral lordosis and posterior pelvic inclination following scoliosis correction.11 until date, there has been no case of nf-1 reported in the literature with pathological fracture of the acetabulum with anterior dislocation of hip attributable to a neurofibroma involving the acetabulum, pubic rami and femoral head . Orthopedic manifestations of nf-1 comprehensive literature review on published cases of hip dislocation / subluxation in nf-1 the various treatment options described for pathological hip dislocations in nf-1 include closed reduction, open reduction, shelf operation with fascia lata tenorraphy, rotational acetabular osteotomy with femoral varus osteotomy, girdle stone resection, total hip replacement with the trochanteric distalisation.791114 the rate of re dislocation is very high in most of the cases, subsequently requiring a secondary surgical procedure for stabilization . Since only a handful of cases have been described in the literature, it is difficult to comment upon the best line of management . In our case, arthrodesis was the best possible option since the bone stock after tumor resection was so inadequate that none of the above mentioned procedures could be tried . Moreover, the fear of redislocation, which might necessitate repeated surgeries, was negated . A combination of ilioinguinal and iliofemoral approach was employed to have a better exposure of the hip joint . The neurofibromatous tumors associated with nf-1 are usually benign; however, there is a 2 - 5% chance of malignant transformation, especially with plexiform neurofibromas.15 plexiform neurofibromas are diffuse, poorly defined nerve sheath tumors arising from multiple nerve fascicles and surrounding tissues . They are more prone for hemorrhage, dysfunction, pain, disfigurement, and malignant transformation.16 there was no clinical or radiological evidence of recurrence or malignant transformation in our patient at 1-year followup . Subtle clinical signs such as skin patches, axillary freckling, and subcutaneous neurofibromas can be easily missed . Orthopedic surgeons must be aware about the various management options available and tailor them as per the needs of their patient . Iliofemoral arthrodesis offered a good functional outcome with improved quality of life in our case.
Ramus can be used to differentiate between male and female strongly expresses univariate sexual dimorphism . When skeletal sex determination is considered, metric analyses on the radiographs are often found to be of superior value owing to their objectivity, accuracy, and reproducibility . After both of these bony areas, mandible remains next in the human which will also help us in the identification of age, sex, and race . Humphrey et al . Emphasized that almost any site of mandibular bone deposition, or resorption, or remodeling for that matter, seems to have a potential for becoming sexually dimorphic . Hence, mandibular condyle and ramus, in particular, are generally the most sexually dimorphic as they are the sites associated with the greatest morphological changes in size and remodeling during growth . Among various radiographic technique the orthopantomography (opg) is still used as one of the measure in determination of sex wherein, the morphology of mandibular ramus is studied . Hence, this study was taken to understand the sexual dimorphism, using digital opg . This study aims to determine the usefulness of mandibular ramus as an aid in sex determination . This study aims to determine the usefulness of mandibular ramus as an aid in sex determination . A short retrospective study was conducted of sixty males and sixty females, using orthopantomographs which were taken using kodak 8000c digital panoramic and cephalometric system (73 kvp, 12 ma, 13.9 s). Patients with a history of extraction, fracture, and any other severe developmental disturbances leading to variation in the size of mandible were excluded from the study . All the mandibular ramus measurements will be carried out using carestream health, inc, 2010, kodak 8000c digital panoramic and cephalometric system (73 kvp, 12 ma, 13.9 s) with master view 3.0 software . The ethical clearance obtained from the institution review board . The following variables were measured using mouse - driven method by moving the mouse and drawing lines using chosen points on the digital panoramic radiograph [figures 1 and 2]. Mandibular ramus measurements with five variables mandibular ramus measurements of female patient on orthopantomograph the sex could be determined from calculations using the equations given below: (table 1) 10 dmale: 185.622 + 1.361 (max . Ramus breadth) + 1.087 (min.ramus breadth) + 2.253 (condylar height) 0.717 (projective height of ramus) + 0.081 (coronoid height) dfemale: 161.761 + 1.276 (max . Ramus breadth) + 0.948 (min.ramus breadth) + 2.220 (condylar height) 0.753 (projective height of ramus) + 0.063 (coronoid height) maximum ramus breadth: the distance between the most anterior point on the mandibular ramus and a line connecting the most posterior point on the condyle and the angle of jawminimum ramus breadth: smallest anterior posterior diameter of the ramuscondylar height / maximum ramus height: height of the ramus of the mandible from the most superior point on the mandibular condyle to the tubercle, or most protruding portion of the inferior border of the ramusprojective height of ramus: projective height of ramus between the highest point of the mandibular condyle and lower margin of the bonecoronoid height: projective distance between coronion and lower wall of the bone . Maximum ramus breadth: the distance between the most anterior point on the mandibular ramus and a line connecting the most posterior point on the condyle and the angle of jaw minimum ramus breadth: smallest anterior posterior diameter of the ramus condylar height / maximum ramus height: height of the ramus of the mandible from the most superior point on the mandibular condyle to the tubercle, or most protruding portion of the inferior border of the ramus projective height of ramus: projective height of ramus between the highest point of the mandibular condyle and lower margin of the bone coronoid height: projective distance between coronion and lower wall of the bone . The data were analyzed by the discriminant function analysis using fischer exact test [table 1]. Mean measurements in males and females descriptive statistics shows mean values were higher in males compared to females . F - statistic values indicated that highest sexual dimorphism was seen with projective height of ramus and least with minimum ramus breadth . Maximum ramus breadth, condylar height, and projective height of ramus were statistically significant with p <0.05 as shown in table 2 . Descriptive statistics sex was accurately determined in 32 cases out of sixty male mandibular measurements with prediction accuracy rate of 53%, and sex was accurately determined in 36 cases out of sixty female mandibular measurements with an accuracy rate of 60% [table 3]. The data were analyzed by the discriminant function analysis using fischer exact test [table 1]. Mean measurements in males and females descriptive statistics shows mean values were higher in males compared to females . F - statistic values indicated that highest sexual dimorphism was seen with projective height of ramus and least with minimum ramus breadth . Maximum ramus breadth, condylar height, and projective height of ramus were statistically significant with p <0.05 as shown in table 2 . Descriptive statistics sex was accurately determined in 32 cases out of sixty male mandibular measurements with prediction accuracy rate of 53%, and sex was accurately determined in 36 cases out of sixty female mandibular measurements with an accuracy rate of 60% [table 3]. Determination of sex by morphological assessment has remained as one of the oldest approaches in forensic anthropology and medico - legal examinations . The method may vary and depend on the available bones and their conditions . The identification of sex is of significance in cases of mass fatality incidents where bodies are damaged beyond recognition . When entire adult skeleton is available for analysis, sex can be determined up to 100% accuracy (pelvis). However, in cases of mass disasters where usually fragmented bones are found, sex determination with 100% accuracy is not possible, and it depends largely on the available parts of skeleton . However, in cases where intact skull is not found, mandible may play a vital role in sex determination as it is the most dimorphic bone of skull . Anthropometry of the face and intraoral regions can help in the field of forensic odontology when common forensic data are unavailable . The disadvantages opg technique is unequal magnification and geometric distortion, which causes many problems . These distortions are the result of the horizontal movement of the film and x - ray source . The position of the movement object is within the focal trough, the size, and shape of the resultant images keeps changing . Panoramic radiographic technique remains as quite sensitive to positioning errors because of relatively narrow image layer . Objects which are outside the focal trough are blurred, magnified or reduced in sizes which are sometimes distorted . A study conducted by kambylafkas et al . Concluded that the evaluation of total ramal height is reliable, and an asymmetry of more than 6% is an indication of atrue asymmetry using panoramic radiograph . In this study, asymmetry noted was 7% using panoramic radiograph . Found mandibular ramus height to be the best parameter in their study, with 75.8% accuracy . In our study, mandibular ramus height found to be the best parameter with statistically significant with p = 0.005 . A study conducted by saini, et al . Showed that coronoid height possessed the best potential for sex determination on indian people with the accuracy of 74.1%, and the combination of it with minimum ramus breadth, maximum ramus breadth, and/or mandibular ramus length will show significant sexual dimorphism with an overall accuracy of 80.2% . In this study, all five variables that is maximum ramus breadth, minimum ramus breadth, condylar height, projective height of ramus, coronoid height showed prediction accuracy of 53% for males and prediction accuracy of 60% for females . Another study conducted by indira et al . On mandibular ramus measurements were subjected to discriminant function analysis . Each of the five variables measured on mandibular ramus using orthopantomograph showed statistically significant sex differences between sexes, indicating that ramus expresses strong sexual dimorphism . The mandibular ramus demonstrated greatest univariate sexual dimorphism in terms of minimum ramus breadth, condylar height, followed by projective height of ramus . In our study, highest sexual dimorphism was seen with projective height of ramus and least with minimum ramus breadth . Maximum ramus breadth, condylar height, projective height of ramus was statistically significant with the p <0.05 . Shivaprakash and vijaykumar conducted a study in diagnosing in the sex by observing the mandibular ramus posterior flexure . Sex was accurately determined in 44 cases out of 55 male mandibles with an accuracy rate of 80%, and sex was accurately determined in 35 cases out of 49 female mandibles with accuracy rate of 71% . In our study, sex was accurately determined in 32 cases out of sixty male mandibular measurements with prediction accuracy rate of 53% and sex was accurately determined in 36 cases out of sixty female mandibular measurements with accuracy rate of 60% . The mandibular ramus height can be considered another valuable tool in gender determination with the help of opg . However, further studies with larger sample and less magnification errors are needed to be taken up in future.
They form an integral part of indian diet, especially for those who have type 2 diabetes for whom white rice is considered less desirable because of its high gi . Chapattis and other flatbreads are popular in europe also where they form a part of daily diet among members of ethnic minority groups who follow traditional dietary patterns . Chapattis are made from whole - wheat flour and cooked on hot flat open griddles . They can also be prepared by substituting wheat flour with other cereal or legume flours at different levels . Incorporation of cereal brans at proportions up to 10% has resulted in good quality chapattis . The color and appearance of chapattis were found to be good with substitution of wheat flour with up to 10% cereal brans . Generally, chapatti is prepared from whole - wheat flour obtained by grinding wheat in a disk mill (locally known as chakki). Chapatti quality can be assessed from its softness and flexibility which may be affected by flour protein quantity and quality . The chapatti quality is also influenced by the dough consistency, which in turn depends mainly on the quantity of water added . Bran is the hard outer layer of cereal grains, rich in a myriad of healthy phytochemicals, namely, phenolics, flavonoids, glucans, and pigments . Unfortunately, these nutrition - rich components are often discarded during milling out of ignorance, organoleptic reasons, and rancidity problems . Knowing the phytochemical constituents and pharmacological profile of bran is expected to give insight to their potential application in promotion of health . Cereal brans, the by - products obtained in large amounts in grain milling industry, considered as inedible material for humans, is mostly used as animal feed . However, brans are concentrated source of dietary fibre and other nutrients (proteins, b - vitamins, and minerals). Brans are generally composed mainly of insoluble cellulose and hemicellulose, with only about 5 percent soluble fibre, and have little hypercholesterolemia effect . Bran contributes a pleasing, sweet, nutty flavour when added as a flavour enhancer in a variety of food products . Commercial wheat flour and oat bran (baggry's india ltd ., new delhi, india) were purchased from local market . Chappati was prepared by the addition of cereal brans (wheat, oat, and rice) singly and in combination (w: r: o:: 2: 1.5: 1.5) to wheat flour at 5 and 10% bran supplementation . Chapatti . Cereal brans singly and in combination at 5 and 10% level were added to wheat flour and required quantity of water which were mixed manually to obtain dough of suitable consistency . The dough was divided into four equal parts and moulded into circular chapattis of 15.0 cm in diameter with rolling pin and board . Traditional home baking procedure was followed to bake chapattis on iron plate (tawa). Chapattis were cooled and comparative evaluation was done using the following criteria which also included observations on dough handling properties . Characteristic score grade dough handling nonsticky sticky slightly sticky very sticky puffing of chapatti full partial nil . The instrument was calibrated with the user supplied black plate calibration standard that was used for zero setting . The instruments were placed on the plate and three exposures at different places were conducted . Readings were displayed as a, b, and l color parameters according to the cielab system of color measurement . The a value ranges from 100 (redness) to + 100 (greenness) and the b value ranges from 100 (blueness) to + 100 (yellowness), while the l value, indicating the measure of lightness, ranges from 0 (black) to 100 (white). Was evaluated by using texture analyser (stable micro systems, model ta - hdi, uk). One strip at a time was placed on the centre of the sample holder and the blade was allowed to cut the chapatti strip . The force (n) required to cut chapatti strip into two pieces was recorded . Bran enriched products such as extruded snacks, breakfast cereal - porridge, and chapatti were evaluated for sensory attributes (appearance, colour, texture, flavor, and overall acceptability) through a panel of semi - trained judges using 9-point hedonic scale . Water activity of bran enriched products was estimated using water activity meter having hygrolab 3 bench - top indicator (rotrogenic company). Standard aoac procedure was followed for free fatty acids determination in cereal bran enriched products . Product sample (5 g) was taken in flask and 50 ml benzene was added and kept for 30 min for extraction of free fatty acids . After extraction, 5 ml extract, 5 ml benzene, 10 ml alcohol, and phenolphthalein as indicator were taken in flask and titrated against 0.02 n koh till light pink colour disappeared: (1)%ffa (% oleic acid) = 2820.02 n kohml of alkali useddilution factor1000wt of sample taken 100, moisture content by method of aacc 2000, total plate count by method of maturin and peeler . Moisture content by method of aacc 2000, total plate count by method of maturin and peeler . Data collected from the aforesaid experiments was subjected to statistical analysis for standard error and duncan's multiple range test using minitab software . The quality evaluation of chapattis prepared by different bran enriched levels is mentioned in table 1 . The pooled scores obtained by the various bran enriched levels of chapattis for appearance, color, texture, and flavor were 7.92, 7.18, 7.68, and 8.10 for wheat bran, rice bran, oat bran, and bran in combination enriched chapattis, respectively . The overall acceptability at 5 and 10 percent level of supplementation was 7.65 and 7.80, respectively . Reported that chapattis, prepared by the addition of 10% bran, showed better performance and were quite comparable with whole - wheat flour regarding the proximate components and sensory attributes . Dough handling characteristics of bran enriched chapattis do not show much variation with respect to type of bran used . Except for rice bran incorporated dough for chapatti (slightly sticky), all others showed nonsticky behavior during dough development . All types of bran enriched chapattis showed full puffing except 10% rice bran enriched chapattis in which partial puffing during chapatti preparation was visualised . The data presented in table 2 depicted color and texture analysis of bran enriched chapatti . Statistically significant (p 0.05) difference was observed in l value of bran enriched chapatti . L value of various cereal bran enriched chapattis was 64.37, 59.12, 60.04, and 61.92 for wheat, rice, oat, and bran in combination, respectively . L value showed decreasing trend with increase in level of supplementation of cereal brans in chapattis . The l value of 66.83, 61.59, and 61.14 was observed at 0, 5, and 10% level of supplementation, which means slightly lower brightness at higher levels of supplementation . A value of wheat, rice, oat, and bran in combination was 4.18, 5.24, 4.71, and 4.28, respectively . With increase in level of supplementation, a value (redness) increased from 3.14 at 0% level of supplementation to 5.11 at 10% level . Altan et al . Stated that, among the color parameters, the l and a values showed marked changes due to addition of tomato pomace . An increase in tomato pomace level decreased the l value of the sample and increased the a value of samples . Also, increasing bran level supplementation resulted in a decrease in the b value of chapattis . A negative correlation was found between a value and b value of the enriched chapattis . Cutting force (n) reflects the texture of the chapattis and it stimulates the biting action of the human teeth on chapattis . Cutting force (n) of various bran enriched chapattis varied as 6.56 n, 5.92 n, 5.30 n, and 5.99 cutting force increased due to presence of more fibres at higher enrichment levels . At 0, 5, and 10 percent level of supplementation, the corresponding cutting force (n) was 5.25, 5.91, and 5.97 n, respectively . Manu and prasada rao reported that cutting force of chapattis prepared from different wheat varieties ranged from 4.22 to 6.96 n. hemalatha et al . Also reported that the cutting force (n) for chapattis made from different wheat varieties ranged between 4.22 and 6.67 . The variation in cutting force might be because of variation in protein and fibre content of brans which determine the resistance offered by the samples . Increase in fibre content might have increased the water holding capacity of chapattis and hence increased moisture content (%) with addition of bran . Reported that moisture content (%) of bran enriched chapattis was 31.0 percent while control had moisture content of 30.2 percent . Maximum water activity was observed in rice bran enriched chapattis (0.462) which was statistically at par with water activity of bran in combination enriched chapattis (0.455). Water activity of oat and wheat bran enriched chapattis was 0.429 and 0.406, respectively . It was observed from data that water activity of samples was positively correlated with moisture content and followed the same pattern . Manthey et al . Reported that water activity of bran / fibre enriched pasta increased with bran supplementation over control . The increase in water activity is correlated with increase in moisture content at higher levels of bran supplementation . The data pertaining to free fatty acids (%) is presented in table 3 . The free fatty acids (%) of bran enriched chapattis ranged from 0.057 to 0.085 . The highest free fatty acids were recorded in rice bran enriched chapattis (0.085%), being the lowest in wheat bran enriched chapattis (0.057). It is also evident from the table that, with increase in level of bran supplementation, free fatty acids increased significantly . The free fatty acids (%) at 5 and 10 percent level of supplementation were 0.067 and 0.079 percent, respectively . Khan et al . Reported similar results regarding free fatty acids while studying development and evaluation of long shelf life ambient stable chapattis . A significant variation (p 0.05) was observed in total plate count of bran extruded chapattis . Total plate count of enriched chapattis varied from 6.5 to 17 10 cfu / g . It is also evident from the table that with increase in bran supplementation level, a slight increase in total plate content was observed . The mean value of total plate content for 5 and 10% level of supplementation was 10.25 10 and 12.5 10 cfu / g, respectively . Frazier and westhoff reported that total plate content increased from 2.3 10 to 3.4 10 cfu / g for flour . Quality characteristics for chapatti revealed that dough handling and puffing of bran enriched chapattis prepared by 5 and 10% level of bran supplementation did not vary significantly . All types of bran enriched chapattis except rice bran enriched chapattis showed nonsticky behavior during dough handling . Rice bran enriched chapatti recorded maximum moisture (%), water activity, and free fatty acids (%). With increase in level of supplementation, moisture, water activity, and free fatty acids increased . The future emphasis can be given on development of functional flatbreads which has got increased demand due to increase in health conscious consumer base.
Glaucoma is a progressive optic neuropathy leading to permanent visual loss that is often associated with elevated intraocular pressure (iop). Primary open - angle glaucoma (poag) is the most common type of glaucoma . Normal tension glaucoma (ntg) is an important subset of poag; while many poag patients have high iop,1 patients with ntg have statistically normal iop.24 the prevalence of ntg is higher among the japanese population than among caucasians, and recent studies reported that 92% of poag patients in japan had ntg.58 the diagnosis of glaucoma is based on a combination of factors including optic nerve damage and specific field defects for which iop is the only treatable risk factor . Ntg, however, tends to be underdiagnosed because of an accompanying high myopia in many cases and the lack of elevated iop . Although relatively higher iop, myopia, and older age are known to be factors associated with the development of ntg,9 they are not pathognomonic and there remains a growing interest in the identification of pathogenetic factors associated with ntg . Glaucoma is genetically heterogeneous and the detection of susceptibility genes could provide useful information for early diagnosis of glaucoma . To date, over 30 genetic loci for glaucoma have been identified by linkage analysis in multiple pedigrees; 1012 14 loci of poag, 3 loci of primary congenital glaucoma (pcg), and 1 locus of pigment dispersion syndrome have been designated glc1a - glc1n, glc3a - glc3c, and gpds1 (with approval from the hugo genome nomenclature committee), respectively . Among them, glc3a harbors cytochrome p450, family 1, subfamily b, polypeptide 1 (cyp1b1).13 cyp1b1 sapans 8.5 kb on chromosome 2p21 with three exons, and mutation of this gene is a major cause of pcg.10,13 most cyp1b1 mutations are genetic insertions, deletions, or missense mutations, indicating that cyp1b1 is relatively susceptible to recombination events.10 cyp1b1 has also been found to be involved in the development of juvenile - onset glaucoma (juvenile open angle glaucoma [joag]).1416 in addition, recent studies have shown that cyp1b1 mutations are related to poag in several ethnic groups,10 and there have been reports of delayed expression of a cyp1b1 mutation and coexistence of pcg and poag in the same pedigree.17,18 these recent studies suggest that cyp1b1 mutations in the glc3a locus may contribute to a broader range of glaucoma phenotypes than the pcg phenotype alone, including ntg . In this study, with prospective snp analysis of the cyp1b1 gene in ntg patients in mind, we performed comprehensive microsatellite mapping in and around the glc3a locus and investigated the disease - susceptibility of this locus in ntg patients . We recruited 243 japanese subjects from the yokohama city university, yamanashi university, gifu university, kobe university, yamaguchi university, kumamoto university, hokkaido university, tokyo university, niigata university, kanazawa university, hiroshima university, tajimi municipal hospital, and tokai university in japan . Of these subjects, 142 were diagnosed with ntg, and 101 were control subjects . The control subjects were of the same age and sex as the ntg patients, and they were not affected by glaucoma or any ophthalmological or systemic diseases that could result in optic nerve or visual field changes . Furthermore, the control cases either had no myopia or had mild myopia with refractive errors of 3.00 d or less . All patients and control subjects were of japanese ethnicity, with similar social backgrounds and residing in the same urban area . Informed consent was obtained from all patients, and the study was conducted in accordance with the declaration of helsinki and subsequent revisions thereof . Ntg patients enrolled in this study were diagnosed as such if the patient had the following conditions: glaucomatous changes in the optic nerve head with or without retinal nerve fiber layer defect and corresponding glaucomatous visual field defects; normal open angle with angle width greater than shaffer grade 2; absence of intraocular pressure greater than 21 mmhg on repeated examination by goldmann applanation tonometry without medication; neurologic, rhinologic, and general medical examinations including magnetic resonance imaging that failed to disclose any pathology responsible for optic nerve change . Glaucomatous optic nerve abnormality was diagnosed when the vertical cup / disc ratio of the optic nerve head was more than or equal to 0.7, or the rim width at the superior portion (111 hour) or inferior portion (57 hour) was less than or equal to 0.1 of disc diameter, or the difference of the vertical cup / disc ratio was greater than or equal to 0.2 between both eyes, or a nerve fiber layer defect was found . Glaucomatous visual field defect was defined on a hemifield basis using a reliable field data examined by the humphrey static visual field analyser (carl zeiss meditec, oberkochen, germany,) c-30 - 2 program according to the anderson and patella s criteria; 19 the hemifield was judged abnormal when the pattern deviation probability plot showed a cluster of three or more nonedge - contiguous points having sensitivity with a probability of less than 5% in the upper or lower hemifield, and in one of these with a probability of less than 1% . The following inclusion and exclusion criteria we excluded individuals who were diagnosed under 20 or over 60 years of age and who had 8.0 d or higher myopic refractive error of spherical equivalence . The selection criteria of hfa mean deviation were stratified depending on the subjects age in order to minimize the effect of aging on retinal ganglion cell loss and subsequent visual field defect(s) (i) no limitation if the patient was diagnosed under 50 years of age (ii) 10.00 db or worse in at least one eye if the patient was diagnosed between 50 and 55 years of age (iii) 15.00 db or worse in at least one eye if the patient was diagnosed above 55 years of age . In this study, the cases exhibiting a comparatively early onset were selected as they suggest that genetic factors may show stronger involvement . During diagnosis, patients whose refraction values had changed due to cataract surgery, refractive surgery, or other intervention were excluded from the study . In cases where a glaucomatous visual field defect was present only in one eye, the refraction value and glaucomatous visual field defect of the affected eye were adopted . In cases where a glaucomatous visual field defect was present in both eyes, the refraction value and glaucomatous visual field defect of the more severely affected eye were adopted . Genomic dna was extracted using the qiaamp dna blood mini kit (qiagen, hilden, germany) or the guanidine method . In this association study, we selected 13 highly polymorphic microsatellite markers that are located in and around the glc3a locus as shown in figure 1 . The markers were determined based on the national center for biotechnology information for fine mapping . Polymerase chain reaction (pcr) was performed in a reaction mixture with a total volume of 12.5 l containing pcr buffer, genomic dna, 0.2 mm dinucleotide triphosphates (dntps), 0.5 m primers, and 0.35 u taq polymerase . The pcr conditions were as follows: 94 c for 5 min, followed by 30 cycles of denaturation at 94 c for 30 sec, annealing at 56 c for 30 sec, extension at 72 c for 1 min, and a final elongation step at 72 c for 10 min . The reaction was carried out in a pcr thermal cycler (geneamp system 9700, applied biosystems, foster city, ca, usa). The forward primer was labelled at the 5 end with 6-fam, vic, pet, or ned (sigma - aldrich, st . Louis, mo, usa) (table 1). To determine the number of microsatellite repeats, the pcr products were denatured at 97 c for 2 min, mixed with formamide, and electrophoresed using an abi3130 genetic analyser (applied biosystems). The number of microsatellite repeats was estimated automatically using the genescan 672 software (applied biosystems) by the local southern method with a size marker of gs500 tamra (applied biosystems). The significance of allelic frequencies between the patient and control groups was evaluated by fisher s exact test . The probability of association was corrected by the bonferroni inequality method, ie, by multiplying the obtained p values with the number of alleles compared . Statistical analyses were performed on a computer using the spss software (version 10.1; spss inc ., haplotype frequencies and linkage disequilibrium (ld) in the multi - locus analyses were calculated using pypop.20 haplotype frequencies were estimated using the iterative expectation - maximization algorithm . We recruited 243 japanese subjects from the yokohama city university, yamanashi university, gifu university, kobe university, yamaguchi university, kumamoto university, hokkaido university, tokyo university, niigata university, kanazawa university, hiroshima university, tajimi municipal hospital, and tokai university in japan . Of these subjects, 142 were diagnosed with ntg, and 101 were control subjects . The control subjects were of the same age and sex as the ntg patients, and they were not affected by glaucoma or any ophthalmological or systemic diseases that could result in optic nerve or visual field changes . Furthermore, the control cases either had no myopia or had mild myopia with refractive errors of 3.00 d or less . All patients and control subjects were of japanese ethnicity, with similar social backgrounds and residing in the same urban area . Informed consent was obtained from all patients, and the study was conducted in accordance with the declaration of helsinki and subsequent revisions thereof . Ntg patients enrolled in this study were diagnosed as such if the patient had the following conditions: glaucomatous changes in the optic nerve head with or without retinal nerve fiber layer defect and corresponding glaucomatous visual field defects; normal open angle with angle width greater than shaffer grade 2; absence of intraocular pressure greater than 21 mmhg on repeated examination by goldmann applanation tonometry without medication; neurologic, rhinologic, and general medical examinations including magnetic resonance imaging that failed to disclose any pathology responsible for optic nerve change . Glaucomatous optic nerve abnormality was diagnosed when the vertical cup / disc ratio of the optic nerve head was more than or equal to 0.7, or the rim width at the superior portion (111 hour) or inferior portion (57 hour) was less than or equal to 0.1 of disc diameter, or the difference of the vertical cup / disc ratio was greater than or equal to 0.2 between both eyes, or a nerve fiber layer defect was found . Glaucomatous visual field defect was defined on a hemifield basis using a reliable field data examined by the humphrey static visual field analyser (carl zeiss meditec, oberkochen, germany,) c-30 - 2 program according to the anderson and patella s criteria; 19 the hemifield was judged abnormal when the pattern deviation probability plot showed a cluster of three or more nonedge - contiguous points having sensitivity with a probability of less than 5% in the upper or lower hemifield, and in one of these with a probability of less than 1% . The following inclusion and exclusion criteria we excluded individuals who were diagnosed under 20 or over 60 years of age and who had 8.0 d or higher myopic refractive error of spherical equivalence . The selection criteria of hfa mean deviation were stratified depending on the subjects age in order to minimize the effect of aging on retinal ganglion cell loss and subsequent visual field defect(s) (i) no limitation if the patient was diagnosed under 50 years of age (ii) 10.00 db or worse in at least one eye if the patient was diagnosed between 50 and 55 years of age (iii) 15.00 db or worse in at least one eye if the patient was diagnosed above 55 years of age . In this study, the cases exhibiting a comparatively early onset were selected as they suggest that genetic factors may show stronger involvement . During diagnosis, patients whose refraction values had changed due to cataract surgery, refractive surgery, or other intervention were excluded from the study . In cases where a glaucomatous visual field defect was present only in one eye, the refraction value and glaucomatous visual field defect of the affected eye were adopted . In cases where a glaucomatous visual field defect was present in both eyes, the refraction value and glaucomatous visual field defect of the more severely affected eye were adopted . Genomic dna was extracted using the qiaamp dna blood mini kit (qiagen, hilden, germany) or the guanidine method . In this association study, we selected 13 highly polymorphic microsatellite markers that are located in and around the glc3a locus as shown in figure 1 . The markers were determined based on the national center for biotechnology information for fine mapping . Polymerase chain reaction (pcr) was performed in a reaction mixture with a total volume of 12.5 l containing pcr buffer, genomic dna, 0.2 mm dinucleotide triphosphates (dntps), 0.5 m primers, and 0.35 u taq polymerase . The pcr conditions were as follows: 94 c for 5 min, followed by 30 cycles of denaturation at 94 c for 30 sec, annealing at 56 c for 30 sec, extension at 72 c for 1 min, and a final elongation step at 72 c for 10 min . The reaction was carried out in a pcr thermal cycler (geneamp system 9700, applied biosystems, foster city, ca, usa). The forward primer was labelled at the 5 end with 6-fam, vic, pet, or ned (sigma - aldrich, st . Louis, mo, usa) (table 1). To determine the number of microsatellite repeats, the pcr products were denatured at 97 c for 2 min, mixed with formamide, and electrophoresed using an abi3130 genetic analyser (applied biosystems). The number of microsatellite repeats was estimated automatically using the genescan 672 software (applied biosystems) by the local southern method with a size marker of gs500 tamra (applied biosystems). The significance of allelic frequencies between the patient and control groups was evaluated by fisher s exact test . The probability of association was corrected by the bonferroni inequality method, ie, by multiplying the obtained p values with the number of alleles compared . Statistical analyses were performed on a computer using the spss software (version 10.1; spss inc ., haplotype frequencies and linkage disequilibrium (ld) in the multi - locus analyses were calculated using pypop.20 haplotype frequencies were estimated using the iterative expectation - maximization algorithm . Patient age range was 2158 years (mean 49.1 9.7); 47.2% were male and 52.8% were female . The mean refraction value was 3.74 3.02 diopters (d), and the mean deviation observed in the humphrey static visual field determination (carl zeiss meditec, oberkochen, germany) was 10.14 8.04 db . We genotyped 13 polymorphic microsatellite markers in and around the glc3a locus in 142 patients and 101 controls (figure 1). The observed and expected frequencies of each genotype for the 13 markers in the case and control subjects were in hardy weinberg equilibrium (data not shown). Only two adjacent markers, d2s0416i and d2s0425i, were significantly positive, as shown in table 2, and the frequency of the 444 allele of d2s0416i and the 258 allele of d2s0425i were decreased in cases compared to controls (p = 0.022, or = 0.59 and p = 0.034, or = 0.42, respectively). However, this statistical significance disappeared (pc> 0.05) when evaluated by bonferroni correction . The magnitude of ld between these two markers was low, with pair - wise d = 0.25, and the comparison of haplotype consisting of two alleles (d2s0416i_444 and d2s0425i_258) rendered no significant difference between cases and controls (cases vs controls = 3.5% vs 7.3%, p = 0.055) (data not shown). The purpose of this study was to investigate whether the glc3a locus is associated with ntg in japanese subjects, based on results from recent studies reporting that the cyp1b1 gene, located at the glc3a locus on chromosome 2p21, could be a causative gene in poag as well as pcg . To this end, we genotyped 13 microsatellite markers in and around the glc3a locus . Here we report a lack of association between the glc3a locus and ntg in japanese patients, suggesting that genetic variation at this locus may not play an important role in the development of ntg . Sarfarazi and colleagues mapped glc3a as a putative pcg locus to 2p21 in 11 turkish families, and the d2s177 microsatellite, located 270 kb telomeric to the cyp1b1 gene, showed a maximal lod score of 9.40.22 bejjani and colleagues also reported that the maximal lod score was 15.76 with d2s177 in 25 saudi arabian pcg families.23 recently, it has been reported that the d2s177 allele was associated with cyp1b1 mutations related to pcg.24 therefore, d2s177 may be potentially useful as a marker for genetic events associated with glaucoma, such as cyp1b1 mutations . In the present study, we did not find any significant association between d2s177 alleles and ntg, suggesting that cyp1b1 mutations may not be a risk factor for ntg . It has been hypothesized that mutations of cyp1b1 cause developmental abnormality in the structure and function of the anterior ocular segment.25 however, ntg patients have statistically normal iop with no abnormality in the structure of anterior ocular segment, suggesting that cyp1b1 is a potential disease susceptibility gene of pcg, poag, and joag, but not ntg . On the other hand, we found a weak association between two adjacent markers, d2s0416i and d2s0425i, and ntg, although this link did not reach statistical significance when corrected . The nearest gene of d2s0416i is the sos1 (son of sevenless homolog 1 [drosophila]) gene, which is about 40 kb from the marker . Sos1 encodes a protein that is a guanine nucleotide exchange factor for ras proteins,26 and mutations in this gene are associated with gingival fibromatosis and noonan syndrome.2729 another marker, d2s0425i, is within the cdkl4 (cyclin - dependent kinase - like 4) gene, a member of the ser / thr protein kinase family . Currently, there are no reports suggesting any connection between these two genes and ntg . Previous studies reported that mutations in genes such as optic atrophy 1 (opa1),3032 apolipoprotein e (apoe),33,34 and optineurin (optn)3537 are implicated in ntg . Recently, wd repeat - domain 36 (wdr36),38 endothelin receptor type a (ednra)39,40 methylenetetrahydrofolate reductase (mthfr),41 and -1-adrenergic receptor (adrb1)42 have also been reported as risk factors for ntg . However, associations between these genes and ntg were not strong or not often replicated in other populations,4347 suggesting that there may be other unknown genetic factors having more powerful effects on the development of ntg than those identified so far . In conclusion, we performed an association analysis of the glc3a locus including the cyp1b1 gene using microsatellite markers in ntg patients, but the marker involved in ntg was not detected in this locus . In ntg patients, diagnosis is usually made after a visual field defect has occurred, due to few subjective symptoms and normal iop . Therefore it is necessary to identify a disease susceptibility gene and elucidate pathogenic mechanisms of ntg for early diagnosis, prevention, and therapeutic development.
Recently, a single center study conducted by oiva and coworkers and published in critical care demonstrated that phospho - specific whole blood flow cytometry could be used to assess activated signaling path - ways in leukocytes isolated from pancreatitis patients . Pancreatitis is an inflammatory process in which pancreatic enzymes are activated leading to autodigestion of the pancreas and adjacent tissues . During pernicious cases, the initial inflammatory response is excessive and can result in tissue injury, organ failure, and death . To counter this inflammation, these steps can include leukocyte apoptosis, anti - inflammatory cytokine production, and anergy . When patients become leukopenic and immunosuppressed, they often become susceptible to infections with organisms to which a healthy patient would normally be resistant . Although therapy aimed at blunting the initial over - exuberant inflammation may be beneficial if applied early, it may also be detrimental by impairing the host's ability to fight infection that is a common cause of death in these patients . To complicate the potential use of immune - modulating therapy, inflammatory and anti - inflammatory processes can occur along different time - lines for the varied leukocyte subsets . For example, during immune response, t lymphocytes might be undergoing apoptosis while neutrophils are primed to produce reactive oxygen species . Thus, during prolonged pancreatitis, leukocyte subsets may be concurrently exhibiting both pro- and anti - inflammatory processes . As noted above, a potential promising therapy of pancreatitis is modulation of the immune response . This is a difficult undertaking as the host response will be affected by a number of variables, including genetic background, co - morbidities, age, gender, and so on . In addition, the patient's immune response will vary during the course of the disease . Thus, what is needed for immunotherapy to be practical is a rapid, robust measure of the host immune system . In the manuscript published by oiva and co - workers, the authors determined the signaling profiles of circulating leukocytes isolated from pancreatitis patients using phospho - specific whole blood flow cytometry . This is a powerful new technology that allows for simultaneous single - cell determination of leukocyte subsets using cell surface markers as well as intracellular protein phosphorylation . This work represents a continuation of work published previously . In this earlier work, oiva and co - workers demonstrated that stimulated monocytes isolated from patients with acute pancreatitis had decreased phosphorylated erk 1/2, nf-b, and stat1 and 3 . The authors concluded that these changes could lead to impaired monocyte recruitment as well as increased susceptibility to infections . Here, changes in activated t lymphocyte p38, nf-b, stat1 and stat6 activity was observed that could be interpreted as being pro- or anti - inflammatory . Interestingly, they determined that patients' lymphocytes exhibited decreased phosphorylated nf-b and stat1 and increased phosphorylation of p38 and stat6, suggesting a transition from a th1 to th2 phenotype . Additionally, this minimally invasive methodology could be used to generate immune status within 6 hours under ideal conditions . Thus, this methodology represents an essential step prior to targeting the underlying immune response to pancreatitis . Although beyond the scope of the report, a potentially necessary step after immune status determination, and prior to treatment with inflammatory altering treatments, would be to first treat the whole blood cells isolated from the patients with the potential therapeutic agent and evaluate the immune effector cell response . Phospho - specific flow cytometry could also be used to determine if the leukocytes responded in a way that would be beneficial . Thus, the minimally invasive, relatively quick methodology developed in this paper could be utilized to determine immune status as well as provide a method to test potential therapies . One unavoidable limitation to the report is that the signaling processes were determined using only peripheral leukocytes . A possibility exists that peripheral leukocytes may not respond similarly to ex vivo stimuli as leukocytes isolated from the inflammatory site(s). When feasible, future studies need to be undertaken to compare the response of peripheral leukocytes to these tissue leukocytes . Rapid results from whole blood phospho - specific flow cytometry during pancreatitis will allow for immune status determination, likely improve early diagnosis and provide a rational basis for immune targeting therapies . This work was supported in part by national institutes of health grants gm44118 and gm55194 (to rsh), gm72760 (to ccc), and the alan a and edith l wolff foundation.
These misconceptions have a significant influence on the day - to - day life including the search for treatment in times of illness . A number of studies have reported that misconceptions and inadequate knowledge present significant barriers to effective management of diabetes . It is imperative for physicians to understand myths and misconceptions in a particular community about a disease to improve patient care, especially when dealing with chronic diseases like diabetes . Diabetes mellitus (dm) is one of the most common noncommunicable diseases in the world and its prevalence is increasing dramatically . Currently, there are around 285 million diabetic patients around the world, and the numbers are predicted to rise to 439 million by 2030, with the largest increase in the developing rather than in the developed world . Its dramatic increase began a few decades ago with the rapid urbanization and development in the country . Studies in the 1980s showed a trend towards an increase among adult saudis especially females . A large study of saudi patients from 1995 to 2000 revealed prevalence of 23.7% . However, a study in 2011 showed a significant rise in prevalence reaching 34.1% in men and 27.6% in women . Correct knowledge about diabetes and its management has an enormous impact on attitude and practice of diabetic patients . A study of diabetic patients in new york, reported that patients with diabetes frequently had beliefs of the disease and medication that were false and even dangerous . It is imperative that awareness of diabetes and its correct management be created and various myths and misconceptions surrounding its course and management be removed . A few excellent studies about prevailing misconceptions about diabetes have already been carried out in the saudi population, more specifically in female diabetic teachers, in the eastern region, western region and qasim region . However, no studies have examined in detail the determinants of the misconceptions of diabetic patients . Therefore, this study was undertaken to identify the determinants of prevailing misconceptions on diabetes and the management of diabetic patients registered at a diabetes clinic of a tertiary care hospital in the eastern region of saudi arabia . This cross - sectional study was carried out at the diabetes clinic of a tertiary care hospital, in eastern saudi arabia . This clinic has a registered patient population of 2000 diabetic patients . At a confidence level of 95%, response distribution of 50% and accepted margin of error of 10%, we calculated a sample size of 200 . A table of random numbers was used to select patients from the medical record numbers of the registered patients of the diabetes clinic . The inclusion criteria were all saudi patients who had been registered at the diabetes clinic of the tertiary care hospital in saudi arabia for 6 months or more since diagnosis and on continuous treatment . No freshly diagnosed patient was included . In the same vein uncooperative or nonconsenting patients, as well as patients with any illness (physical or psychological) that was likely to influence reliable, valid responses to the interviewer's questions were excluded . The same interviewer spoke to all the selected patients on their scheduled follow - up visits after taking their informed consent . When a prospective participant refused to take part, the next patient on the list of random numbers was asked until the sample size was completed . All attendees were interviewed in standardized conditions with prior information to ensure valid reliable responses . The data collection instrument was an interviewer - filled questionnaire prepared in accordance with brief illness perception questionnaire and belief about medicines questionnaire . The demographic and classification data included age categories (<20, 21 - 40, 41 - 60 or> 60 years), gender (male and female), area of residence (urban or rural), education (primary, middle, high school, graduate and above), family history of diabetes (present or absent), type of diabetes (type 1 or type 2), time since diagnosis (<5, 6 - 10, 11 - 15 or> 15 years), type of treatment (oral hypoglycemic or insulin), self - monitoring (yes or no) and diet control as per doctor's advice (yes or no). In order to establish validity and reliability of the questionnaire, a pilot study was conducted in a sub - sample of attendees who were not included in the study proper . Necessary changes were made in the questionnaire as well as the interview style as necessary . Questions on the commonest reported misconceptions relating to etiology, types, pathogenesis, day - to - day life, diet and treatment of diabetes were included after a review of studies from saudi arabia, usa, india, nepal, and pakistan . An answer in yes to a misconception question was considered a misconception and a score of one was given . The total number of misconception questions was 36; therefore, the possible maximum score for any patient was 0 - 36 . Low (scores 0 - 12), moderate (scores 13 - 24), and high (scores of 24 - 36). Chi - square test was used to determine the association of the socio - demographic variables with the three categories of misconception scores . Stepwise logistic regression analysis was applied to the socio - demographic variables showing significant association with the misconception score by chi - square test and values of -coefficient, chi - square, odds ratio, and confidence interval were reported . A total of 200 responses for each item in the questionnaire were recorded from 200 subjects . The distribution of subjects in the age groups was 32 (16%) in <20 years, 62 (31%) in 21 - 40 years, 81 (40.5%) between 41 and 60 years and 25 (12.5%) in> 60 years . The subjects from urban areas were 168 (84%) and those from rural areas were 32 (16%). Those with the highest level of education were 7 (3.5%) who had up to postgraduation, 36 (18%) were university graduates, 76 (38%) high school, 43 (21.5%) middle, 29 (14.5%) primary and 9 (4.5%) illiterate . There was a family history of diabetes in 147 (73.5%) and no history in 53 (26.5%). Those with type 1 diabetics were 78 (39%) while 122 (61%) had type 2 diabetes . The time since diagnosis was <5 years in 73 (36.5%), 6 - 10 years in 72 (36%), 11 - 15 years in 21 (10.5%) and> 15 years in 34 (17%) subjects . The type of treatment was insulin in 105 (52.5%) and oral hypoglycemics in 95 (47.5%) indicating that many type 2 diabetic patients were also on insulin . Self - monitoring of blood glucose was done by 124 (62%) of the subjects while 76 (38%) did not . Diet control was used by 112 (56%) subjects while 88 (44%) did not . A total of 167 (83.5%) had received formal diabetes awareness education while 33 (16.5%) had not . The total misconception score was low (0 - 12) in 115 (57.5%), moderate (13 - 24) in 77 (38.5%) and high (24 - 36) in 8 (4%) respondents (n = 200). Table 1 demonstrates the frequency distribution of respondents according to misconceptions about etiology, types and pathogenesis of diabetes . The most common misconceptions identified on etiology, types and pathogenesis were that overweight causes diabetes (89%), diabetes is only a hereditary disease (80.5%) and eating too much sugar causes diabetes (69%). In addition, this table shows the distribution of subjects with reference to misconceptions about daily life . The most frequent misconceptions about the daily life of diabetics were that diabetics cannot lead a normal social life frequency of respondents with misconceptions about etiology, types, pathogenesis and day - to - day life of diabetes (n=200) table 2 illustrates the distribution of respondents according to misconceptions about diet . The second misconception on treatment was that there is no need to take medicines when blood glucose is normal table 3 shows the association of the selected socio - demographic variables on the misconception score . It shows that female gender, living in a rural area, little or no education, <5 or> 15 years since diagnosis, lack of self - monitoring, poor compliance with dietary control and no diabetes education were significantly (p <0.05) associated with moderate or high misconception score . However, age, family history of diabetes, type of diabetes and type of treatment were not significantly associated with the misconception score . Frequency of respondents with misconceptions about diet and treatment (n=200) association of misconception score category with sociodemographic variables table 4 gives stepwise logistic regression to determine the predictive value of independent variables [showing significant association as per table 3] with the misconception score (dependent variable). It indicates that diabetes education, gender, education and time since diagnosis are good predictors of misconception score . Every chronic disease necessitates long - term commitment from the patient, family and health care professionals . Health care is a full package that includes the proper education of society in general and the patient in particular, about all aspects of the disease in question . A label of no definitive cure on chronic diseases generates many myths and misconceptions . These misconceptions are affected by socio - demographic factors and are propagated by opportunists who take advantage of patients to market their products . Understanding the myths and misconceptions about a disease, like dm, is important for the provision of excellent care and health education to both patients and healthy individuals . These myths and misconceptions are generally about forbidden foods, the use of herbs, life - style changes, side - effects of treatment and so - called dependence on medicines . They usually interfere with self - management plans for diabetic patients leading to undesirable complications . The frequency of common misconceptions reported by other studies is more or less similar to what our report presents . A previous study in eastern saudi arabia that was carried out on adult male attendees of primary health centers found misconceptions about the etiology of diabetes in 21.2% of those studied, about general concepts of diabetes in 13.8% and diet in 10.7% of the patients . However, our study population was different, in that all of them were diabetics . A study in western saudi arabia reported that the top misconceptions that diabetic patients had were that oral medications might be more effective than insulin, medications might cause habituation and serious complications, the efficacy of medications depends on their cost, cure is expected following a short course of treatment and a diabetic could eat anything as long as medications were taken . The prevailing myths reported in the population of qassim region of saudi arabia were that consuming sugar results in diabetes, diabetics should avoid sweets, some type of dates do not increase sugar level, honey intake doesn't increase sugar level, and diabetes in its early stages can affect sexual performance . Determinants of myths and misconceptions are the factors that are directly related to generation or propagation of these wrong beliefs . Knowledge of the determinants of any misconception, attitude or behavior is very important to the management of disease . Our results indicate that around 42.5% of the subjects had a moderate to high misconception score, indicating that there was much room for improvement in our patients' education . The mean misconception score was 10.29 4.92, while 38.5% had moderate (1324) and 4% high (24 - 36) misconception scores . The factors which had a significant association with high misconception scores were female gender, rural residence, little or no education, <5 years or> 15 years since diagnosis, no self - monitoring, poor diet control and no prior education about diabetes . The relationships of different determinants of misconceptions studied in our study are discussed below . Female gender was found to be significantly (p <0.05) associated with moderate to high misconception score . This was not a totally unexpected finding due to social norms of a conservative society with significantly less exposure of women to information . This finding gave us a clear task to target our female patients to provide them with the correct knowledge about diabetes and its management . A study carried out on female school teachers of alkhobar showed that their understanding of diabetes was inadequate . Similarly, a sudanese study also found poor compliance to therapy, poor glycemic control and poor knowledge about diabetes in women as compared to men . This association was also reported in misconception studies carried out in new york and in india . However, a study carried out in qasim did not find gender to be associated with myths and misconceptions . The proportion of patients from rural areas was very low in our study as a result of the saudi government's policy of ensuring that patients access health facilities nearest to their homes . However, the proportion of patients with moderate to high misconception scores was higher in patients from rural areas . This shows that certain socio - demographic factors render people from those areas more vulnerable to misconceptions . However, there are other associated factors that can modify beliefs of urban populations as well . In a study carried out in a low income population of new york, a survey carried out in the 4 largest city of india, chennai, on around 26000 subjects concluded that even though there had been diabetes education campaigns from as far back as 1948, the level of lack of knowledge and misconceptions was unacceptable . As expected, the proportion of subjects with moderate to high misconception was highest in the illiterate group, and this decreased as the level of education rose . The study by sabra et al . In eastern saudi arabia found high misconception scores in 23.5% of illiterate or those who could only read - and - write . A study in new york found that participants with less than a high school education were more likely to have misconceptions . A study of myths and misconceptions in the qasim region also found significant differences in responses according to educational status . This necessitates the identification of the less educated from the beginning in order to design special educational programs that suit their respective levels of education . Time since diagnosis was found to be significantly associated (p <0.05) with misconception scores . It also had a high predictive value for the misconception score . Patients who had been diagnosed with diabetes> 15 years before were more prone to have a moderate to high misconception score, and the chances of having misconceptions decreased in categories as times since diagnosis lessened . This might be due to better current diabetes awareness programs as well as the ease of access to information for freshly diagnosed diabetics who are curious . The knowledge of the subjects presenting for the 1 time has generally been reported to be inadequate and other studies have indicated this relationship . The patients who reported that they were controlling their diabetes by self - monitoring of blood glucose had low misconception scores . This group comprised 62% of the total number of patients, about two - thirds of whom had low misconception scores . On the other hand, two - thirds of those who were not self - monitoring had moderate to high misconception scores . This shows that making the patient take charge of his or her diabetes control develops an interested attitude . In addition, a clear picture of alterations in blood sugar by medicines, diet control and life - style changes help in removing of unfounded baseless misconceptions . No other study has reported this association . However, a study from western saudi arabia has shown an association of discontinuity of treatment with a high level of misconception . Noncompliance to the recommended diet control was found to be a major determinant of misconceptions . Surprisingly, most of patients with the highest misconception scores (7 out of 8) reported to have little or no diet control . This reflects a general carefree attitude of these patients to all aspects of life - style changes associated with the management of diabetes . However, most of the patients using the recommended diet control were found to have less misconception scores . This suggests that we must identify patients with a carefree attitude from the beginning and design proper education programs, with psychotherapy sessions if possible for them . The majority of the patients (83.5%) reported some formal instruction by health care professionals about diabetes and its management . As expected most of the subjects who had undergone a proper education about diabetes (presentation, management and life - style changes) had low misconception scores . Not surprisingly, this was the most significant single determinant of removing misconceptions about diabetes . The results also indicated that there was room for improvement in the education of the large proportion of patients with moderate to high misconception scores . In our study, family history of diabetes, type of diabetes and type of treatment were not found to have any significant association with misconception scores . As in our study similarly, the age of our subjects did not show a significant association with misconception score . However, the proportion of subjects with moderate to high misconception scores was highest in the categories of the youngest and the oldest patients (<20 and> 60 years). Though not statistically significant, it still shows that we must make an extra effort to educate these two vulnerable age groups about their disease . Suboptimal knowledge and beliefs are potentially modifiable and are logical targets for educational interventions to improve diabetes self - management . Knowledge of all the identified determinants of moderate to high misconception scores will help in streamlining the awareness programs for patients in accordance with these factors . Similarly, it will act as a guideline for other units in our region to develop better patient education programs . If the patients are given proper guidance and education on diabetes care, there would be a significant improvement in their life - style which would in turn help in producing good glycemic control . We conclude that myths and misconceptions about diabetes and its management are common in our patients . The strongest determinants of the misconceptions in our study population are female gender, rural area of residence, illiteracy or little education, <5 or> 15 years since diagnosis, no self - monitoring of blood glucose, poor diet control and no education about diabetes . Therefore, diabetes educational programs should focus on individuals with one or more of these predictors.
Thoracic injury can result in a wide range of clinical manifestations depending on the structures involved like chest wall, diaphragm, mediastinum, trachea, lungs parenchyma etc . The non penetrating trauma to the lungs manifests as contusions, lacerations of pulmonary parenchyma, pneumatocele formation, hematomas, and fractures of trachea and bronchi . While pulmonary contusion is commonly associated with blunt thoracic trauma, appearance of cavitary lesions variably described as traumatic pulmonary pesudocysts, (tpp), traumatic pneumatoceles, traumatic lung cysts are rare, developing in less than 3% of patients with pulmonary parenchymal injuries . A 26 year old unmarried male was referred to us with history of left sided chest pain for two days . He had consulted at local hospital immediately after trauma . A skiagram chest taken there [figure 1] showed non homogenous opacity with central lucency in the region of left hilum . On examination, the patient gave history of trauma - a tractor wheel hitting him on the anterior chest wall, while he was lying down; following which he developed the pain . There was no history of breathlessness, fever, cough, hemoptysis, cough or expectoration . There was no history suggestive of bronchial asthma or exposure to pets at home or work place . The margins of the lesion were now thinned out and smooth [figure 2]. Chest radiograph on day 1 - pa view showing non homogenous opacity with central translucency near left hilum day 3 - chest radiograph pa view showing multiple cavities near left hilum his sputum for afb was negative . Other lab investigation revealed haemoglobin 14.4, leukocyte count 7900, and differential count of 78% polymorphs and 19% lymphocytes . His serum was non reactive for hiv antibodies . A ct scan of thorax done on 03 - 09 - 2011 showed a single smooth walled cavity abutting left chest wall with surrounding areas of ground glass haziness, probably suggestive of lung contusion [figure 3]. Computed tomogram of chest showing smooth walled cavity in left upper lobe with surrounding ground glass opacity of lung contusion fibre optic bronchoscopy was performed to achieve a microbiological diagnosis and to inspect the trachea - bronchial tree in view of previous reports being inconclusive . Bronchoscopy revealed a bleeding spot in left main bronchus, but no active bleeding was seen . Bronchial brushings taken from left upper lobe were negative for malignant cells and afb, and the lavage fluid also was sterile . In view of the patient's history of ancedent recent trauma, negative past and present history for any infectious process, and negative microbiological reports, a diagnosis of post traumatic pulmonary pseudo cyst (tpp) was made . Serial follow up chest skiagram showed rapid resolution of the cavities [figure 4] without any antibiotics, thus confirming our diagnosis of tpp . Among the various names given to the cavitary appearance of pulmonary lacerations, the term traumatic pulmonary pseudo cysts (tpp) appears to be the best nomenclature because the wall of these lesions are formed by the inter lobar interstitial connective tissue and shows no epithelial lining or bronchial wall elements . The rarity of the entity can be estimated by the fact that there have been only around 10 large case series with 8 or more patients published since 1967 . Tpp following blunt or non penetrating trauma develops through a mechanism that allows transmission of high compressive forces to the lung parenchyma . Retraction of normal lung elastic tissue from contusion induced cavities permits the escape of air and fluid into it . Laceration of pulmonary parenchyma and appearance of cavity may also occur if there is closure of the glottis at the moment of injury which may prevent fast exit of the air from compressed lung segment . The parenchyma and/or interstitium get lacerated in a bursting manner resulting in a cavity formation . Resolution of a pulmonary hematoma or drainage into a bronchus may result in development of tpp at a later stage and it is called secondary tpp . Tpp can occur at any age, but they are more often seen in children and young adults, probably because of greater compliance of chest wall which permits a larger transmission of force of impact to the parenchyma . Sorsdahl and powell reported that 85% of the patients with tpp were under the age of 30 years with male predominance . Impact velocity and degree of chest wall displacement may play an important role in the development of tpp after blunt trauma to the chest . A high velocity impact causes peripheral tpps while a low velocity high displacement impact produces central parenchymal and major bronchial disruption . Tpp is a clinical entity that manifests itself with minor clinical and major radiological signs . The usual clinical manifestations include hemoptysis, occurring in about half of the cases, cough, dyspnoea, chest pain, fever and leucocytosis . Tpps may appear immediately or within a few hours after injury and their sizes range from 2 to 14 cm in diameter tpp can be differentiated from cavitating hematomas on the basis of radiographic appearance of air within the tpp's within 48 hours . Tpps may be identifiable on chest radiography but ct scans are superior for detecting them . Unlike other cystic lesions and cavities, the size, shape, and nature of wall of tpps change relatively quickly . Therefore, serial skiagrams of chest, done over several days can help to differentiate pseudo cysts from other lesions . Found a greater resolution time in blood filled tpps and those with> 2 cm in size . They may be seen on the site of injury or on the opposite side secondary to countre coup effects . The majority of tpps are found in the lower lobes, the ct appearance of single or multiple thin walled cystic lesions with air space consolidation of the surrounding lung parenchyma in the backdrop of antecedent trauma is diagnostic . Differential diagnosis includes ruptured oesophagus, or herniation of viscera, post pneumonia pneumatocele, tuberculosis cavity, cavitating bronchial carcinoma, lung abscess, bronchogenic cysts, and pulmonary sequestration . A history of trauma, rapid sequential changes over days on chest skiagram, and presence of contusion on the base of lesion usually delineates any confusion . If the cavitary lesion does not decrease with time other aetiology must be considered . Conservative management is recommended as long as evidence of a decrease in size of the lesion occurs within 6 weeks after trauma in adults and 3 - 4 months in children . The use of antibiotics is controversial and may be used more to provide simple reassurance that the pseudo cyst wo nt get infected . Tpp's can be complicated and may require surgery . They may rupture and cause a secondary pneumothorax that may require tube thoracostomy . The indications for diagnostic and therapeutic bronchoscopy are endobronchial bleeding, thick sputum, large air leak, mediastinal emphysema and lobar collapse . The approach to an infected pseudo cyst is similar to that for a lung abscess . If an infected pseudo cyst is larger than 2 cm, or there are unremitting signs of sepsis after 72 hours of antibiotics, the pseudo cysts may be drained percutaneously . Early lobectomy may be considered for complex tpp's with extensive lung abscess surrounded by necrotic parenchyma, failure of bronchoscopic treatment of massive airway bleeding, infected pseudo cyst more than 6 cm in size, or no response to more conservative treatment . Video assisted thoracoscopic surgery (vats) may be considered for managing a persistent air leak, hemothorax due to pseudo cyst rupture, failure of lung expansion, progressive enlargement of pseudo cyst and compression of lung parenchyma . Late thoracotomy has been reported (lobectomy, cystotomy, capitonnage) up to 6 months after trauma because of pneumonic infiltration and persistent cavitary size . Our patient presented with few abrasions on anterior chest wall and history of blunt trauma to chest wall of one day duration . There was no previous history of any respiratory illness or unconsciousness during or after trauma . The immediate chest skiagram revealed an area of consolidation with central area of lucency around left hilar region . There was no associated cough, fever, expectoration, hemoptysis or breathlessness which clinically ruled out an infective process . Serial skiagrams over next 24 hours showed a rapid development of a cavity which regressed spontaneously and significantly over next 7 days without any antibiotics . Ct thorax done was also suggestive of tpp, in view of a cavity adjacent to anterior chest wall with surrounding contusion . Exclusion of other infectious diseases like tuberculosis, lung abscess, by absence of relevant clinical history, sputum negativity on zn staining, sterile pyogenic culture, negative mantoux test, negative microbiology of bal, and cytology of bronchial brushing led us to the diagnosis of tpp which was well supported by the available literature . It is generally a self limiting benign condition, diagnosed by excluding other conditions which may present with similar radiological manifestations . A typical history of blunt trauma to chest, rapid radiological changes of the cavity and exclusion of other cavitary pulmonary diseases by adequate laboratory work helps in diagnosing this rare condition . The disease at times might become complicated and becomes life threatening; hence, should be closely monitored and followed up.
. However, cerebral phaeohyphomycosis (cp) caused by darkly pigmented fungi appears to be a common exception to this rule because about one - half of this fungal infection occurred in patients with no underlying disease or risk factors . Cp is a very rare cause of brain abscess, but is often a fatal disease regardless of immune status14,17,23). The authors illustrate a 75-year - old, immunocompetent male patient who had a single brain abscess from dematiaceous fungi . To the authors' knowledge, this is the first case of cp in korea . A 75-year - old male, a resident of a rural area and a farmer by occupation, visited our outpatient clinic with the symptoms of poor cognition and memory decline over 2 weeks . He denied any history of fever, headache, blurred vision, vomiting or seizure . There were no laboratory abnormalities including leukocytosis or c - reactive protein rising . Upon the neurologic examination, he was conscious and there were no neurologic deficits except intermittent expressive dysphasia and disorientation . Brain magnetic resonance imaging (mri) was performed because of suspicion of some type of dementia . It showed a 20 mm sized nodular enhancing mass with peritumoral edema in the left frontal lobe . High - grade glioma or metastatic tumor was initially presumed based on his age and progressive symptoms . The lesion appeared to be white and took the form of a relatively hard mass with a clear boundary, permitting radical excision of the mass (fig . . Septated hyphae and melanin pigments were confirmed at fontana - masson stain consistent with cp (fig . The patient was started on intravenous amphotericin b at a dose of 68 mg daily . After 10 days, he was switched to 270 mg of intravenous voriconazole twice a day because of the elevation of serum creatinine . He took the injection for 8 weeks, followed by oral voriconazole 200 mg twice a day for 2 months . A follow - up brain mri 3 weeks after surgical excision demonstrated a significant resolution of the edema . Ongoing resolution of the lesion was found on the latest follow - up mri (fig . He showed dramatic improvement in his symptoms including disorientation and memory disturbance after completion of surgery and antifungal therapy . Cp is a rare infection caused by darkly pigmented fungi, namely dematiaceous fungi23). Dematiaceous fungi represent a group of filamentous molds that contain melanin pigment in their cell walls3,6,14,17). Rhinocladiella mackenziei (formerly ramichloridium mackenziei) is the second most common cause of cp, which is exclusively endemic in the middle east area17,22). Because of this, occupational predisposition has been reported in agricultural workers, especially farmers due to risk of soil exposure17,23). Cp commonly occurs in the second and third decades of life with male predominance, except rhinocladiella mackenziei which affects adults with a median age of 62 years without male predominance3,13,23). The most unique characteristic of cp is its occurrence irrelevant to the immune status of the host15,17,19,23). Even though immunodeficiency may play a role as a risk factor, there are many reports of this infection in immunocompetent individuals similar to the patient in this report12,15,17,19,25). The portal entry to brain is unclear, although several possible routes have been suggested, such as hematogenous dissemination of inhaled spores or accidental skin inoculation as well as direct extension from adjacent paranasal sinuses or ears2,6,10,14,15,19,22,23). Pathogenesis of cp is associated with the presence of melanin as a virulence factor that provides advantages in evading host defense and crossing the blood - brain barrier by binding to hydrolytic enzyme14,16,17). Clinical spectrum of phaeohyphomycosis was listed as a variable, ranging from solitary subcutaneous nodules to a life - threatening infection5,11,16,17,18). In the central nervous system (cns) about 70 - 80% of cases typically manifest as a single brain abscess particularly on the frontal lobe (52%) like in our case, while multiple brain abscesses can be seen in immunocompromised patients3,6,17,19). The diagnosis of cp can be difficult because dematiaceous fungi are often considered contaminants when identified in culture . Furthermore, the pathogen cannot always be cultured and isolated from the serum or cerebrospinal fluid (csf)12,21,24). No molecular techniques are available to speedily identify these fungi even to the genus level17). Only the tissue examination can be useful to identify irregularly swollen hyphae with yeast - like structure and to confirm the presence of dematiaceous hyphae in melanin - specific fontana - masson stain14,17). Unfortunately in this case, fungus was not identified in the culture of surgical specimen, therefore, the species that causes cp could not be detected . Meanwhile, the brain mri reveals a ring - enhancing lesion with a low - attenuation core, suggesting the presence of necrosis or pus10,19). In cases where high - grade glioma or metastasis is mimicked by irregular and variably contrast - enhancing lesions, imaging findings of this patient were more suggestive of a glioma than an abscess, because nodular heterogeneity on contrast injection mimicked the images seen in high - grade tumors . Consequently, surgical biopsy is essential for the diagnosis of cp . Because of the rarity of the cases complete excision of brain lesions may provide better results than simple aspiration unless the lesion is multiple or is located within the eloquent area of the brain3,17). Antifungal agents are generally used in combination of amphotericin b, 5-flucytosine and itraconazole because it is associated with improved survival rates3,14,17,20). Voriconazole can be used as alternative to itraconazole because of its good penetration into both csf and brain tissue17,22). Duration of taking the medications is still unknown because most reported patients expired during treatment except a few survivors who received voriconazole for about 12 months8,19). In addition, posaconazole may be a potent drug when pathogen is rhinocladiella mackenziei1,3,8). In this case, amphotericin b has fatal side effects such as nephrotoxicity, therefore, close observation on kidney function is needed . Mortality rate approaches 100% in untreated patients, while that of treated cases as high as 65% to 73% despite the aggressive treatment6,9,10,17,19,23). Interestingly, mortality rate did not differ significantly between immunocompromised and immunocompetent patients (75% vs. 71%)12,17). Fortunately, the patient reported here had a good response to surgery and chemotherapy and showed fine recovery without any sequela . Solitary lesion and the good general condition of the patient, together with an aggressive therapeutic approach, are therefore inferred to contribute to a favorable outcome . Further studies are necessary to find more potentially useful antifungal regimen for these refractory infections and to investigate more detailed pathophysiology and prognostic factors to increase the survival rate . Cp is rare disease, but challenging one with high mortality rate, particularly when the cns is affected . As shown in this report, complete resection and adequate antifungal therapy are the most recommended modality for patients with cp - related abscess to this time.
Cephalopelvic disproportion (cpd) in labour occurs when there is a mismatch between the size of the fetus and the dimensions of the maternal pelvis . The factors which mainly influence the outcome of the delivery can be summarised as the three ps of the labour: passageway, passenger, and power of the uterus . The passageway component of this trinity has been investigated by pelvimetry which measures the maternal bony pelvic dimensions, with very little emphasis on its shape or pelvic floor muscles . During the last decades, the use of pelvimetry has been discouraged, but at present, no replacing methods to evaluate the maternal pelvis have been introduced . The benefits of vaginal deliveries are well known when no risk factors are present even after previous cesarean section (cs) [5, 6]. On the other hand, unplanned interventions during labour such as acute or emergency cesarean sections as well as operative vaginal delivery increase both maternal and fetal morbidities as does a prolonged second stage of the delivery . The safety and the accuracy of the measurements obtained in pelvimetry have improved in the era of the mri technology [9, 10]. It is also in the interest of the mother and her physician to minimize the number of unplanned interventions during labour . The purpose of this observational cohort study was to evaluate whether pelvic measurements, especially pelvic outlet, displayed any association with operative vaginal deliveries and the duration of the second stage of the delivery . This retrospective study was approved by the ethical committee of north - carelian central hospital . It investigated caucasian women, that had been examined by x - ray or mri pelvimetry during 20002008 in north - carelian central hospital . Eligibility criteria included that pelvimetric and fetal measurements had been recorded . In the operative delivery group, there were no signs of fetal distress in cardiotocography, inertia was not diagnosed, and there was no malpresentation . Originally, 915 women were screened for possible inclusion, but 429 women were excluded because of breech presentation . A total of 486 patients with the fetus in the cephalic presentation were screened in the study, but those 234 women that went through elective or acute cesarean section were excluded from the analysis . The clinical indication for pelvimetry was breech presentation, or if the fetus was in cephalic presentation, the indication was suspected cephalopelvic disproportion in clinical examination . The findings that referred to cpd in clinical examination were clinically small pelvis, unengaged presentation, or suspected macrosomia . Pelvimetric measurements were found in all patients, as required by the inclusion criteria . There were 252 participants with fetal cephalic presentation delivered vaginally, of whom 184 women delivered spontaneously and 68 women went through operative vaginal delivery with vacuum extraction . Of this latter group of women, in 26 patients, the vacuum extraction was undertaken primarily because of fetal distress and inertia, and these patients were excluded from the final analysis, leaving 42 women in the operative vaginal delivery group . Thus, the total number of participants evaluated in the final stage of this study was 226 . The obstetric and radiologic data were collected from patients' medical records by the author (uk) and transferred into a commercially available worksheet (excel, microsoft 2003, ireland). The following pelvimetric parameters were recorded: in the pelvic inlet, anteroposterior (conjugata vera) and transverse diameters and in outlet, interspinous diameter and sagittal diameter from the surface of the pubic symphysis to the surface of the sacrum measured at the spinous level . Pelvic inlet and outlet circumferences were calculated from the pelvic anteroposterior and transverse diameters using the formula (ap + dt 1.57). Until the year of 2003, all pelvimetries were performed with an x - ray technique, and from the year 2004 onwards, they were performed with magnetic resonance imaging (mri). During the transition period, both x - ray and mri pelvimetries were performed to verify the repeatability of the measurement results . Already at the beginning of 1990, in order to minimize the variability in pelvimetric measurements, they were centralized so that instead of being conducted by several radiologists, they were conducted by trained obstetricians . When the mri pelvimetry was taken into clinical practice, there was one radiologist with previous experience of mri pelvimetry, and during a two - year period (20042006), three radiologists and further three obstetricians were also trained to measure the images . In this evaluation of the diagnostic accuracy of the pelvimetry in vaginal deliveries, patients were divided into subgroups according to the size of the fetus and also by the parity to evaluate the variability reflecting differences in patient groups . For statistical analysis, we used spss 17.0 (spss inc ., 2009, receiver operating characteristic (roc) curves were established, and the area under curve (auc) values with significances were calculated . Pelvimetric measurements were found in all patients, as required by the inclusion criteria . In the spontaneous vaginal delivery group, most of the nulliparous patients were sent to maternity clinics consultation because of suspected disproportion . In the multiparous group, 24% had delivered by cs and 37% by operative vaginal delivery . In the operative vaginal delivery group, of the nine multiparous patients, six had delivered by cs in their previous pregnancy and two had had previous vaginal operative delivery . The demographic data of these 226 patients subdivided according to the route of delivery are shown in table 1 . Patients were further subdivided into two subgroups according to the infant's weight and the mode of delivery . The maternal pelvic inlet and outlet sizes and duration of the first and second stages of the delivery by the mode of delivery in infant weight subgroups are shown in table 2 . The mean maternal outlet (sd) was 3613 (20) mm in all, 351 (17) mm in infant weight <3700 g, and 369.5 (17.7) mm in infant weight 3700 g groups . No clinically or statistically significant differences in the pelvic sizes were found between the modes of delivery within the subgroups . Between the subgroups, the size of the maternal pelvic size was 4%-5% larger in the mothers with infant weight 3700 g. the duration of the second stage of the delivery was 54 minutes longer (p <0.01) in the operative vaginal delivery group amounting to a 45-minute longer duration (p = 0.01) in infant weight <3700 g and 62 minutes longer (p <0.01) in infant weight 3700 g group . The one - minute apgar scores were above 8 in all groups with the exception of those with infant weight less than 3700 g in the operative vaginal group, where the mean of apgar score at one minute was 7.8 1.8 . The receiver operating characteristic curve analysis for pelvic inlet and outlet as a diagnostic test for the mode of vaginal delivery is shown in figures 2(a) and 2(b). The area under the curve (auc) for the pelvic inlet was 0.566 with the p value of 0.18 and 95% confidence interval (ci) of 0.4650.667 . For pelvic outlet, the main finding of this study was that the maternal bony pelvic dimensions displayed virtually no correlation to the need for operative vaginal deliveries . The indications for intervention in vaginal deliveries were chosen on clinical grounds as evidenced by the fact that there was an association between the duration of the second stage of the delivery and the size of the pelvic outlet . If the delivery had reached the second stage, it was probable that the uterine power played a more significant role in the overall outcome than either the passageway or the passenger . On the other hand, pelvic floor muscles, the three - dimensional shape of the bony pelvis, or other soft tissues the pelvimetry was performed in most of the patients because of suspected disproportion, or an intervention had been required in a previous labour . Of those patients that had previous cs and were now exposed to the trial of labour, over 80% delivered spontaneously, and less than 20% required an operative vaginal delivery . This is in agreement with previous studies [6, 13]. Of those women that had had a previous operative vaginal labour, only 5% underwent repeated vaginally assisted delivery . This may have been due to the fact that the patients were chosen for the trial of labour correctly irrespective of the previous operative delivery . There were no statistically significant differences between the size of the maternal inlet or outlet in the spontaneous and the operative vaginal delivery groups . When patients were divided into subgroups according to the infant weight, the maternal inlet was 4.7% and the outlet was 5.1% larger in the infant weight 3700 g subgroup among those who delivered spontaneously compared to those vaginally assisted . The duration of the first stage of the delivery was longer in the smaller infant group, whereas the second stage was shorter than in the larger infant group . In the two delivery subgroups, the duration of the second stage of the delivery was significantly longer in operative vaginal delivery group than in spontaneous vaginal delivery group . The apgar scores were acceptable in all delivery groups referring to the fact that both spontaneous and operative vaginal deliveries were uncomplicated and severe shoulder dystocia was not present . The apgar 1-minute scores were lower in the operative vaginal delivery group than in spontaneous vaginal delivery group when infant weight was <3700 g. these results refer to the fact that operative vaginal delivery increases the time of the second stage of the delivery and decreases the apgar 1-minute scores . The roc curve analysis for maternal pelvic inlet and outlet revealed that both inlet and outlet had only a fair prognostic value in predicting the mode of the vaginal delivery . The poor predictive value of pelvimetry to predict protracted labor is a well - known fact from previous studies, whereas the evidence on the need of vaginal operative deliveries is less extensively evaluated . The data did not reveal in detail the fluency of the operative deliveries . As mentioned earlier, therefore, it was not possible to evaluate the influence of the pelvic dimensions on the severe dystocia . For that kind of study, the cohort examined here study is too small due to the rare incidence of severe dystocia . Accordingly, due to the retrospective nature of the study, no blinding was present, and the caregivers were aware of the pelvimetric measurements during the labour . Furthermore, the study women were at high risk of operative deliveries due to the inclusion criteria, but in clinical care, this would be the group eligible for pelvic assessment before delivery . In conclusion, our study revealed that maternal bony pelvic dimensions, either pelvic inlet or outlet, were not associated with the need for operative vaginal deliveries . It was more likely that other factors related to the maternal perineal soft tissue, maternal resources, and the passenger were the reasons leading to operative vaginal deliveries . Subsequently, we cannot recommend that caregivers use pelvimetric measurements to predict the outcome of the second stage of the labour . Observational studies with larger cohorts would be needed, if one wished to investigate whether the maternal bony pelvic size has any effect on severe dystocia . In addition, the three - dimensional shape of the bony pelvis and the soft tissues are worth considering in future studies.
A close relationship between tmj and structures of the middle ear110 has been explained by the presence of the anterior mallear ligament (aml), the sphenomandibular ligament (sml) and the discomallear ligament (dml).2,3,8,1113 in the past, aml was accepted as an initial ligament lying between the middle ear and articular disc (ad) of tmj.13 however, further investigations discovered that two distinct ligaments form the aml.12 the anatomic relations and functional properties of dml, aml, and sml are described comprehensively in well - documented studies.5,8,12,14 on the other hand, different opinions exist about histological characteristics of these ligaments.6,8,9,12,1417 various investigators have recognized aml to be consisted of muscular fibers, fibrotic tissue bands, fibro - elastic bands and lastly collagenous fibers since the last century.3,6,14,16,17 the dml is a free ligament, lying between the malleus and the tmj,5,8,12,18 which was shown to have a total structural difference from the aml.19 these ligaments were exposed to various traction and stretching tests in the cadaveric studies to discover whether they were responsible for mallear mobility by experimental simulation of discal or condylar translation.6,9,1719 a number of researches supposed that these ligaments were comprised of collagenous fibres9,12 whereas the others supported that only fibro - elastic fibres could transmit traction forces of tmj and cause mobility of the mallear ossicle.6,8 for that reason, we conducted a histological study on the ligaments between the ad and the malleus to clarify the weight of histological properties on mobility of the mallear ossicle in the middle ear . 15 adult skulls, fixed in a formalin solution, with undamaged tmjs were used in the presented study . The cadavers were obtained from gulhane military medical academy, department of anatomy, ankara, turkey . Then malleus and incus were explored by entering middle ear cavity through the arcuate eminence . With protection of malleus and incus, petrous part of the temporal bone the components of middle ear, the ligaments lying anterior to malleus, the ad, and the capsule were explored . Anterior surface of the petrous part was removed subsequently to follow posteromedial section of tmj . Little forceps were used to collect bone remnants . By this way, posteromedial and retrodiscal tissues of tmj were completely exposed . Aml and dml were excised and the intact ones were processed for histologic examination to evaluate their histologic structure and relations with surrounding tissues . The ligaments, which were already fixed in 10% buffered formal - saline solution were sectioned longitudinally (710 m thickness) for examination under light microscopy . The sections were stained with verhoff s van gieson s stain (vvg) (for elastic fibres). The sections were visualised at x2.5 and x10 magnifications under a leica dmi 6000 microscope (leica, germany) using the software leica application suite (leica, germany). By vvg stain, the elastic fibres are observed in blue colour and the collagen fibres in red colour . Histologically, the sml and the aml were composed of collagen bundles and fibrocytes between them . The collagenous fibres were running as filaments in some part of the stained specimens, or they were dispersed in denser fashion as thick bands (figure 1). However, no evidence of elastic fibres was found in none of the examined sections . Nuclei of fibrocytes and collagenous filaments straddling perpendicularly were observed clearly in the thicker collagenous bands . In addition, lumens of capillary vessels were observed particularly between collagenous bands (figure 1). The dml was constituted of rich collagenous fibres in analysed sections (figures 3 and 4). Interestingly, one specimen, that was harvested between petrotympanic fissures and at the insertion sites of dml to articular disc - capsular ligament unit, contained elastic fibres dispersed as cotton - bowls amid the collagenous filaments (figures 3 and 4). However, no evidence of elastic structure was found in the part of the dml spanning between the malleus and petrotympanic fissure . The collagenous fibres were running as filaments in some part of the stained specimens, or they were dispersed in denser fashion as thick bands (figure 1). However, no evidence of elastic fibres was found in none of the examined sections . Nuclei of fibrocytes and collagenous filaments straddling perpendicularly were observed clearly in the thicker collagenous bands . In addition, lumens of capillary vessels were observed particularly between collagenous bands (figure 1). The dml was constituted of rich collagenous fibres in analysed sections (figures 3 and 4). Interestingly, one specimen, that was harvested between petrotympanic fissures and at the insertion sites of dml to articular disc - capsular ligament unit, contained elastic fibres dispersed as cotton - bowls amid the collagenous filaments (figures 3 and 4). However, no evidence of elastic structure was found in the part of the dml spanning between the malleus and petrotympanic fissure . Mammalian meckel s cartilage, which is derived from the first brachial arch, consists of calcified and uncalcified segments.20 it is well known that the anterior portion that is anterior to the mental foramen and the auricular portion of meckel s cartilage contribute to bone formation of the mandible, the malleus and incus by endochondral ossification.20 by contrast, the midportion of meckel s cartilage, in the soft tissue, eventually forms the sml.21 aml and dml were demonstrated, in many studies, lying from malleus to petrotympanic fissure.8,12,14 such a relationship of the ligaments with adjacent anatomic structures set a hypothesis about possible damage to the middle ear ossicles and the tympanic membrane.12,19 that hypothesis was based on the fact that mallear movement would be responsible for such a dilemma that might be caused by invasive tmj surgery or anterior displacement of the ad.19,22 another hypothesis for this phenomenon was role of extreme distraction of mandibular ramus that incorporated in stretching of aml by intense traction of sml.19 the previous studies mentioned that application of strain forces on dml caused mobility of mallear ossicle.6,9,23 mallear movement by sml stretching was observed in specimens among 50 cadavers.12 kim stretched dml and found no movement; in contrary, noteworthy mobility of the malleus was obtained by aml stretching.17 the same findings were proved in our studies in which we detected movement in one third of 15 cadavers.19 in the light of these studies, histologic properties of these ligaments were thought to have a possible role on the mallear movement . At the same time the stretching tests are partially doubtful, since the adherences or the bony impingements in the petrotympanic fissure are removed during cadaveric dissections to free these ligaments . Therefore, the reported studies may be suspicious to some extent since mallear movement would not have occurred unless full unearthing or bony dissection for these ligaments was accomplished in the middle cranial fossa . That important fact made us to investigate the ligaments histologically to establish a theory for explaining mallear movement by grounds of the previous stretching tests and histological properties of the filaments . The most recent view about histologic properties of aml and dml was that they consisted of collagenous filaments.15 in our study, we found exactly the same findings . The elastic fibres were dispersed throughout the stroma of ligament as cotton - bowl appearance . The ligaments of any filament composition connected to the malleus can initiate mallear mobility without taking into account the adherences in the petrotympanic fissure . However, the abundant elastic fibres in the dml may be responsible for compensation of strain forces on this ligament and may reflect minor forces to the mallear head . The collagenous ligaments are lacking such a compensatory mechanism and may reproduce major movement of malleus by direct transformation of strain forces . This hypothesis could be accepted when histological characteristics of the ligaments are judged . Accordingly, the same mechanism was observed in the first part of this study that covered anatomic and functional aspects of these ligaments . The aml forms pulling effect on the structures which it is inserting by initiation and termination of a traction force . Theoretically, this special configuration is not expected to form the same strain forces as the aml does which means that the dml has a less disimpaction effect on the malleus.
A key issue in biomedicine and biotechnology is to control processes in cells with the highest specificity without unwanted side effects of the intervening agents . Gene transcription is universally controlled by structural elements within the gene, cisacting regulatory elements, which specifically bind transcription factors.1 among these cisacting elements, the core promoters bind general transcription factors of the general transcription machinery, whereas enhancer elements and proximal promoter elements bind sequencespecific transcription factors, which activate transcription by stimulating transcription initiation at the core promoter . Targeting these enhancer and proximal promoter elements with, for example, synthetic sequencespecific transcription factors, represents a way to activate the transcription of only the genes harboring these elements . Yet however, unlike enhancer elements, which act independently of their distance to the core promoter, the effect of proximal promoter elements depends on the distance between the transcription factor binding site and the core promoter.2, 3 transcription factors are modular proteins composed of a dna binding domain (dbd), which specifically recognizes the response element, and a transactivation domain (tad), which directly or indirectly interacts with the general transcription machinery at the core promoter . Therefore, if one could engineer an artificial transcription factor with a fixed distance between the dbd and the tad that matches the distance between the proximal promoter element and the core promoter of a particular gene (figure 1), one would potentially have a much more specific tool to selectively activate the transcription of this gene . A) a dnabinding domain (dbd) and transactivation domain (tad) are separated by a rigid don of a length matching the distance between the transcription factor binding site (tfbs) and the core promoter of gene a. upon binding of the dbd moiety to the tfbs, the tad would be ideally positioned to activate the transcription of gene a. the length of the don does not match the distance between the tfbs and core promoter in gene b. therefore, gene b is predicted not to be activated by the construct . B) as a test case, a rigid, tubular don (don1) derivatized with the suicide ligands benzylguanine (bg) at one end and chlorohexane (ch) at the other end is functionalized with a bgreactive, snaptagged version of the dbd of the yeast gal4 transcription factor and with a chreactive, halotagged version of the vp16 tad to generate don gv . Don gv is then microinjected in zebrafish embryos together with a luciferase reporter gene construct, in which five copies of the consensus binding site for gal4 (5uas) are separated from the core promoter by a spacer sequence predicted to match the length of the don . They are readily available by folding a long singlestranded dna (ssdna) molecule with a designed set of short synthetic staplestrand oligonucleotides.5, 6, 7 dna origami nanostructures (dons) have typical dimensions in the 10100 nm regime, which perfectly match those of large supramolecular protein complexes, such as the molecular machinery involved in gene regulation, cell signaling, or cell division.8 more importantly, these dimensions are within the range of the typical distances between proximal promoter elements and core promoters . Furthermore, dons have unique structuredirecting properties, because they can be used as scaffolds for the precise arrangement of nonnucleic acid components such as proteins or colloidal nanoparticles.9, 10 it is, therefore, not surprising that there is increasing activity to explore dons as tools to manipulate and analyze cellular functions.11 along this line, efforts are underway to study the stability and function of dons under in vitro and in vivo conditions . For instance, it has been demonstrated that dons are stable reagents under cell culturing conditions.12, 13, 14 dons are functional as immuneactivating programmable adjuvants,15, 16 and they can be used as drugdelivery vehicles to circumvent drug resistance.17, 18, 19, 20 these studies have suggested that intracellular don uptake is usually dependent on endocytosis.18 furthermore, dons can bind to the outside of the cells to stimulate cellular transmembrane receptors.21, 22, 23 despite these exciting reports, the exploitation of dons for the analysis and manipulation of cells is still in its infancy, and there is a strong demand for further insights into the complex interactions of these synthetic biomacromolecules with the machinery of living systems . Towards this goal, we report here the microinjection of a proteinfunctionalized don construct into zebrafish embryos (figure 1). Our working hypothesis is that using a don to control the distance between a dbd and a tad in order to match the distance between a proximal promoter element and a core promoter would enable the selective activation of a downstream gene . As a test system, we took advantage of the gal4uas system . This binary expression system has been widely used in many model species to drive the expression of genes . The first component of the binary system consists of a synthetic transcription factor composed of the dbd of the yeast gal4 transcription factor fused to the tad of the herpes simplex vp16 protein . The second component is comprised of a synthetic gene with a tandem array of the dna binding sites for the galvp16 chimera, called the upstream activating sequence or uas (cgg agtac t gtcct ccg),24 upstream of a minimal core promoter . This uas promoter controls the expression of downstream cloned genes with dependence on the gal4vp16 chimeric transcription factor.25 it is known that gal4vp16 fusion proteins generate robust expression from the uas responder genes in transient and stable activation assays in zebrafish.26 to allow for a quantitative assessment of transcription, we chose the firefly luciferase reporter gene as a regulated gene . To test the effect of the distance between the uas and the core promoter in the reporter plasmid, they were separated from each other by a variable spacer (grey region of the reporter gene, figure 1). The reporter plasmid was constructed by gibson cloning27 by sequentially aligning five copies of the uas, a spacer, a minimal cmv (cytomegalie virus) promoter and the firefly reporter gene28 (detailed plasmid map shown in figure s1). We reasoned that, upon injection into zebrafish embryo, the level of expression of the firefly luciferase reporter should then depend on the distance between the dbd (gal4) and the tad (vp16) physically connected to each other through a don spacer in the supramolecular construct don to prepare the don gv construct, we assembled a threedimensional 811148 nm rigidrod tubular don1 (figure 2 a) by using the singlestranded 5438 nt template 109z5, prepared as previously described.29 design and analysis of dna origami nanostructures . B) position (top) and chemical structure (bottom) of the staple strands used for the incorporation of the suicide ligands benzylguanine (bg) and chlorohexane (ch) at 5 ends of the selected staple oligonucleotides . C) ethidium bromidestained 0.75% agarose gel of don samples before (not purified, np) and after (purified, p) peg purification . The supernatant (s) removed after precipitation contains the nonincorporated staple strands (red arrow). E) height profiles obtained from afm images along the crosssections are marked in blue and red in panel (d). The boxwhisker plots show the results of the statistical analysis of 60 particles to determine their dimensions along their axes . The values for x and y were determined from the crosssections, whereas the z values were determined from the particle's heights . To enable the selective attachment of the gal4dbd and vp16 protein at the two ends of the rod, we incorporated at the appropriate positions pairs of the smallmolecule suicide ligands benzylguanine (bg) and chlorohexane (ch), to which snap and halotagged proteins, respectively, can be ligated.30 the bg and chmodified staple strands were prepared from nhsactivated precursors, as previously described,30, 31 and characterized by using gel electrophoresis (figure s2). The assembly of don1 was achieved by temperaturedependent annealing . In brief, the singlestranded dna plasmid and the staple strands at a 1:20 molar ratio were heated to 95 c and the annealing was performed by decreasing the temperature from 85 to 65 c at 1 c per cycle with each step held for 10 min and, subsequently, from 65 to 25 c at 1 c per cycle with each step held for 15 min . The assembled don1 samples were purified through polyethylene glycol precipitation32 and their integrity and correct folding was confirmed by gel electrophoresis (figure 2 c), fretdependent annealing studies (figure s3), and afm analysis (figure 2 d). Statistical analysis of the afm images (figure 2 e) revealed that the observed length (1465 nm), thickness (173 nm), and height (71 nm) were in agreement with the theoretically calculated values of 148 nm length, 11 nm thickness, and 8 nm height for the rods, as depicted in figure 2 a. to synthesize the regulatory proteindecorated construct don gv (figure 1), the required proteins were produced by recombinant expression . We cloned gal4dbd cterminally fused to the snaptag through a flexible (ggggs)3 linker (figure s4). The corresponding protein gal4snap was overexpressed in e. coli and purified to homogeneity by fast protein liquid chromatography (fplc, figure s5). As native gal4 functions as a homodimer furthermore, the functionality of the novel gal4snap fusion protein to bind the uas recognition sequence was demonstrated by using a microbeadbased pulldown assay (figure s7). The vp16 tad was cloned with the halotag fused to its nterminus (figure s4). The corresponding halovp16 protein was overexpressed in e. coli, purified to homogeneity by fplc, and its identity was verified with western blot analysis (figure s8). Having both regulatory proteins available as pure and bioconjugatable reagents (figure 3 a) to this end, a cy5labeled don1 (don1) was prepared as described above and mixed with 25 molar equivalents of gal4snap and halovp16 . Several controls, which included don2, a construct identical to don1 but lacking the bg and ch ligands, and samples lacking one of the two proteins were prepared in a similar manner . The reaction products were characterized by using gel electrophoresis (figure 3 b), which clearly indicated that don the experiments also indicated the absence of unspecific binding between gal4snap, halovp16, and the don . A) electrophoretic analysis of the conjugation of the two ch and bgmodified staple strands with the recombinant proteins halovp16 and gal4snap to be used as tad and dbd, respectively, in don gv (see figure 2 b). This is a 16% sds gel, 20 v cm, 1 h, stained with coomassie; m: page ruler prestained protein ladder . The appearance of a second band with lower electrophoretic mobility upon incubation with the ligandconjugated dna oligonucleotides (sample 2, 3, 5, and 6) indicates the successful conjugation of the protein with the respective staples . B) halovp16 and gal4snap do not bind to don2, a construct lacking ch and bgmodified staple strands (lanes 15). In contrast, don1 containing the ch and bg modifications can bind both of the proteins, as indicated from the shift of the bands in lanes 79 . Note that the conjugation with halovp16 (lane 8) induces only slight electrophoretic mobility shifts, presumably owing to net negative charge of vp16 (pi=4.4). This is a 0.75% agarose gel, 70 v, 3 h, visualized by cy5 fluorescence imaging (top) and ethidium bromide staining (bottom). We then started to analyze the behavior of the dons in vivo in the zebrafish embryo . To this end, we used a zebrafish line stably expressing gfpfused histone h2a33 that labels the cells nuclei and, thereby, facilitates the determination of the dons subcellular localization . Don1 (approximately 500 pl, 250 nm) was injected into the yolk of onecellstage embryos and the embryos were imaged 6 h later (figure 4 a). Onecell stage embryos from a zebrafish line stably expressing gfpfused histone h2a were used,33 which allows the direct localization of the cell nuclei (green channel). The cy5labeled constructs (red channel) were microinjected into the yolk with don1 alone (a), the proteindecorated don (don1gv; b) or don1gv along with the reporter plasmid (c). Then, at 6 h postfertilization, the embryos were embedded in agarose and analyzed by using confocal fluorescence microscopy . Three channels were recorded: the brightfield image (i), the signal from the gfphistone h2 in green (ii), and the cy5 signal from the dons in red (iii). The highintensity signal in the red channel shows that the large dna constructs were internalized at a high rate from the yolk . In addition, the high magnification (bottom images) clearly indicates that the internalized dons are mostly cytosolic (orange arrow), with very little to no nuclear localization (white arrow). Note that the presence of neither the proteins nor the reporter plasmid influenced the internalization or the subcellular distribution of the dons . Scale bars: 30 m and 10 m for low and high magnification, respectively . The injected embryos survived and developed normally, suggesting that the introduced nanomaterial was well tolerated . This result shows that don1 has no obvious deleterious effects on early embryonic development and is indeed efficiently internalized from the yolk (figure 4 a, panel iii). However, comparison of the nuclei position (gfp signal, panel ii) with the don fluorescent signal (panel iii) indicates that the don localizes mainly to the cytosol . Gv injected alone or together with the reporter plasmid (figures 4 b and 4 c, respectively). The don gv also had no obvious effect on early embryonic development and was internalized by the cells . Furthermore, it was only detected in the cytosol, irrespective of the presence of the reporter plasmid (figures 4 b and 4 c). Gv had no effect on the expression of the reporter gene, as compared to the don1 (data not shown). Spherical particles with a diameter below 26 nm freely diffuse into the nucleus, whereas larger particles require a nuclear localization signal (nls) to actively translocate through the nuclear pore complex, with an upper size limit of about 39 nm.34 the gal4dbd used to functionalize the don gv harbors an intrinsic nuclear localization signal.35 however, it was apparently not sufficient to drive the don gv to the nucleus . Recently, it was shown that functionalization with an nls increases the nuclear uptake of 510 nm100300 nm rodshaped polymeric gv is unlikely because of its size, but rather to a low efficiency of the gal4dbd nls in the context of the construct . Stronger nls as a peptide covalently attached to the don might increase its nuclear uptake . We report here on an ambitious concept for controlling gene transcription in a distancedependent manner in the developing zebrafish embryo, taking advantage of designed protein dna origami nanostructures . Although we could clearly demonstrate the successful synthesis of our target construct, applications in vivo require further developments . Nonetheless, we believe that our study represents an important contribution to the exploitation of dons for the analysis and manipulation of cells in vivo . We demonstrated, for the first time, that microinjection in the yolk of the zebrafish embryo enables intracellular distribution in most of the cells of large origami constructs harboring active proteins precisely arranged in a 3d fashion . Furthermore, we demonstrated that the don constructs are not toxic for the embryos and allowed normal development, at least in the early stages . We foresee that this technology could be applied to deliver complex multiprotein constructs to the cytosol and maybe also to the nuclei by means of strong nuclear localization signals . As a service to our authors and readers, this journal provides supporting information supplied by the authors . Such materials are peer reviewed and may be reorganized for online delivery, but are not copyedited or typeset . Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.
Visfatin, also known as nicotinamide phosphoribosyltransferase (nampt) as well as pre - b - cell colony - enhancing factor, is a multifaceted protein with suggested enzymatic, immunological, and metabolic properties . Visfatin has been analyzed in hypo- and hyperthyroidism in in vitro and in vivo studies, but results are inconclusive . In addition, nampt level was found to be elevated in many autoimmune diseases, that is, rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel diseases, and psoriasis [25]. Visfatin also positively correlates with activity and severity of rheumatoid arthritis and psoriasis [2, 5]. We have recently found an overexpression of nampt in leukocytes of patients with graves' ophthalmopathy with corresponding increased serum concentration (accepted manuscript). Our findings suggest that visfatin might be involved in autoimmune processes in thyroid diseases . In our opinion, the controversial findings of visfatin in thyroid hormone deficiency may arise from the heterogeneity of thyroid dysfunction . We hypothesized that regulation of visfatin in hypothyroidism might be altered by coexisting chronic autoimmune thyroiditis, since high visfatin levels were observed in other autoimmune diseases . To answer the question about the influence of coexisting chronic autoimmune inflammation on visfatin level, we analyzed its serum concentration among hypothyroid patients with chronic autoimmune thyroiditis and in patients after thyroidectomy, who were negative for thyroid antibodies . This is a prospective case - control study of 118 subjects . The autoimmune study group (ait) consisted of 39 patients newly diagnosed with hypothyroidism in a course of chronic autoimmune thyroiditis . The nonautoimmune study group (tt) consisted of 40 patients thyroidectomized due to the differentiated thyroid cancer staged pt1 . Tt patients were evaluated five years after radioiodine remnant ablation and were negative for thyroglobulin and radioiodine uptake in a whole body scintigraphy (wbs). They achieved endogenous tsh stimulation and became hypothyroid after l - t4 withdrawal for at least 4 weeks . The control group comprised 39 healthy volunteers adjusted for age, sex, and bmi with normal thyroid function and negative thyroid antibodies . Exclusion criteria consisted of other autoimmune diseases, active neoplastic disease, diabetes mellitus, and infection, which were reported to alter visfatin level . Fasting blood samples were taken for visfatin, tsh, free thyroxine (ft4), free triiodothyronine (ft3), antithyroperoxidase antibodies (tpoab), antithyroglobulin antibodies (tgab), glucose, and insulin levels . In tt group tsh, ft4, and ft3 were measured using the electrochemiluminescence technique in cobas e 601 (norm ranges: tsh 0.274.2 tpoab and tgab were measured by radioimmunoassay (norm range: <34 iu / ml and <115 iu / ml, resp . ). Glucose level was assessed with the use of hitachi cobas e601 chemiluminescent analyzer (roche diagnostics) and insulin concentration was assessed using elisa kit from phoenix pharmaceuticals . The estimate of insulin resistance by homeostasis model assessment (homa - ir) was calculated . The study was approved by the local ethics committee, and informed consent was signed by every subject . Statistical analysis was performed with medcalc version 12.1.3.0 (medcalc software, mariakerke, belgium). Variables with normal distribution were compared between three groups with one - way analysis of variance . If data did not follow normal distribution, comparison of the analyzed parameters between three groups was performed with the kruskal - wallis test . Simple regression analysis was used to test for the relationships between them . Before inclusion to this statistical analysis, furthermore, stepwise multiple regression analysis was employed to investigate the influence of various parameters on visfatin serum concentration [age, bmi, ft3, autoimmunity (yes / no), homa - ir]. Variables were entered into the model if their associated p - values were less than 0.05 and then sequentially removed if their associated p - values became greater than 0.2 . Tests were considered to be statistically significant if p - value was lower than 0.05 . Clinical and laboratory data of the study groups and the control group are shown in table 1 . The highest visfatin serum concentration was in ait group, and healthy controls had visfatin level higher than tt (p = 0.0001) (figure 1). Three groups did not differ in age, sex, bmi, fasting glucose, and insulin levels, homa - ir . They had statistically different tsh, ft4, ft3, and tgab levels (table 1). Simple linear regression analysis revealed that visfatin serum concentration was significantly associated with autoimmunity (= 0.1014; p = 0.003), ft4 (= 0.05412; p = 0.048), ft3 (= 0.05242; p = 0.038), and tpoab (= 0.0002; p = 0.0025) (figure 2). There was no association between visfatin and age, sex, bmi, tsh, tgab, fasting insulin and glucose levels, and homa - ir (table 2). In the stepwise multiple regression analysis we confirmed the association between serum visfatin level and autoimmunity (coefficient = 3.8461; p = 0.0001), and ft3 (coefficient = 0.4198; p = 0.0441), whereas age, bmi, and homa - ir did not contribute significantly . In separate stepwise multiple regression analysis we confirmed the association of serum visfatin concentration with autoimmunity (coefficient = 4.1105; p = 0.0001) and ft4 (coefficient = 0.1397; p = 0.038), whereas age, bmi, and homa - ir did not enter the model . Similarly, association of visfatin with tpoab (coefficient = 0.0057; p = 0.0163) was observed with adjustment for age, bmi, ft3, and homa - ir in multivariate regression analysis . To date, visfatin serum concentration in hypothyroidism has been analyzed in a few studies . Reported elevated level of this adipocytokine in hypothyroidism with further increase after restoration of thyroid function . Ozkaya et al . Observed that visfatin level decreased after recovery . In those articles the etiology of hypothyroidism varied from chronic autoimmune thyroiditis, postpartum thyroiditis to thyroid function insufficiency after radioiodine treatment or after thyroidectomy . Hence, to date autoimmune status of studied patients has not been taken into consideration . We hypothesized that these controversial findings might result from heterogeneity of study groups . To answer the question, whether coexisting autoimmune inflammation influences visfatin level in hypothyroid patients, we analyzed its serum concentration in chronic autoimmune thyroiditis and thyroidectomized patients negative for thyroid antibodies . Since we have previously proved that visfatin mrna expression is increased in thyroid malignancies and is correlated with tumor stage, we recruited only those patients who did not have any features of active neoplastic disease . We also recruited the control group adjusted for age, sex, and bmi . To the best of our knowledge, this is the first study addressing the changes in the release of visfatin in thyroid autoimmunity . We came to interesting results indicating that visfatin serum concentration in hypothyroid patients is associated with both autoimmunity and free thyroid hormones level (ft4, ft3). Visfatin has been recognized as a cytokine with a broad range of immune and inflammatory activities, including induction of inflammatory cytokines, and regulation of macrophage and lymphocyte proliferation . Visfatin stimulates the production of proinflammatory cytokines (il-6, tnf-, and il-1) and potentially acts as a chemotactic factor for monocytes . Furthermore, its expression is upregulated by il-6, tnf-, and il-1 [1012]. Enhanced mrna expression of visfatin was observed in inflamed mucosa of patients with inflammatory bowel disease (ibd). Further analysis identified that antigen presenting cells (i.e., macrophages, dendritic cells) might be a main source of this protein in ibd . Visfatin has also potency for activation of t cells by upregulation of costimulatory molecules (cd40, cd54, and cd80) on monocytes . We observed the positive association between visfatin and tpoab, and the latter is considered the best serological marker of chronic autoimmune thyroiditis . Furthermore, tpoab contribute to thyroid destruction through antibody- and complement - dependent cell - cytotoxicity [13, 14]. The first mechanism is associated with mononuclear cell infiltration of thyroid stroma . In addition, th1-derived cytokines (il-2, tnf-, and inf) were found to be elevated in patients with chronic autoimmune thyroiditis . Also, il-1 and tnf- have been recently reported to discriminate chronic autoimmune hypothyroid children from healthy controls . Altogether, association of visfatin with tpoab observed in our study further supports our hypothesis that visfatin might be involved in the pathogenesis of chronic autoimmune thyroiditis . Our findings about the association of visfatin with ft3 levels are in accordance with the results of in vitro and in vivo studies . However, controversial results whether t3 stimulates or downregulates the production of visfatin were found . In contrast, caixs et al . Did not find any relationship between visfatin and free thyroid hormones . In vitro experiment showed the nonlinear regulation of visfatin mrna expression in the 3t3-l1 cell culture model affected by t3 . Since our study groups significantly differed with free thyroid hormones levels, we were able to analyze visfatin concentration in a broad spectrum of ft3 and ft4 . Therefore, we might suggest that pattern of visfatin changes varies in different ft3 concentration . According to our observations, as well as other authors, visfatin did not reflect insulin resistance assessed by homa - ir [18, 19]. Our study might then prove that visfatin in hypothyroidism depends on thyroid hormones level and coexisting autoimmunity . We may assume that these two factors should be taken into consideration to assess visfatin level in patients with thyroid dysfunction . In addition, the possible involvement of visfatin in pathogenesis of chronic autoimmune thyroiditis needs further research . The main limitation of our study is its cross - sectional design that does not enable us to reveal the causal pathways of relationship between visfatin and autoimmune thyroiditis . However, we used very strict criteria of exclusion to limit the possible influence of other known factors such as diabetes mellitus, other autoimmune processes, infection, and active neoplastic disease . Our nonautoimmune group with hypothyroidism had been thyroidectomized at least 5 years earlier and did not have any clinical nor laboratory features of active thyroid cancer.
Diastolic heart failure (dhf) is a clinical syndrome characterized by the symptoms and signs of heart failure, a preserved ejection fraction and abnormal diastolic function . The percentage of patients with dhf in epidemiological studies ranges from 4071% (mean 56%), but in hospital - based cohort studies it is slightly lower, ranging from 2455% (mean 41%). Elderliness, hypertension with left ventricular (lv) hypertrophy, pathologies such as diabetes, obesity, coronary artery disease, new onset atrial fibrillation, and others are commonly associated with dhf [24]. Cardiotrophin-1 (ct-1) is a member of the interleukin (il)-6 cytokine family that shares the transmembrane signaling protein, glycoprotein (gp) 130, as a receptor . Ct-1 mrna is expressed in adult human heart, skeletal muscle, ovary, colon, prostate and testis and in fetal kidney and lung . Ct-1 has hypertrophic and cytoprotective actions on the cardiac myocytes and may play an important role in other organ systems . The plasma concentration of ct-1 is increased in various cardiovascular diseases such as congestive heart failure, hypertension, valvular heart disease, acute coronary syndrome, and cardiomyopathies [818]. Although, the prognostic importance of ct-1 in various cardiovascular diseases, including congestive heart failure, is well - known, there is limited data about ct-1 in patients with dhf . The purpose of the present study was to determine if ct-1 levels are significantly different in dhf patients compared to controls and to investigate the relationship between ct-1 and echocardiographic parameters . Fifty - seven consecutive patients (mean age 578 years, 24 (42%) males) diagnosed with dhf in our clinic and 33 controls (mean age 557 years, 12 (36%) males) were included in the study . Dhf was diagnosed when symptoms (dyspnea not associated with any other cause) and signs (rale or peripheral edema) of heart failure were observed along with a preserved lv ejection fraction (lvef) (50%) and evidence of diastolic dysfunction . The control group was formed from volunteer subjects admitted to our clinic who did not have heart failure symptoms and signs and who had a preserved lvef . In order to exclude other causes of dyspnea, all patients underwent physical and laboratory examinations, including serum hemoglobin and thyroid hormones, chest radiogram and spirometry . Patients with systolic heart failure, moderate or severe valvular stenosis or regurgitation, congenital heart disease, atrial fibrillation, chronic obstructive pulmonary disease, malignancy, and other extracellular fluid - increasing diseases, such as hypothyroidism and liver cirrhosis, were excluded from the study . All of the study participants underwent echocardiographic evaluation (2.5 mhz transducer, philips envisor c, bothell, washington, usa). Standardized projections and measurements were performed for the evaluation of cardiac anatomy, ventricular function and valve competence . Lv mass index was obtained by dividing the lv mass by the body surface area . The following conventional mitral inflow pulse wave doppler parameters were measured: peak velocity of early diastolic filling (e), late filling (a), and deceleration time (dt) of the e - wave velocity and isovolumetric relaxation time (ivrt). These parameters were obtained from the apical four - chamber view with a 1 to 3 mm sample volume placed between the mitral leaflet tips during diastole . Pulmonary venous flow parameters were also measured: peak systolic velocity (ps), peak anterograde diastolic velocity (pd) and the ps / pd ratio . These parameters were obtained from the apical four - chamber view with a 2 to 3 mm sample volume placed 1 cm into the pulmonary vein . Tissue doppler parameters were measured: peak systolic mitral annular velocity (sm) and early diastolic mitral annular velocity (em) and late diastolic mitral velocity (am). These parameters were obtained from the apical four - chamber view with a 2 to 5 mm sample volume placed 1 cm within the septal and lateral insertions of the mitral leaflets . The mean of 3 or more measurements was used for analysis of the doppler data . The ratio of mitral peak velocity of early diastolic filling to early diastolic mitral annular velocity (e / em) was calculated for the lateral and septal annulus and the mean of the lateral and septal e / em were also determined . As previously described, the formula (1.24(e / em)+1.9) was used to estimate pulmonary capillary wedge pressure (pcwp). Patients with mean e / em 8 or a change in e / a ratio with the valsalva maneuver of <0.5 were excluded from the study . Diastolic dysfunction was defined as em <am if em was less than 10 cm / sec in lateral mitral annulus or less than 8 cm / sec in septal mitral annulus . Blood samples were obtained during admission for routine chemistry, including ct-1 and n - terminal pro - b - type natriuretic peptide (nt - probnp) following an overnight fast . Ct-1 values were measured with a sandwich enzyme immunoassay method (organon teknika microwell system reader 230 s, germany) in our hospital laboratory . Nt - probnp analyses were made by the electrochemiluminescence immunoassay method (cobas 6000 analyzer, roche diagnostics gmbh, mannheim, germany). The cockcroft - gault formula was used to calculate creatinine clearance . According to kolmogorov - smirnov normality test, 2 independent sample t tests were used to compare the normally distributed independent variables between the 2 groups, and mann - whitney u test was used to compare the non - normally distributed independent variables between the 2 groups . Normally distributed continuous data were expressed as mean standard deviation (sd); non - normally distributed continuous variables were presented as median and interquartile range (iqr) [quartile l to quartile 3]. Linear regression analyses were used to determine the effect of age, creatinine clearance, systolic blood pressure, left atrium diameter, and lv mass index on log ct-1 and log nt - probnp . Statistical analysis was performed by using commercial software (ibm spss statistics 19, spss inc . Fifty - seven consecutive patients (mean age 578 years, 24 (42%) males) diagnosed with dhf in our clinic and 33 controls (mean age 557 years, 12 (36%) males) were included in the study . Dhf was diagnosed when symptoms (dyspnea not associated with any other cause) and signs (rale or peripheral edema) of heart failure were observed along with a preserved lv ejection fraction (lvef) (50%) and evidence of diastolic dysfunction . The control group was formed from volunteer subjects admitted to our clinic who did not have heart failure symptoms and signs and who had a preserved lvef . In order to exclude other causes of dyspnea, all patients underwent physical and laboratory examinations, including serum hemoglobin and thyroid hormones, chest radiogram and spirometry . Patients with systolic heart failure, moderate or severe valvular stenosis or regurgitation, congenital heart disease, atrial fibrillation, chronic obstructive pulmonary disease, malignancy, and other extracellular fluid - increasing diseases, such as hypothyroidism and liver cirrhosis, were excluded from the study . All of the study participants underwent echocardiographic evaluation (2.5 mhz transducer, philips envisor c, bothell, washington, usa). Standardized projections and measurements were performed for the evaluation of cardiac anatomy, ventricular function and valve competence . Lv mass index was obtained by dividing the lv mass by the body surface area . The following conventional mitral inflow pulse wave doppler parameters were measured: peak velocity of early diastolic filling (e), late filling (a), and deceleration time (dt) of the e - wave velocity and isovolumetric relaxation time (ivrt). These parameters were obtained from the apical four - chamber view with a 1 to 3 mm sample volume placed between the mitral leaflet tips during diastole . Pulmonary venous flow parameters were also measured: peak systolic velocity (ps), peak anterograde diastolic velocity (pd) and the ps / pd ratio . These parameters were obtained from the apical four - chamber view with a 2 to 3 mm sample volume placed 1 cm into the pulmonary vein . Tissue doppler parameters were measured: peak systolic mitral annular velocity (sm) and early diastolic mitral annular velocity (em) and late diastolic mitral velocity (am). These parameters were obtained from the apical four - chamber view with a 2 to 5 mm sample volume placed 1 cm within the septal and lateral insertions of the mitral leaflets . The mean of 3 or more measurements was used for analysis of the doppler data . The ratio of mitral peak velocity of early diastolic filling to early diastolic mitral annular velocity (e / em) was calculated for the lateral and septal annulus and the mean of the lateral and septal e / em were also determined . As previously described, the formula (1.24(e / em)+1.9) was used to estimate pulmonary capillary wedge pressure (pcwp). Patients with mean e / em 8 or a change in e / a ratio with the valsalva maneuver of <0.5 were excluded from the study . Diastolic dysfunction was defined as em <am if em was less than 10 cm / sec in lateral mitral annulus or less than 8 cm / sec in septal mitral annulus . Blood samples were obtained during admission for routine chemistry, including ct-1 and n - terminal pro - b - type natriuretic peptide (nt - probnp) following an overnight fast . Ct-1 values were measured with a sandwich enzyme immunoassay method (organon teknika microwell system reader 230 s, germany) in our hospital laboratory . Nt - probnp analyses were made by the electrochemiluminescence immunoassay method (cobas 6000 analyzer, roche diagnostics gmbh, mannheim, germany). According to kolmogorov - smirnov normality test, 2 independent sample t tests were used to compare the normally distributed independent variables between the 2 groups, and mann - whitney u test was used to compare the non - normally distributed independent variables between the 2 groups . Normally distributed continuous data were expressed as mean standard deviation (sd); non - normally distributed continuous variables were presented as median and interquartile range (iqr) [quartile l to quartile 3]. Linear regression analyses were used to determine the effect of age, creatinine clearance, systolic blood pressure, left atrium diameter, and lv mass index on log ct-1 and log nt - probnp . Statistical analysis was performed by using commercial software (ibm spss statistics 19, spss inc . There were no significant differences between the patient and control groups with regard to age, sex, hypertension, diabetes, cad, smoking, medications, body mass index, fasting blood glucose, thyroid status, lipid profile, creatinine clearance, serum creatinine and hemoglobin levels (table 1). The patient group had a higher lv posterior wall thickness and a larger left atrial size, but differences in lvef, chamber sizes, and mass index remained insignificant (tables 1 and 2). Arterial blood pressures were also not different between the 2 groups . Ct-1 and nt - probnp were significantly higher in the patient group (ct-1: 11.30 [8.0916.51] fmol / ml vs. 17.5 [8.9528.74] fmol / ml, p=0.017; nt - probnp: 64 [27.595] pg / ml vs. 82 [55.5241] pg / ml, p=0.009) (table 1). The mitral e, lateral, septal and mean e / em and the pcwp estimated from each of the e / em measurements were all significantly higher, whereas ps / pd, lateral and septal em and lateral and septal em / am were significantly lower in the patient group (table 2). Ct-1 positively correlated with nt - probnp (p=0.001, r=0.349), mean e / em (p=0.003, r=0.307), and estimated mean pcwp (p=0.001, r=0.308) (figure 1). According to bivariate linear regression analyses, log ct-1 was predicted by left atrium diameter and log nt - probnp was predicted by age, creatinine clearance, and left atrium diameter (table 3). In multiple linear regression analysis, log ct-1 and log nt - probnp were predicted only by left atrium diameter (table 4). The data of this study shows that the ct-1 is elevated in dhf patients and is associated with nt - probnp and estimated lv filling pressures in dhf patients . The ct-1 protein expression is constitutive not only in the heart, but also in the pulmonary, renal, gastrointestinal, cerebral, and muscular tissues . Cardiac myocytes and cardiac fibroblasts may produce ct-1 in situations of biomechanical stress and under exposure to humoral factors such as angiotensin ii and norepineprine [2325]. Ventricular stretch caused by pressure or volume overload is thought to be the major stimulus of myocardial ct-1 release . Previous studies have found that ct-1 promotes myocardial structural changes, later participating in the progression of lv remodeling, which results in lv failure in various diseases such as hypertensive heart disease, coronary artery disease, aortic stenosis, and dilated cardiomyopathy . Both bnp and ct-1 have a beneficial effect not only on the myocardium but also on hemodynamic variables . Therefore, ongoing stimulation of bnp and ct-1 caused by ventricular stretch and circulating cytokines, promote structural remodeling and may become maladaptive with the progression of heart failure . Ct-1 levels correlate with the severity of the heart failure and has been shown to be an independent predictor of mortality in chronic heart failure . Plasma ct-1 has high diagnostic efficacy for heart failure (at concentration of 68 fmol / ml, sensitivity and specificity were 95% and 82.5%, respectively). Talwar et al found that plasma ct-1 levels measured shortly after an acute myocardial infarction serve as a strong independent predictor of lv systolic dysfunction . In another study, talwar et al . Found that changes in ct-1 levels may reflect early changes in ventricular physiology that occur in the early part of the heart failure process before they can be detected echocardiographically . It was also found that ct-1 is significantly increased in patients with moderate to severe mitral regurgitation despite having normal lv systolic function and no hypertrophy . They were unable to demonstrate a similar increase in subjects with tricuspid or aortic regurgitation . In our study, we showed ct-1 elevation in a group of dhf patients with normal lv systolic function and no significant hypertrophy, but having more dilated left atrium than controls . Considering these results, it may be speculated that left atrial wall stretch is a more important and earlier stimulation for ct-1 secretion rather than ventricular wall stretch . It has already been shown that ct-1 mrna was detected in both atria and ventricles . In a series of studies performed by lopez et al, they concluded that ct-1 is a more sensitive and specific biomarker than nt - pro bnp for detection of the inappropriateness of lv mass and lv dysfunction in hypertension, and nt - pro bnp remains a useful diagnostic tool for hypertensive heart disease only when lv systolic dysfunction is present . It was also shown that elevated ct-1 levels represent an earlier stage of the same neurohumoral cascade that results in elevated plasma bnp ., both ct-1 and nt - pro bnp were found to be more elevated in dhf patients than in control subjects, and there was a positive correlation between ct-1 and nt - pro bnp . Previous studies show that elevated nt - probnp values is diagnostic for dhf and associated with elevated lv filling pressures . It has been found that there is a strong correlation between nt - probnp and e / em, and a threshold of 269.1 pg / ml of nt - probnp predicted an e / em> 15 with 90% sensitivity and 73% specificity in dhf . The positive correlation between elevated ct-1 and nt - probnp, e / em and pcwp in our study implies that pressure overload represented as increases in lv filling pressures could be the main underlying mechanism for ct-1 secretion in patients with dhf . A series of studies performed by lopez et al concluded that ct-1 is associated with systolic and diastolic dysfunction in hypertensive patients . In the other study they found that the ratio of peak velocity of early diastolic filling to peak velocity of late filling of mitral inflow (e / a) is the only parameter that differed in the hypertensive group, while dt and ivrt remained unchanged . Significant association was found between normalization of ct-1 and regression of lv hypertrophy and increment of e / a . In their studies they only used conventional doppler parameters in order to evaluate diastolic function, and e / a was the only parameter found to be different from the control group . In our study chronic ct-1 treatment resulted in the development of insulin resistance as judged by a decrease in insulin - stimulated glucose uptake . We found high glucose levels in dhf patients, but this difference was insignificant, probably due to small sample size . Second, despite the evidences that e / em is an effective noninvasive predictor of lv filling pressures, we did not measure lv pressures directly . It was already known that adipose tissue can be recognized as a source of ct-1, which could account for the high circulating levels of ct-1 in patients with metabolic syndrome . Finally, ct-1 mrna is expressed in other organs, and potential additional sources of circulating ct-1 cannot be excluded in patients with dhf . This increase was associated with nt - probnp and estimated lv filling pressures in dhf patients . Our results suggest that diastolic dysfunction and subsequent pressure increase at the left side of the heart may be responsible for ct-1 increase in dhf patients . However, further studies are required to elucidate the underlying mechanism for the ct-1 increase in these patients.
Helicobacter pylori is a stomach bacterium that colonizes ~50% of people globally.1 h. pylori is the primary risk factor for gastric cancer the third highest cause of global cancer morbidity.2 h. pylori infection rates are highly dependent on socioeconomic status; ~80% of those living in low socioeconomic areas of latin america, asia, and eastern europe are infected, compared with <20% of asymptomatic caucasians in the usa.3 h. pylori infection is treatable with different regimens of antibiotics,4 and eradication of h. pylori is a recognized way to lower incidence of gastric cancer.5 however, recurrence of infection is variable,6,7 and the emergence of antibiotic resistance compromises treatment efficacy . Thus, determining the best course of treatment is important to improve treatment efficacy and to reduce recurrence of h. pylori infection . Unfortunately, there is no broad consensus about an optimal antibiotic therapy for the treatment of h. pylori . For example, meta - analyses of european and asian clinical data compared the standard triple therapy (amoxicillin, clarithromycin, and a proton - pump inhibitor for 714 days) with 5- or 10-day quadruple therapy regimens (adding metronidazole or tinidazole to the triple therapy) and found that quadruple therapies are both significantly more effective and cheaper than the triple therapy.810 however, we previously published a study comparing eradication therapies in seven sites of six latin american countries that showed that the 14-day triple therapy was superior to the 5-day concomitant quadruple therapy, and no different than the 10-day sequential quadruple therapy.11,12 these inconsistencies reflect localized differences in antibiotic use practices, such as the use of clarithromycin for upper respiratory infections.13 the differences in efficacy of antibiotic therapy are supported by primary antibiotic resistance data . For example, h. pylori resistance to amoxicillin varied widely between africa (65.6%), europe (0.5%), asia (11.6%), and the americas (2.2%).12 even in the same region, patterns of resistance differ: within central and latin america, reported average metronidazole resistance varies from 30% in argentina to 83% in columbia, and tetracycline resistance varies from 2% in brazil to 33% in columbia.14 as such, characterizing local resistance patterns is important for selecting therapies with the highest likelihood of success . Our research focused on peru, where gastric cancer is the leading cancer killer in men and women combined.15 thus, we searched the literature for reports of primary antibiotic resistance to h. pylori in peru . Three studies were identified, which reported 36.9% resistance to levofloxacin,16 an average of 66% resistance to metronidazole,17,18 50% resistance to clarithromycin,17 and 0% resistance to tetracycline.17 unfortunately, there were no data on amoxicillin, and the reported results of other antibiotics were based on small sample sizes, so whether their results are generalizable is unknown . As successful eradication of h. pylori infection is an important step toward prevention of gastric carcinoma,5,19 we assessed primary h. pylori antibiotic resistance among 76 isolates from a cohort of patients recruited in lima, peru, measuring resistance to metronidazole, amoxicillin, tetracycline, clarithromycin, levofloxacin, and rifampicin to cover the gamut of antibiotics used from initial through second- and third - line therapies.11,20 our data showed significant primary antibiotic resistance to first- and second - line antibiotics among h. pylori isolates from a clinical setting in lima, peru.21 the study protocol was approved by the ethics committee of the universidad peruana cayetano heredia in lima, peru, and the institutional review board of the university of michigan in ann arbor, mi, usa . The cohort of patients from whom h. pylori isolates were obtained has been previously described.21 all experiments were conducted under the registered clinical trial gob nct015128, and swog clinical trial s1119 . Briefly, patient recruitment occurred between september 2011 and august 2013 at the clinical facilities of the universidad peruana cayetano heredia hospital in metropolitan lima . Signed, informed patient consent for procedures, antibiotic treatment, follow - up, and downstream molecular analyses were obtained before enrollment in the trial . Study participants were aged 2070 years and had symptoms of dyspepsia for at least 6 months . Six biopsies per patient were obtained: four for histologic studies and two for culture, which were stored in 1.5 ml of 1 phosphate - buffered saline (pbs) with 20% glycerol at 80c until processing . Following endoscopy, infected patients were treated with esomeprazole, amoxicillin, and clarithromycin twice a day for 14 days . Patients were followed up 1 year after treatment to check for h. pylori infection status via the urea breath test.21 gastric biopsy samples were thawed on ice and then homogenized using omni probes at maximum speed (omni international, kennesaw, ga, usa). A volume of 50 l of the homogenized sample was plated onto both 5% sheep s blood tryptic soy agar plates (remel, columbus, oh, usa) and on h. pylori selective media (columbia blood agar base with 10% horse blood, 10 mg / l vancomycin, 5 mg / l trimethoprim, 5 mg / l cefsulodin, 5 mg / l amphotericin b, 300 mg / l urea, and 3500 u polymyxin b / l).22 plates were incubated at 37c in microaerobic conditions for 37 days . Presumptive h. pylori isolates were subcultured, then confirmed by morphology and checked for urease activity using a urease indicator broth (0.33 m urea, 0.2% phenol red, 0.02% nan3, 0.01 m napo4 buffer [ph 6.5]). Glycerol stocks of each isolate were prepared in brucella broth (remel) with 15% glycerol . H. pylori isolated dna was tested for the presence of h. pylori caga and vaca genes by polymerase chain reaction (pcr) using previously described primers and the takara pcr kit (clontech, mountain view, ca, usa). For caga, primers f1 (5-gataacaggcaagcttttgagg-3) and b1 (5-ctgcaaaagattgtttggcaga-3) were used to amplify a 349 base pair product.23 primers vag - f (5-caatctgtccaatcaagcgag) and vag - r (5-gcgtcaaaataattccaagg) were used to amplify the m1/m2 subunits of the vaca gene, yielding a 570 or 645 base pair product.24 pcr products were visualized on a 1.5% agarose gel . Using a protocol adapted from the university of michigan health system clinical microbiology laboratory and biomrieux s (durham, nc, usa) instructions, h. pylori isolates were tested for susceptibility to amoxicillin, clarithromycin, levofloxacin, metronidazole, rifampicin, and tetracycline using e - test . Isolates were subcultured and then grown on mueller hinton agar supplemented with 5% sheep s blood (remel, columbus, ohio, usa). Colonies were collected and suspended in 1 pbs and visually compared with a 3.0 mcfarland turbidity standard . Cell suspensions were then spread on mueller hinton agar with 5% sheep s blood (mh) and stored for 15 minutes in microaerobic conditions, allowing the suspension to dry on the plate . Then, e - test strips were placed on the plates with sterile forceps, and the plates were incubated for 72 hours at 37c in microaerobic conditions . Results were interpreted per the european committee on antimicrobial susceptibility testing.24 the minimum inhibitory concentrations (mic) of amoxicillin, clarithromycin, levofloxacin, metronidazole, rifampicin, and tetracycline were measured according to the manufacturer s instructions . Atcc strain 43504 (h. pylori) and atcc 25922 (escherichia coli) were used as quality control strains . Atcc 43504 was prepared and treated in the same way as unknown isolates and was run simultaneously with each batch of isolates against clarithromycin, amoxicillin, metronidazole, and tetracycline . Atcc 25922 was plated from a glycerol stock 48 hours onto an mh plate before testing, and subcultured onto an mh plate 24 hours before testing . Colonies were suspended in 1 pbs to a visual density of a 0.5-mcfarland standard and were run simultaneously with each batch of isolates against levofloxacin and rifampicin . Qc results were typically within range, though interestingly, our reference strain of atcc 43504 was completely resistant to metronidazole, and consistently had tetracycline mics between 0.047 and 0.25 (slightly lower than usual). H. pylori plates were read after 72 hours of incubation in microaerobic conditions . Mics of strains were interpreted according to eucast (european committee on antimicrobial susceptibility testing) standards, which are based on epidemiological cut - off values, which distinguish wild - type isolates from those with reduced susceptibility.25 antibiotic resistance mics were examined using descriptive statistics . Student s t - tests were used to examine whether isolates with the m1 versus m2 subunit of the vaca gene were resistant to different numbers of antibiotics . The study protocol was approved by the ethics committee of the universidad peruana cayetano heredia in lima, peru, and the institutional review board of the university of michigan in ann arbor, mi, usa . The cohort of patients from whom h. pylori isolates were obtained has been previously described.21 all experiments were conducted under the registered clinical trial gob nct015128, and swog clinical trial s1119 . Briefly, patient recruitment occurred between september 2011 and august 2013 at the clinical facilities of the universidad peruana cayetano heredia hospital in metropolitan lima . Signed, informed patient consent for procedures, antibiotic treatment, follow - up, and downstream molecular analyses were obtained before enrollment in the trial . Study participants were aged 2070 years and had symptoms of dyspepsia for at least 6 months . Six biopsies per patient were obtained: four for histologic studies and two for culture, which were stored in 1.5 ml of 1 phosphate - buffered saline (pbs) with 20% glycerol at 80c until processing . Following endoscopy, infected patients were treated with esomeprazole, amoxicillin, and clarithromycin twice a day for 14 days . Patients were followed up 1 year after treatment to check for h. pylori infection status via the urea breath test.21 gastric biopsy samples were thawed on ice and then homogenized using omni probes at maximum speed (omni international, kennesaw, ga, usa). A volume of 50 l of the homogenized sample was plated onto both 5% sheep s blood tryptic soy agar plates (remel, columbus, oh, usa) and on h. pylori selective media (columbia blood agar base with 10% horse blood, 10 mg / l vancomycin, 5 mg / l trimethoprim, 5 mg / l cefsulodin, 5 mg / l amphotericin b, 300 mg / l urea, and 3500 u polymyxin b / l).22 plates were incubated at 37c in microaerobic conditions for 37 days . Presumptive h. pylori isolates were subcultured, then confirmed by morphology and checked for urease activity using a urease indicator broth (0.33 m urea, 0.2% phenol red, 0.02% nan3, 0.01 m napo4 buffer [ph 6.5]). Glycerol stocks of each isolate were prepared in brucella broth (remel) with 15% glycerol . H. pylori isolated dna was tested for the presence of h. pylori caga and vaca genes by polymerase chain reaction (pcr) using previously described primers and the takara pcr kit (clontech, mountain view, ca, usa). For caga, primers f1 (5-gataacaggcaagcttttgagg-3) and b1 (5-ctgcaaaagattgtttggcaga-3) were used to amplify a 349 base pair product.23 primers vag - f (5-caatctgtccaatcaagcgag) and vag - r (5-gcgtcaaaataattccaagg) were used to amplify the m1/m2 subunits of the vaca gene, yielding a 570 or 645 base pair product.24 pcr products were visualized on a 1.5% agarose gel . Using a protocol adapted from the university of michigan health system clinical microbiology laboratory and biomrieux s (durham, nc, usa) instructions, h. pylori isolates were tested for susceptibility to amoxicillin, clarithromycin, levofloxacin, metronidazole, rifampicin, and tetracycline using e - test . Isolates were subcultured and then grown on mueller hinton agar supplemented with 5% sheep s blood (remel, columbus, ohio, usa). Colonies were collected and suspended in 1 pbs and visually compared with a 3.0 mcfarland turbidity standard . Cell suspensions were then spread on mueller hinton agar with 5% sheep s blood (mh) and stored for 15 minutes in microaerobic conditions, allowing the suspension to dry on the plate . Then, e - test strips were placed on the plates with sterile forceps, and the plates were incubated for 72 hours at 37c in microaerobic conditions . Results were interpreted per the european committee on antimicrobial susceptibility testing.24 the minimum inhibitory concentrations (mic) of amoxicillin, clarithromycin, levofloxacin, metronidazole, rifampicin, and tetracycline were measured according to the manufacturer s instructions . Atcc strain 43504 (h. pylori) and atcc 25922 (escherichia coli) were used as quality control strains . Atcc 43504 was prepared and treated in the same way as unknown isolates and was run simultaneously with each batch of isolates against clarithromycin, amoxicillin, metronidazole, and tetracycline . Atcc 25922 was plated from a glycerol stock 48 hours onto an mh plate before testing, and subcultured onto an mh plate 24 hours before testing . Colonies were suspended in 1 pbs to a visual density of a 0.5-mcfarland standard and were run simultaneously with each batch of isolates against levofloxacin and rifampicin . Qc results were typically within range, though interestingly, our reference strain of atcc 43504 was completely resistant to metronidazole, and consistently had tetracycline mics between 0.047 and 0.25 (slightly lower than usual). Mics of strains were interpreted according to eucast (european committee on antimicrobial susceptibility testing) standards, which are based on epidemiological cut - off values, which distinguish wild - type isolates from those with reduced susceptibility.25 antibiotic resistance mics were examined using descriptive statistics . Student s t - tests were used to examine whether isolates with the m1 versus m2 subunit of the vaca gene were resistant to different numbers of antibiotics . Seventy - six h. pylori strains were isolated from the gastric biopsies and were tested for primary antibiotic resistance (table 2). Metronidazole was the antibiotic to which isolates were most commonly resistant (61.8%), while isolates showed least resistance to tetracycline (3.9%). About one - third of isolates were resistant to either clarithromycin or amoxicillin, which are typically used for the standard triple therapy, and 10.5% were resistant to both (table 3). Further, 40.1% of strains were resistant to> 3 of the tested antibiotics . By pcr, all 76 strains were positive for the caga pathogenicity island, 57 (75%) were positive for vaca m1 and 19 (25%) were positive for vaca m2 . No differences were seen between the presence of vaca m1/m2 and the mean number of antibiotics to which isolates were resistant . By pcr, all 76 strains were positive for the caga pathogenicity island, 57 (75%) were positive for vaca m1 and 19 (25%) were positive for vaca m2 . No differences were seen between the presence of vaca m1/m2 and the mean number of antibiotics to which isolates were resistant . To our knowledge, this is the first study characterizing h. pylori primary antibiotic resistance to amoxicillin and rifampicin in peru . When comparing our study results to published studies, we found that the mic cutoffs were inconsistent between studies . Using a mini - well agar dilution method to determine antibiotic resistance, vasquez et al used a clarithromycin mic of 0.125 and a metronidazole mic of 4 mg / l,16 rather than the eucast cutoffs of 0.5 and 8, respectively . Our study showed comparable primary antibiotic resistance among h. pylori isolates to metronidazole, and slightly higher resistance to clarithromycin and levofloxacin.15,26 we conducted a brief meta - analysis compiling all primary antibiotic resistance data in peru from ours and other reports from the literature (table 4). This study demonstrates a high incidence of primary h. pylori antibiotic resistance in lima, peru, to antibiotics used in the standard triple therapy . Inference from our results is limited due to our small sample size and that our patient population is likely not generalizable to peru . However, we noted some important trends in our data and resulting meta - analysis . First, the small percentage of isolates resistant to tetracycline is worth examining in future studies to see if this trend holds . Second, virtually all clinical isolates tested were resistant to one or more of the antibiotics commonly used to treat this infection, including amoxicillin, clarithromycin, levofloxacin, and metronidazole . This may contribute to the lower than anticipated response to h. pylori therapy observed in other parts of latin america.11,26 this overall pattern of antibiotic resistance suggests that it may be worth considering treatment alternatives for h. pylori infection in lima, peru . We suggest that clinicians consider testing the antibiotic resistance profile of clinical isolates from patients with treatment - resistant infection as a way to guide their treatment decisions . An emerging appearance of h. pylori antibiotic resistance has also been reported from other parts of the world, including asia, europe, and the americas.12,1517,19,2628 this observation, coupled with reports of h. pylori reinfection after successful antibiotic treatment,6,7 makes h. pylori treatment more challenging . Meanwhile, gastric cancer remains one of the most common and most lethal cancers in men and women combined in peru.2,14 after accounting for emerging patterns of antibiotic resistance of h. pylori, it might be useful to reconsider present treatment practices while investigating new therapies and considering testing of h. pylori clinical isolates for antibiotic sensitivity in certain regions of the world, such as peru . We show high rates of primary antibiotic resistance to h. pylori clinical isolates in lima, peru . More studies are needed to confirm this finding to optimize the clinical treatment of h. pylori infection in peru.
Using in vivo time - lapse confocal and two - photon imaging of the visual center optic tectum in living larval zebrafish, we first monitored changes in the morphology of microglia under resting state . Similar to the typical morphological properties of resting microglia in the mouse cortex, resting microglia in zebrafish are also highly branched with dynamic processes ended with either stick - like or bulbous tips (fig . 1). These two differently shaped tips can be inter - converted as the process moves around . The bulbous ending forms rapidly through expansion from a stick - like ending, and stalls on the contacted neuronal soma for several minutes before gradually shrinking back again . The existence of resting microglia - neuron contact is further confirmed by using 3-dimensional reconstruction and transmission electron microscopy technique . Series images of a resting microglial cell acquired every 5 with in vivo time - lapse confocal imaging of the optic tectum in a 6-dpf tg(apo - e: egfp) zebrafish larva, in which egfp is specifically expressed in microglia . Images at different time points are shown in different colors . In previous studies, global increase or decrease of neural activity in vivo by using the gabaa receptor inhibitor bicuculline or the voltage - gated sodium channel blocker ttx, respectively, could oppositely regulate the dynamics of resting microglia . In zebrafish, we further proved that neural activity plays an instructive role in steering the motility of resting microglial processes and the formation of microglia - neuron contact . Using glutamate uncaging, we locally upregulated neuronal activity in a small brain region of the intact zebrafish larvae . Glutamate uncaging, a non - invasive approach, can efficiently induce a local increase of neural activity . We applied repetitive glutamate uncaging about 20 m away from microglial soma, and found that the processes of resting microglia can gradually navigate toward the uncaging side at about 10 min after the uncaging onset . Furthermore, more bulbous tips are formed at the uncaging side, indicating an elevated formation of microglia - neuron contact in responding to the local increase of neuronal activity in vivo . Different from the microglia in culture and mammalian brain slices, we found no ionotropic glutamate receptors expressed on microglia in the optic tectum of zebrafish larvae using in vivo whole - cell recording, excluding the possibility that uncaged glutamate directly acts on resting microglia . Importantly, the formation of resting microglial bulbous endings can also be regulated by natural sensory inputs, e.g., visual stimuli, which can globally increase neuronal activity in the optic tectum . However, after global downregulation of neural activity via pre - incubation of larvae with ttx, the number of bulbous endings is markedly decreased . All these results further confirm an instructive role of neuronal activity in regulating the dynamics of resting microglial processes and the formation of microglia - neuron contact . Having shown that local elevation of neuronal activity can induce the formation of microglial bulbous endings wrapping neuronal somata, we next asked what might be mechanisms by which neurons with high activity talk to resting microglia . We found that this process requires the membrane depolarization - activated pannexin-1 hemichannels on tectal neurons and the atp / p2 purinergic receptor signaling between neurons and microglia . Pannexin-1, a large pore - like hemichannel, is widely expressed in the central nervous system . The opening of pannexin-1 is gated by some cellular signals, such as membrane depolarization, intracellular calcium and so on . Small molecules, including atp, nad and pge2, can be released through these hemichannels . Using whole - mount in situ hybridization and in vivo whole - cell recording, we found that the functional pannexin-1 hemichannels are expressed in tectal neurons but not microglia . After impairing the function of pannexin channels by drug treatment or morpholino - mediated genetic downregulation, glutamate uncaging - induced orientated movement of microglial processes and formation of bulbous contacts were prevented . Similar results were observed when we applied the atp - hydrolyzing enzyme apyrase or the p2 purinergic receptor blocker suramin . Neuronal activity steers resting microglial processes by facilitating the redistribution of the cytoskeleton protein small rho gtpase rac . Rac is known to be required for the membrane protrusion and migration of many types of cells, including zebrafish germ cells . Using confocal fret imaging, we found that the rac fret intensity is significantly increased in microglial processes, especially at bulbous endings, at the uncaging side after repetitive glutamate uncaging . It is immediately followed by the oriented movement of microglial processes and the formation of bulbous endings . However, if we genetically inhibited endogenous rac activity in microglia, those neuronal activity - induced microglial morphological changes could not be observed . Taken together, we identify the underlying mechanisms by which neurons with high activity signal to resting microglia . Considering the facilitated formation of microglia - neuron contact induced by neuronal activity increase by simultaneously monitoring changes in both the morphology of microglial processes and ca activity of tectal neurons, we found that microglial processes preferentially contact with neurons that exhibit high levels of spontaneous activity before contact . We then mosaically overexpressed the human inward rectifier k channel kir2.1 (kir), which is used to reduce the excitability of neurons, and a non - conducting mutant version of kir2.1 (mkir) in tectal neurons . In comparison with mkir - expressing neurons, these results further indicate that resting microglia preferentially contact neurons with higher activities, consistent with the data obtained with glutamate uncaging . While accumulating evidence shows that resting microglia respond to neural activity, we surprisedly noticed that resting microglia can also in turn regulate neuronal activity . In the zebrafish optic tectum, we found that such resting microglia - neuron contact can downregulate both spontaneous and visually evoked activities in contacted neurons . Interestingly, the decreases in both the frequency and magnitude of spontaneous ca activity are positively correlated with the duration of microglia - neuron contact . What is the consequence on neuronal firing after loss of resting microglia? In response to two - photon laser - induced focal injury, some microglia quickly trans - locate to the injury site . Consistent with the speculation based on our previous findings, the spontaneous activity of neurons within microglia pre - existing territories is significantly elevated after the translocation of microglia to the injury site . Meanwhile, in other cases with the similar injury but no microglial translocation, the neuronal activity is not significantly changed . Thus, our study reveals a functional regulation of neuronal activity by resting microglia under physiological conditions, offering a new way for homeostatic regulation of neuronal activity . Collectively, our work brings forth and demonstrates a novel reciprocal regulation between resting microglia and neurons in vivo . We not only demonstrate an instructive role of neuronal activity in resting microglial motility, but also reveal, for the first time, a previously unappreciated function of microglia in homeostatic regulation of neuronal activity in the healthy brain (fig . 2). Considering the bi - directional modulation between neurons and microglia, this study also represents a new perspective in understanding the physiological function of resting microglia . Highly active neurons attract resting microglial processes and induce the formation of microglia - neuron contact via atp signaling (top). Such neuronal activity - driven microglia - neuron contact in turn using in vivo time - lapse confocal and two - photon imaging of the visual center optic tectum in living larval zebrafish, we first monitored changes in the morphology of microglia under resting state . Similar to the typical morphological properties of resting microglia in the mouse cortex, resting microglia in zebrafish are also highly branched with dynamic processes ended with either stick - like or bulbous tips (fig . 1). These two differently shaped tips can be inter - converted as the process moves around . The bulbous ending forms rapidly through expansion from a stick - like ending, and stalls on the contacted neuronal soma for several minutes before gradually shrinking back again . The existence of resting microglia - neuron contact is further confirmed by using 3-dimensional reconstruction and transmission electron microscopy technique . Series images of a resting microglial cell acquired every 5 with in vivo time - lapse confocal imaging of the optic tectum in a 6-dpf tg(apo - e: egfp) zebrafish larva, in which egfp is specifically expressed in microglia . In previous studies, global increase or decrease of neural activity in vivo by using the gabaa receptor inhibitor bicuculline or the voltage - gated sodium channel blocker ttx, respectively, could oppositely regulate the dynamics of resting microglia . In zebrafish, we further proved that neural activity plays an instructive role in steering the motility of resting microglial processes and the formation of microglia - neuron contact . Using glutamate uncaging, we locally upregulated neuronal activity in a small brain region of the intact zebrafish larvae . Glutamate uncaging, a non - invasive approach, can efficiently induce a local increase of neural activity . We applied repetitive glutamate uncaging about 20 m away from microglial soma, and found that the processes of resting microglia can gradually navigate toward the uncaging side at about 10 min after the uncaging onset . Furthermore, more bulbous tips are formed at the uncaging side, indicating an elevated formation of microglia - neuron contact in responding to the local increase of neuronal activity in vivo . Different from the microglia in culture and mammalian brain slices, we found no ionotropic glutamate receptors expressed on microglia in the optic tectum of zebrafish larvae using in vivo whole - cell recording, excluding the possibility that uncaged glutamate directly acts on resting microglia . Importantly, the formation of resting microglial bulbous endings can also be regulated by natural sensory inputs, e.g., visual stimuli, which can globally increase neuronal activity in the optic tectum . However, after global downregulation of neural activity via pre - incubation of larvae with ttx, the number of bulbous endings is markedly decreased . All these results further confirm an instructive role of neuronal activity in regulating the dynamics of resting microglial processes and the formation of microglia - neuron contact . Having shown that local elevation of neuronal activity can induce the formation of microglial bulbous endings wrapping neuronal somata, we next asked what might be mechanisms by which neurons with high activity talk to resting microglia . We found that this process requires the membrane depolarization - activated pannexin-1 hemichannels on tectal neurons and the atp / p2 purinergic receptor signaling between neurons and microglia . Pannexin-1, a large pore - like hemichannel, is widely expressed in the central nervous system . The opening of pannexin-1 is gated by some cellular signals, such as membrane depolarization, intracellular calcium and so on . Small molecules, including atp, nad and pge2, can be released through these hemichannels . Using whole - mount in situ hybridization and in vivo whole - cell recording, we found that the functional pannexin-1 hemichannels are expressed in tectal neurons but not microglia . After impairing the function of pannexin channels by drug treatment or morpholino - mediated genetic downregulation, glutamate uncaging - induced orientated movement of microglial processes and formation of bulbous contacts similar results were observed when we applied the atp - hydrolyzing enzyme apyrase or the p2 purinergic receptor blocker suramin . Neuronal activity steers resting microglial processes by facilitating the redistribution of the cytoskeleton protein small rho gtpase rac . Rac is known to be required for the membrane protrusion and migration of many types of cells, including zebrafish germ cells . Using confocal fret imaging, we found that the rac fret intensity is significantly increased in microglial processes, especially at bulbous endings, at the uncaging side after repetitive glutamate uncaging . It is immediately followed by the oriented movement of microglial processes and the formation of bulbous endings . However, if we genetically inhibited endogenous rac activity in microglia, those neuronal activity - induced microglial morphological changes could not be observed . Taken together, we identify the underlying mechanisms by which neurons with high activity signal to resting microglia . Considering the facilitated formation of microglia - neuron contact induced by neuronal activity increase, we wondered what the physiological function of such contact is . By simultaneously monitoring changes in both the morphology of microglial processes and ca activity of tectal neurons, we found that microglial processes preferentially contact with neurons that exhibit high levels of spontaneous activity before contact . We then mosaically overexpressed the human inward rectifier k channel kir2.1 (kir), which is used to reduce the excitability of neurons, and a non - conducting mutant version of kir2.1 (mkir) in tectal neurons . In comparison with mkir - expressing neurons, these results further indicate that resting microglia preferentially contact neurons with higher activities, consistent with the data obtained with glutamate uncaging . While accumulating evidence shows that resting microglia respond to neural activity, we surprisedly noticed that resting microglia can also in turn regulate neuronal activity . In the zebrafish optic tectum, we found that such resting microglia - neuron contact can downregulate both spontaneous and visually evoked activities in contacted neurons . Interestingly, the decreases in both the frequency and magnitude of spontaneous ca activity are positively correlated with the duration of microglia - neuron contact . What is the consequence on neuronal firing after loss of resting microglia? In response to two - photon laser - induced focal injury, some microglia quickly trans - locate to the injury site . Consistent with the speculation based on our previous findings, the spontaneous activity of neurons within microglia pre - existing territories is significantly elevated after the translocation of microglia to the injury site . Meanwhile, in other cases with the similar injury but no microglial translocation, the neuronal activity is not significantly changed . Thus, our study reveals a functional regulation of neuronal activity by resting microglia under physiological conditions, offering a new way for homeostatic regulation of neuronal activity . Collectively, our work brings forth and demonstrates a novel reciprocal regulation between resting microglia and neurons in vivo . We not only demonstrate an instructive role of neuronal activity in resting microglial motility, but also reveal, for the first time, a previously unappreciated function of microglia in homeostatic regulation of neuronal activity in the healthy brain (fig . 2). Considering the bi - directional modulation between neurons and microglia, this study also represents a new perspective in understanding the physiological function of resting microglia . Highly active neurons attract resting microglial processes and induce the formation of microglia - neuron contact via atp signaling (top). Such neuronal activity - driven microglia - neuron contact in turn
Lung cancer is prevalent worldwide, accounting for 1.6 million new cases and 1.4 cancer - related deaths in 2008 . Histologically, lung cancer can be divided into non - small cell lung cancer (nslcl) and small cell lung cancer, and the former accounts for approximately 80% of all lung cancer cases . Nsclc is most often diagnosed at an advanced stage, when surgical resection of the tumor lesion is no longer an option . Therefore, patients are likely to receive chemoradiotherapy, although more recently targeted therapies have provided a novel strategy for the control of advanced nsclc . Ionizing radiotherapy is used to treat advanced non - resectable nsclc in an estimated 64.3% of patients, but the outcome of this treatment varies significantly . Ionizing radiation induces cell death mainly through generation of short - lived but highly reactive dna radicals that evolve into stable, long - lived dna lesions, such as dna double strand breaks or through interactions with the cell membrane . However, dramatic differences in survival outcomes have been reported even in nsclc patients with tumors of a similar pathological or clinical stage that received identical treatments, suggesting that the patient s sensitivity to radiotherapy could play an important role in nsclc prognosis . Biomarkers for the prediction and monitoring of treatment responses and survival are urgently sought in order to appropriately tailor treatment regimens to patients . Aberrant expression or single nucleotide polymorphisms (snps) in certain genes may influence host responses to chemoradiotherapy . For example, altered expression of dna repair proteins and their polymorphisms has been reported to modulate sensitivity to radiotherapy [811]. Other studies reported that aberrant expression of other genes, including cell proliferation - related genes, could also influence sensitivity to radiotherapy . To this end, we have focused on the influence of top2a and dusp6 on radiotherapy outcomes . Demonstrated that top2a expression was associated with clinicopathological parameters and tumor cell proliferation in lung adenocarcinoma . Dusp6 is a putative negative feedback regulator of the ras - erk pathway and plays a physiologically important role in maintenance of cell homeostasis in response to growth factors [1315]. Disruption of this feedback loop could result in neoplastic, and even malignant, transformation . Okudela et al . Reported that levels of dusp6 expression decreased as both growth activity and histological grade of lung cancer increased . Moreover, dusp6 is highly polymorphic, and functional dusp6 snps have been identified and demonstrated to regulate dusp6 expression . Given the important role of top2a and dusp6 in carcinogen metabolism and cell proliferation, it is conceivable that genetic variations in top2a and dusp6 that alter gene expression and/or protein production may act as markers of radiotherapy response . Thus, in this study, we assessed the frequency of top2a and dusp6 snps in a cohort of 140 chinese nsclc patients, and probed the association between top2a and dusp6 snps with radiotherapy response and overall survival . We aimed to identify top2a and dusp6 snp biomarkers that predict treatment response and survival of patients with advanced stage nsclc . All procedures performed in studies involving human participants were in accordance with the ethical standards of our university and with the 1964 helsinki declaration and its later amendments or comparable ethical standards . A total of 140 consecutive patients with histologically confirmed nsclc were recruited from the first affiliated hospital of china medical university between june 2009 and december 2012 . The inclusion criteria were: i) patients were pathologically diagnosed with nsclc and computed tomography (ct) scan revealed progressive lesions; ii) patients were not eligible for surgery and nsclc was defined as non - resectable; iii) patients had a who - eastern cooperative oncology group (ecog) performance status 2; iv) patients did not show evidence of pleural effusion; and v) patients could be encompassed within a tolerable radiotherapy treatment volume . Thirty - four patients had intrathoracic recurrent disease (recurrence after previous surgery), and 106 patients had previously untreated primary nsclc . Treatments were selected based on physical examination, medical history, complete blood test, routine blood chemistry, and ct scan of the chest and abdomen . All demographic and clinical data, including gender, age, clinical stage, ecog performance status, tobacco smoking status, treatment management, and weight loss within six months after the initial hospital visit, were collected from patient s medical records . Patients were categorized as never - smokers if they had smoked <100 cigarettes in a lifetime . Former smokers were those that had quit tobacco smoking at least one year before study enrollment, and current smokers were those that continued smoking or quit smoking less than one year before study enrollment . The patients were followed up on regularly, and the last follow - up was conducted in january 2014 . The median duration of follow - up was 26 months, and ranged from two to 62 months . Radiotherapy was administered to each patient using three - dimensional conformal radiotherapy (3d - crt). Specifically, patients were placed in the radiotherapy position on a dedicated ct - simulator with both arms above the head, using a dedicated immobilization and patient laser marker system . Ct images were retrieved and saved in a pinnacle(3) radiation treatment planning system (philips, best, the netherlands). The pulmonary gross tumor volume (gtv) was set as the primary tumor in the lung window (width 1,600 hu, length 800 hu) and defined by radiation oncologists . The volumes were generated for each phase within the treatment planning system . Only the primary tumor volume was evaluated, unless a nodal volume was the dominant or only lesion . The clinical target volume (ctv) was routinely created by expanding both the gtv and the metastatic mediastinal lymph nodes (gtv - n) by 0.60.8 cm . Where necessary, the planning target volume (ptv) was also created by expanding the ctv by 0.5 cm to compensate for any setup errors and respiratory motion . The radiotherapy protocol was designed to deliver a prescribed dose of 40~65 gy in 2030 fractions to the ptv . The treatments were delivered with a primus 6-mv x - ray linear accelerator (siemens corporation, munich, germany). All patients were treated for five days per week with 2 gy daily fractions . After receiving a total dose of 40 gy/20 fractions in four weeks, each patient was repeatedly scanned in the radiotherapy position with the dedicated ct - simulator, and the new ct data set was compared to the previous ct data set . However, the target volume was again contoured on the basis of the new ct data set according to indications of tumor regression . The new 3d - crt plan was then calculated using a prescribed dose of 7.5~25 gy administered to the new ptv in 3~10 fractions of 2.5 gy / fraction . Tumor size, in terms of gtv of radiotherapy, was calculated by the radiotherapy plan system before radiotherapy was initiated and in the middle of the radiotherapy period (40 gy/20 fraction/4 weeks). The formula r = gtv - gtvsgtv was used to calculate the ratio of alterations in tumor size after radiotherapy, where r was the relative decrease in size of the tumor, gtv was the tumor size pre - radiotherapy, and gtvs was the tumor size after 40 gy/20 fraction/4 weeks of radiotherapy . Meanwhile, chemotherapy, including cisplatin (or carboplatin) in combination with a third - generation drug (e.g., etoposide, gemcitabine, vinorelbine, or taxanes), was given to most patients . Overall, 15 (10.7%) patients received radiotherapy alone, 53 (37.9%) patients received sequential chemoradiotherapy and 72 (51.4%) concurrent chemoradiotherapy . To select candidate gene snps for genotyping, we searched the international hapmap database (http://hapmap.ncbi.nlm.nih.gov/) and identified sequences that included 1 kb upstream and downstream of each gene in snp selection . We then downloaded genotype data restricted to those of the han population from beijing, china (chb), and identified two snps (rs471692 in top2a and rs2279574 in dusp6) with minor allele frequencies (maf) above 1% and with a minimum value of 0.8 for the r parameter using the tagger algorithm implemented in haploview ver . 4.2 . In particular, dusp6 rs2279574 is localized in exon 1, and causes an amino acid change from val to leu, while top2a rs471692 is localized in an intron, which does not alter any amino acids of the top2a protein . To genotype these two snps, we collected 5 ml of peripheral blood from each participant into a sodium citrate tube, then extracted patient genomic dna using a standard proteinase k digestion, followed by phenol - chloroform extraction and ethanol precipitation . These dna samples were then genotyped using a taqman snp genotyping assay kit (affymatrix inc ., cleveland, oh, usa) in an applied biosystems 7500 fast real - time pcr system (foster city, ca, usa). The primers and probes used for top2a rs471692 were 5-atc aca aac cta aaa aag aaa tcc a-3 and 5-aat tat tta cct ggg tcc atg ttc t-3; for dusp6 rs2279574, 5-agc ttc ttg agc agcagc ccg agc a-3 and 5-cga ctc gcc gcc cgt att ctc gtt c-3. The taqman universal pcr master mix and predesigned snp - genotyping assay mixture containing pcr primers and probes were purchased from abi . The pcr amplification contained 25 l master mix (applied biosystems), 10 l of dna, 2.5 l of probe, and 12.5 l of ddh2or which was subjected to an initial denaturing step at 95c for 10 minutes, followed by 47 cycles of 92c for 30 seconds, 60c for one minute, and a final extension at 60c for one minute . To ensure accuracy of pcr amplification, we included three positive controls and two negative controls in each 96-well plate and randomly repeated 10% of the samples for quality control purposes . A further 60 samples were randomly selected for direct dna sequencing to confirm the taqman results, and dna sequence data indicated 100% concordance . The hardy - weinberg equilibrium was determined using a goodness - of - fit test . The two - sided test was performed to assess statistically significant differences between the demographic and clinical features of patients with recurrent and primary nsclc . Overall survival was calculated as the length of time between histological diagnosis and death from any cause, or the date of the last follow - up . Differences in mean relative tumor size between pairs of groups were analyzed using an independent sample t - test . Association between the genotype and survival rate was estimated by the kaplan - meier curve and then the log - rank test . A multivariate cox proportional hazards model was used to estimate the hazard ratios (hrs) and their 95% confidential intervals (cis) for overall survival . All statistical analyses were performed using spss 13.0 software (spss inc ., chicago, il, usa) and a p<0.05 of a two - side test was considered statistically significant . All procedures performed in studies involving human participants were in accordance with the ethical standards of our university and with the 1964 helsinki declaration and its later amendments or comparable ethical standards . A total of 140 consecutive patients with histologically confirmed nsclc were recruited from the first affiliated hospital of china medical university between june 2009 and december 2012 . The inclusion criteria were: i) patients were pathologically diagnosed with nsclc and computed tomography (ct) scan revealed progressive lesions; ii) patients were not eligible for surgery and nsclc was defined as non - resectable; iii) patients had a who - eastern cooperative oncology group (ecog) performance status 2; iv) patients did not show evidence of pleural effusion; and v) patients could be encompassed within a tolerable radiotherapy treatment volume . Thirty - four patients had intrathoracic recurrent disease (recurrence after previous surgery), and 106 patients had previously untreated primary nsclc . Treatments were selected based on physical examination, medical history, complete blood test, routine blood chemistry, and ct scan of the chest and abdomen . All demographic and clinical data, including gender, age, clinical stage, ecog performance status, tobacco smoking status, treatment management, and weight loss within six months after the initial hospital visit, were collected from patient s medical records . Patients were categorized as never - smokers if they had smoked <100 cigarettes in a lifetime . Former smokers were those that had quit tobacco smoking at least one year before study enrollment, and current smokers were those that continued smoking or quit smoking less than one year before study enrollment . The patients were followed up on regularly, and the last follow - up was conducted in january 2014 . The median duration of follow - up was 26 months, and ranged from two to 62 months . Radiotherapy was administered to each patient using three - dimensional conformal radiotherapy (3d - crt). Specifically, patients were placed in the radiotherapy position on a dedicated ct - simulator with both arms above the head, using a dedicated immobilization and patient laser marker system . Ct images were retrieved and saved in a pinnacle(3) radiation treatment planning system (philips, best, the netherlands). The pulmonary gross tumor volume (gtv) was set as the primary tumor in the lung window (width 1,600 hu, length 800 hu) and defined by radiation oncologists . The volumes were generated for each phase within the treatment planning system . Only the primary tumor volume was evaluated, unless a nodal volume was the dominant or only lesion . The clinical target volume (ctv) was routinely created by expanding both the gtv and the metastatic mediastinal lymph nodes (gtv - n) by 0.60.8 cm . Where necessary, the planning target volume (ptv) was also created by expanding the ctv by 0.5 cm to compensate for any setup errors and respiratory motion . The radiotherapy protocol was designed to deliver a prescribed dose of 40~65 gy in 2030 fractions to the ptv . The treatments were delivered with a primus 6-mv x - ray linear accelerator (siemens corporation, munich, germany). All patients were treated for five days per week with 2 gy daily fractions . After receiving a total dose of 40 gy/20 fractions in four weeks, each patient was repeatedly scanned in the radiotherapy position with the dedicated ct - simulator, and the new ct data set was compared to the previous ct data set . However, the target volume was again contoured on the basis of the new ct data set according to indications of tumor regression . The new 3d - crt plan was then calculated using a prescribed dose of 7.5~25 gy administered to the new ptv in 3~10 fractions of 2.5 gy / fraction . Tumor size, in terms of gtv of radiotherapy, was calculated by the radiotherapy plan system before radiotherapy was initiated and in the middle of the radiotherapy period (40 gy/20 fraction/4 weeks). The formula r = gtv - gtvsgtv was used to calculate the ratio of alterations in tumor size after radiotherapy, where r was the relative decrease in size of the tumor, gtv was the tumor size pre - radiotherapy, and gtvs was the tumor size after 40 gy/20 fraction/4 weeks of radiotherapy . Meanwhile, chemotherapy, including cisplatin (or carboplatin) in combination with a third - generation drug (e.g., etoposide, gemcitabine, vinorelbine, or taxanes), was given to most patients . Overall, 15 (10.7%) patients received radiotherapy alone, 53 (37.9%) patients received sequential chemoradiotherapy and 72 (51.4%) concurrent chemoradiotherapy . To select candidate gene snps for genotyping, we searched the international hapmap database (http://hapmap.ncbi.nlm.nih.gov/) and identified sequences that included 1 kb upstream and downstream of each gene in snp selection . We then downloaded genotype data restricted to those of the han population from beijing, china (chb), and identified two snps (rs471692 in top2a and rs2279574 in dusp6) with minor allele frequencies (maf) above 1% and with a minimum value of 0.8 for the r parameter using the tagger algorithm implemented in haploview ver . 4.2 . In particular, dusp6 rs2279574 is localized in exon 1, and causes an amino acid change from val to leu, while top2a rs471692 is localized in an intron, which does not alter any amino acids of the top2a protein . To genotype these two snps, we collected 5 ml of peripheral blood from each participant into a sodium citrate tube, then extracted patient genomic dna using a standard proteinase k digestion, followed by phenol - chloroform extraction and ethanol precipitation . These dna samples were then genotyped using a taqman snp genotyping assay kit (affymatrix inc ., cleveland, oh, usa) in an applied biosystems 7500 fast real - time pcr system (foster city, ca, usa). The primers and probes used for top2a rs471692 were 5-atc aca aac cta aaa aag aaa tcc a-3 and 5-aat tat tta cct ggg tcc atg ttc t-3; for dusp6 rs2279574, 5-agc ttc ttg agc agcagc ccg agc a-3 and 5-cga ctc gcc gcc cgt att ctc gtt c-3. The taqman universal pcr master mix and predesigned snp - genotyping assay mixture containing pcr primers and probes were purchased from abi . The pcr amplification contained 25 l master mix (applied biosystems), 10 l of dna, 2.5 l of probe, and 12.5 l of ddh2or which was subjected to an initial denaturing step at 95c for 10 minutes, followed by 47 cycles of 92c for 30 seconds, 60c for one minute, and a final extension at 60c for one minute . To ensure accuracy of pcr amplification, we included three positive controls and two negative controls in each 96-well plate and randomly repeated 10% of the samples for quality control purposes . A further 60 samples were randomly selected for direct dna sequencing to confirm the taqman results, and dna sequence data indicated 100% concordance . The hardy - weinberg equilibrium was determined using a goodness - of - fit test . The two - sided test was performed to assess statistically significant differences between the demographic and clinical features of patients with recurrent and primary nsclc . Overall survival was calculated as the length of time between histological diagnosis and death from any cause, or the date of the last follow - up . Differences in mean relative tumor size between pairs of groups were analyzed using an independent sample t - test . Association between the genotype and survival rate was estimated by the kaplan - meier curve and then the log - rank test . A multivariate cox proportional hazards model was used to estimate the hazard ratios (hrs) and their 95% confidential intervals (cis) for overall survival . All statistical analyses were performed using spss 13.0 software (spss inc ., chicago, il, usa) and a p<0.05 of a two - side test was considered statistically significant . Clinicopathological data from these 140 consecutive patients with histologically confirmed nsclc are summarized in tables 1 and 2 . In brief, of these patients, 105 (75.0%) were male, and 35 (25.0%) were female, and the median age was 60 years (range 31 to 82 years); 51 (36.4%) had adenocarcinoma, 89 (63.6%) squamous cell carcinoma, while 106 (75.7%) patients had primary nsclc and 34 (24.3%) patients had an intrathoracic recurrent tumor after previous surgery . According to the ajcc 6th edition stage grouping criteria, six patients had stage i, 36 patients stage ii, 76 patients stage iii, and 22 patients stage iv disease, while 28 patients scored 0 on the ecog performance status according to the world health organization (who) recommendations (zubrod ecog / who scores) and 112 patients scored 1 . Furthermore, there were no statistically significant differences in the gender, weight loss, tobacco smoking status, ecog, location of tumor, histology, clinical stage, tumor stage, and lymph node metastasis of patients with primary tumors and recurrent tumors (p>0.05). Radiotherapy was delivered to both primary and recurrent nsclc patients for a median dose of 56.0 (ranged from 47.5~65 gy), while chemotherapy included cisplatin (or carboplatin) in combination with a third - generation drug (e.g., etoposide, gemcitabine, vinorelbine, or taxanes), administered for 26 cycles to 53 (37.9%) patients as sequential chemoradiotherapy and 72 (51.4%) as concurrent chemoradiotherapy; 15 (10.7%) patients only received radiotherapy . After a median radiation dose of 56.0 gy and/or 26 cycles of platinum - based doublet chemotherapy, the mean tumor regression was 0.34 cm during the course of radiotherapy, while the gtvs after 20 fractions of radiotherapy were slightly higher than the basal gtv in three patients . In comparison to the 15 patients treated with radiotherapy only, chemoradiotherapy achieved similar treatment responses . There was no statistically significant difference between radiotherapy response and clinicopathological characteristics of nsclc patients (table 2). The genotype frequency of top2a rs471692 was 47.9% for c / c, 45.7% for c / t, and 6.4% for t / t, and the genotype frequency of dusp6 rs2279574 was 10.7% for c / c, 42.9% for c / a and 46.4% for a / a . However, the genotype frequency of dusp6 rs2279574 or top2a rs471692 was not significantly associated with clinicopathological characteristics, such as gender, age, weight loss, ecog grade, location of tumor, clinical stages, lymph node metastasis, pathological type, or tobacco smoking status (p>0.05; table 3). There was no significant difference in the tumor regression observed in the top2a rs471692 c / c, c / t, and t / t genotypes (0.3550.169 cm, 0.3380.226 cm, and 0.4570.135 cm, respectively) (f=1.455 p=0.237). Tumor regression also did not differ significantly between the cc and combined c / t and t / t genotypes of top2a (t=1.634, p=0.105). Similarly, there was no significant difference in the tumor regression observed in the dusp6 rs2279574 c / c, a / c, and a / a genotypes (0.3690.181 cm, 0.3440.200 cm, and 0.3600.199 cm, respectively) (f=0.152 p=0.859). Tumor regression also did not differ significantly between the cc and combined a / c and a / a genotypes (t=0.313, p=0.755; tables 3, 4). We further compared overall survival of patients with stage i iii nsclc to assess predictors of the effect of radiotherapy and chemoradiotherapy . A total of 87 patients were analyzed, of which four were lost to follow - up (95.4% were thus included in data analysis). By the last follow - up, 53 patients had died, the majority as a result of lung cancer due to local disease progression, distant metastases, or both . One death was attributed to cardiac arrest and another to a cerebrovascular accident, but no treatment - related deaths occurred during this study . The overall one-, two-, and three - year survival rates were 74.7%, 52.6%, and 33.9%, respectively, with a median survival of 26 months (range: 2 to 62 months). Univariate analysis indicated that tumor lymph node metastasis (n stage) and clinical stage were significantly associated with overall survival of patients (table 5), while multivariate analyses indicated the following predictors of survival after definitive radiotherapy or chemoradiotherapy: tumor regression (p=0.009, 95% ci 2.454 [1.2564.796]), weight loss (p=0.043, 95% ci 2.791 [1.0327.548]), clinical stage (p=0.004, 95% ci 1.707 [1.1892.451]), and cigarette smoking (p=0.025, 95% ci 1.561 [1.0562.306]); table 5). However, there was no statistical significance between overall survival in top2a and dusp6 snp groups (p>0.05). Thus, we stratified the patients according to clinicopathological variables and analyzed tumor regression in each group (table 6). We found that tumor regression 0.34 cm was associated with poor survival of stage iii nsclc patients (log - rank p=0.007; figure 1). However, stratifying patients for weight loss, tumor histology, and ecog performance status did not indicate any significant associations with survival (data not shown). Clinicopathological data from these 140 consecutive patients with histologically confirmed nsclc are summarized in tables 1 and 2 . In brief, of these patients, 105 (75.0%) were male, and 35 (25.0%) were female, and the median age was 60 years (range 31 to 82 years); 51 (36.4%) had adenocarcinoma, 89 (63.6%) squamous cell carcinoma, while 106 (75.7%) patients had primary nsclc and 34 (24.3%) patients had an intrathoracic recurrent tumor after previous surgery . According to the ajcc 6th edition stage grouping criteria, six patients had stage i, 36 patients stage ii, 76 patients stage iii, and 22 patients stage iv disease, while 28 patients scored 0 on the ecog performance status according to the world health organization (who) recommendations (zubrod ecog / who scores) and 112 patients scored 1 . Furthermore, there were no statistically significant differences in the gender, weight loss, tobacco smoking status, ecog, location of tumor, histology, clinical stage, tumor stage, and lymph node metastasis of patients with primary tumors and recurrent tumors (p>0.05). Radiotherapy was delivered to both primary and recurrent nsclc patients for a median dose of 56.0 (ranged from 47.5~65 gy), while chemotherapy included cisplatin (or carboplatin) in combination with a third - generation drug (e.g., etoposide, gemcitabine, vinorelbine, or taxanes), administered for 26 cycles to 53 (37.9%) patients as sequential chemoradiotherapy and 72 (51.4%) as concurrent chemoradiotherapy; 15 (10.7%) patients only received radiotherapy . After a median radiation dose of 56.0 gy and/or 26 cycles of platinum - based doublet chemotherapy, the mean tumor regression was 0.34 cm during the course of radiotherapy, while the gtvs after 20 fractions of radiotherapy were slightly higher than the basal gtv in three patients . In comparison to the 15 patients treated with radiotherapy there was no statistically significant difference between radiotherapy response and clinicopathological characteristics of nsclc patients (table 2). The genotype frequency of top2a rs471692 was 47.9% for c / c, 45.7% for c / t, and 6.4% for t / t, and the genotype frequency of dusp6 rs2279574 was 10.7% for c / c, 42.9% for c / a and 46.4% for a / a . However, the genotype frequency of dusp6 rs2279574 or top2a rs471692 was not significantly associated with clinicopathological characteristics, such as gender, age, weight loss, ecog grade, location of tumor, clinical stages, lymph node metastasis, pathological type, or tobacco smoking status (p>0.05; table 3). There was no significant difference in the tumor regression observed in the top2a rs471692 c / c, c / t, and t / t genotypes (0.3550.169 cm, 0.3380.226 cm, and 0.4570.135 cm, respectively) (f=1.455 p=0.237). Tumor regression also did not differ significantly between the cc and combined c / t and t / t genotypes of top2a (t=1.634, p=0.105). Similarly, there was no significant difference in the tumor regression observed in the dusp6 rs2279574 c / c, a / c, and a / a genotypes (0.3690.181 cm, 0.3440.200 cm, and 0.3600.199 cm, respectively) (f=0.152 p=0.859). Tumor regression also did not differ significantly between the cc and combined a / c and a / a genotypes (t=0.313, p=0.755; tables 3, 4). We further compared overall survival of patients with stage i iii nsclc to assess predictors of the effect of radiotherapy and chemoradiotherapy . A total of 87 patients were analyzed, of which four were lost to follow - up (95.4% were thus included in data analysis). By the last follow - up, 53 patients had died, the majority as a result of lung cancer due to local disease progression, distant metastases, or both . One death was attributed to cardiac arrest and another to a cerebrovascular accident, but no treatment - related deaths occurred during this study . The overall one-, two-, and three - year survival rates were 74.7%, 52.6%, and 33.9%, respectively, with a median survival of 26 months (range: 2 to 62 months). Univariate analysis indicated that tumor lymph node metastasis (n stage) and clinical stage were significantly associated with overall survival of patients (table 5), while multivariate analyses indicated the following predictors of survival after definitive radiotherapy or chemoradiotherapy: tumor regression (p=0.009, 95% ci 2.454 [1.2564.796]), weight loss (p=0.043, 95% ci 2.791 [1.0327.548]), clinical stage (p=0.004, 95% ci 1.707 [1.1892.451]), and cigarette smoking (p=0.025, 95% ci 1.561 [1.0562.306]); table 5). However, there was no statistical significance between overall survival in top2a and dusp6 snp groups (p>0.05). Thus, we stratified the patients according to clinicopathological variables and analyzed tumor regression in each group (table 6). We found that tumor regression 0.34 cm was associated with poor survival of stage iii nsclc patients (log - rank p=0.007; figure 1). However, stratifying patients for weight loss, tumor histology, and ecog performance status did not indicate any significant associations with survival (data not shown). The responses of nsclc patients to conformal radiotherapy have been previously reported, but since the responses of individual patients vary dramatically, the role of conformal radiotherapy in nsclc remains controversial [2125].thus, in this study, we assessed the association of snps in top2a and dusp6 with chemoradiotherapy responses and prognosis in 140 chinese nsclc patients . In this group we found no statistically significant differences between the response of patients with top2a rs471692 and dusp6 rs2279574 polymorphisms or clinicopathological variables to chemoradiotherapy . We also did not find top2a and dusp6 snps to be associated with overall survival . Multivariate analyses indicated that tumor regression, weight loss, clinical stage, and cigarette smoking were all independent prognostic predictors of response to chemoradiotherapy . Our data, thus, suggests that top2a rs471692 and dusp6 rs2279574 snps are not associated with chemoradiotherapy response, whereas tumor regression, weight loss, clinical stage, and cigarette smoking are independent prognostic predictors of chemoradiotherapy response in chinese nsclc patients . Indeed, kupelian et al . Previously reported that in ten nsclc patients treated with external beam radiotherapy, tumor regression, detected by serial megavoltage ct images, occurred at a rate of 0.6% to 2.3% per day (average 1.2% per day). Woodford et al . Reported that in 17 nsclc patients that received 30 fractions of radiotherapy via helical tomotherapy, a gtv change of between 12 and 87% (average 38%) was observed, and gtv change was significantly associated with patient s physical or tumor features . Fox et al . Evaluated sequential ct scans to quantify reduction of tumor volume after a course of conventional radiotherapy in 22 patients with stage i iii nsclc, 15 of whom received concurrent chemotherapy . The median gtv reduction did not differ significantly between patients receiving chemoradiotherapy (24.7%, range, 0.3% to 61.7%) and radiotherapy (44.3%, range 0.2% to 81.6%). We found no patient, treatment, or tumor characteristics to be associated with tumor regression, although there was a tendency toward a greater volume reduction in patients with a gtv> 91 cm (gtv> 91 cm versus <91 cm) (p=0.075). The rate of tumor volume shrinkage varied during the course of radiotherapy observed in our current study, and we also observed that after four weeks of radiotherapy, the gtv of three patients had increased slightly . In addition, accelerated repopulation with rapid proliferation and a high growth index may have contributed to this phenomenon . Genetic variants have been reported to influence response to radiotherapy, and have thus improved our understanding of the effect of radiation on human tumors . Polymorphic genetic variants, such as snps, can alter the sensitivity of different cancers to radiotherapy [811]. However, to date, no cell proliferation associated genes have been reported to affect radiotherapy responses . In this study, we identified top2a and dusp6 snps and investigated their association with nsclc regression after chemoradiotherapy . Mutations of top2a have been associated with chemotherapy resistance, and several anticancer agents have been developed to target top2a activity . Furthermore, dusp6 is a member of the dual specificity protein phosphatase subfamily and functions to negatively regulate the activity of the mitogen - activated protein (map) kinase superfamily (mapk / erk, sapk / jnk, or p38), thus regulating cell proliferation and differentiation [2931]. Previous studies have shown that lost dusp6 expression was associated with cancer progression and resistance . However, our current data did not reveal any association between top2a and dusp6 snps and chemoradiotherapy responses or survival of nsclc patients . However, we did find that tumor regression, weight loss, clinical stage, and cigarette smoking were all independent predictors of chemoradiotherapy responses in these patients . Thus, future study will focus on the role of these variants in chemoradiotherapy of advanced stage nsclc patients . Top2a rs471692 and dusp6 rs2279574 snps did not associate with chemoradiotherapy response, whereas tumor regression, weight loss, clinical stage, and cigarette smoking were independent prognostic predictors for these chinese patients with nsclc.
A 50-year - old male (182.9 cm, 78.5 kg) was scheduled for arthroscopic debridement of the left shoulder . The patient had a history of old pulmonary tuberculosis and tb pleurisy (20 years ago) and had no previous surgeries . His mallampati class was 2 and he had been npo for more than 8 hours . 1) showed a lesion suspicious for tracheal stenosis, but it's formal decipher by radiologist was no active lung lesion . So, no further evaluation of airway have been done before surgery . In the operating room, electrocardiogram (lead ii), pulse oximetry, non - invasive blood pressure, end - tidal carbon dioxide concentration (etco2) and bispectral index were monitored . Following 3 minutes of preoxygenation, general anesthesia was induced with lidocaine 60 mg, propofol 120 mg, rocuronium 60 mg and remifentanil 0.5 - 1.0 mcg / kg / min . On direct laryngoscopy, cormack and we tried to insert a 7.5 mm reinforced endotracheal tube with a stylet, but this procedure failed . We then attempted to perform a blind intubation by inserting a 6.5 mm reinforced endotracheal tube with a stylet by another anesthesiologist, but we felt resistance under the vocal cords . We decided to try intubation with optiscope pm 201 (video assisted system, kyung won medical, seoul, korea), but this attempt failed because a 6.0 mm reinforced endotracheal tube stopped at subglottic level . 2) was chosen, and the device was inserted without difficulty on the first attempt . It was inserted blindly, until mild resistance was felt, as recommended by the manufacturer . The patient's lungs were ventilated with a tidal volume of 650 ml at a rate of 12 breaths per minute . The i - gel provided a good seal, and allowed for controlled mechanical ventilation with acceptable peak pressures (12 - 15 mmhg) while the patient was in the beach - chair position . The operation time was 1 hour 40 minutes and the time under anesthesia time was 3 hours . After neuromuscular recovery was demonstrated, the patient was awakened, and the i - gel was removed . Many anesthesiologists use a narrow endotracheal tube . However, with this method, there is an associated risk of failed intubation . The procedure can also lead to complications such as tracheal mucosal trauma, perforation or bleeding . In some patients with tracheal stenosis, airway control might be achieved by the use of an endotracheal tube positioned above the stenosis . If the stenotic segment is in the proximal trachea, however, proper fixation of the tube is difficult . Trauma, neoplasm, inflammation, wegener's granulomatosis and prolonged endotracheal intubation are the causes of the tracheal stenosis . In the case radiography can be very useful, but chest radiographs can also appear normal in patients with tracheal stenosis . Sads have advantages such as easy insertion and high effectiveness in securing a difficult airway . The i - gel is a single - use non - inflatable supraglottic airway device which is launched throughout the uk in january 2007 . It is composed of styrene ethylene butadiene styrene, which is soft and covers the larynx without cuff inflation . The seal pressure improves over time due to the thermoplastic properties of the gel cuff which may form a more efficient seal around the larynx after warming to body temperature . When correctly inserted, the tip of the i - gel will be located in the upper esophageal opening . It is designed to achieve a mirror of the supraglottic anatomy (pharynx and larynx) and the noninflatable cuff is semirigid . Donaldson and michalek reported a successful use of i - gel for subglottic stenosis patient who was undergoing laparoscopic cholecystectomy . The i - gel has an integral bite block and may decrease rotation within the oropharynx . Suggested that the anatomic position of the i - gel does not vary with head and neck position and that ventilation with the i - gel can be effectively performed with the head and neck in the extended or rotated position . In this report, we have presented a case of failed intubation in a patient with subglottic stenosis successfully managed with the i - gel. We suggest that the i - gel is a suitable choice in situations in which endotracheal tube insertion is difficult or has failed.
Bladder cancer (bc) is the ninth most common malignancy in the united states with 74,000 new cases and 16,000 deaths estimated for 2015 . Since radical cystectomy (rc) is the standard treatment for clinically localized muscle - invasive bladder cancer many patients undergo surgery with curative intention . Unfortunately, though the majority of patients are rendered disease - free after surgery, a significant proportion go on to develop bc recurrence and to ultimately succumb to the disease . For patients undergoing cancer surgery, there has been recent interest in identifying perioperative factors that may modulate recurrence and cancer - specific survival after surgery . It has been suggested that perioperative blood transfusion (bt) may be one such factor [35]. Blood transfusions represent the top five most frequently overused therapeutic procedures in the united states [6, 7]. Unfortunately, a clinically significant number of patients (3075%) with bc receive blood products during and after rc [810]. Although bts can be life - saving in some clinical perioperative circumstances, there are adverse events associated with their administration including transfusion - related immune suppression (trim). Trim is one proposed mechanism by which bts may be linked to poor oncologic outcomes . Several retrospective studies have demonstrated that perioperative bts are independently predictive of poor survival in patients with bladder cancer [1214]. A meta - analysis by wang and colleagues demonstrated the association between bts and decreased recurrence - free survival (rfs) and overall survival (os). However, three recent studies that included more than 6,500 patients in aggregate were recently published and are not part of that meta - analysis [1618]. We sought to assess the impact of bt on cancer - related outcomes and mortality in patients who had rc for muscle - invasive bladder cancer . We conducted a systematic review of the literature and meta - analysis to test for an association between perioperative bts and recurrence - free, cancer - specific, and overall survival in patients undergoing rc . We searched ovid medline and embase, pubmed, cochrane library, and the clinicaltrials.gov databases from inception to june 2015, with no limits of language or publication type . To identify additional studies, we also searched the 20102015 meeting abstracts of the american society of clinical oncology, the american urological association, and the european association of urology . Database search strategies included controlled vocabulary (e.g., medical subject headings) and keyword terms to find studies addressing perioperative transfusions or related procedures (such as blood salvage or hemodilution) of whole blood or blood components in bladder cancer patients . Outcomes sought by the search strategies included blood loss (intraoperatively or postoperatively), cancer - specific outcomes (e.g., recurrence, metastasis, and disease progression), and survival . All searches were performed by a medical librarian (greg pratt) who has contributed to more than 50 systematic reviews and meta - analyses . The primary outcomes of interest were recurrence - free survival, cancer - specific survival, and overall survival . We defined a perioperative bt as any amount of prbc within one month before and one month after rc . We included randomized controlled trials (rcts), prospective cohorts, and retrospective studies that evaluated the impact on any (allogeneic versus autologous versus intraoperative recovered cell saved) packed red blood cells (prbcs) in patients with bc who underwent rc . We excluded studies considering patients with distant metastases at surgery; those in which recurrence - free survival, cancer - specific survival, or overall survival were not indicated; and abstracts or poster presentations . Studies with a score of 6 or lower in the ottawa - newcastle scale were also excluded from any statistical analysis . For studies with overlapping patient populations, we calculated the pooled hazard ratio (hr) estimates and 95% confidence intervals by random effects model using the method of dersimonian and laird (d + l). To derive pooled estimates, the d + l method calculates weights by taking the inverse of a combination of within - study and between - study variability, which provides a larger variance compared with the variance produced from fixed effects analyses and thus wider confidence intervals . Cochran's q - test was used to test the null hypothesis of no significant heterogeneity across studies . Cochran's q - statistic follows distribution with (k 1) degrees of freedom, where k is the number of studies . I or the percentage of variation in the measures of association across studies due to heterogeneity was also calculated . I is the equivalent to the quantity of cochran's q minus its degrees of freedom divided by cochran's q, or i = (q df)/q . The value of i ranges between 0% and 100%, where 0% indicates no observed heterogeneity and larger values indicate increasing heterogeneity . The summary effect measure on hazard ratio for intraoperative transfusion on the time - to - event endpoints (overall survival, cancer - specific survival, and recurrence - free survival) was obtained . Lastly, a sensitivity analysis was conducted to test whether the results of the meta - analysis were sensitive to restrictions on any of the included studies . All statistical analyses were performed using r software (version 3.0.2, the r foundation for statistical computing). The initial search identified 14 potential studies that underwent full review (figure 1). Of these, 6 studies were excluded and 8 studies were included in the analysis . Abel's study included data from 2 different institutions; thus the 2 substudies were considered separately for statistical analysis . Only 5 studies clearly stated that patients were transfused with allogeneic blood; the remaining studies did not specify the type of blood . The leukoreduced status of the blood units was not clarified in any of the included studies . Two studies differentiated between intra- and postoperative blood transfusion and found that patients transfused intraoperatively but not postoperatively had worse survival [10, 17]. Eight studies including a total of 15,655 patients reported overall mortality as an outcome (table 1(a)). Of those patients, the 2 studies that did not identify bt as an independent risk factor of os did observe an important trend to worse os [9, 10]. As shown in figure 2, perioperative bts were associated with a 27% (or [95% ci]: 1.27 [1.151.40], p <0.05) increased risk in mortality (figure 2(a)). The i test demonstrated moderate to substantial heterogeneity (68.3%, p = 0.0014) across the studies . Seven studies including a total of 14,878 patients estimated cancer - specific survival in the statistical analysis (table 1(b)). The rate of transfusion in this pool of patients was 38% (n = 5,618). Five of the 7 studies (n = 6,521) demonstrated a negative impact of bt . As shown in figure 3, the risk of dying from cancer after perioperative bt was 29% (or [95% ci]: 1.29 [1.131.46], p <0.05) (figure 2(b)). The i test demonstrated moderate heterogeneity (60%, p = 0.012) across the studies . Five studies including a total of 8,778 patients estimated recurrence - free survival (table 1(c)). Three of the 5 studies (n = 4,910) showed a significant association between perioperative blood transfusions and poor survival [10, 17, 20]. In abel's study patients the association was present for the mayo clinic's population of patients but not for university of wisconsin's patients . As shown in figure 2, perioperative bts were associated with a significant increased risk in reduced rfs (or [95% ci]: 1.12 [1.121.31], p <0.05) (figure 2(c)). The i test demonstrated low heterogeneity (0%, p = 0.549) across the studies . The sensitivity analysis demonstrated that none of the studies included in the meta - meta - analysis was very influential, as the hr ranged from 1.20 to 1.30, 1.24 to 1.34, and 1.18 to 1.26 for os, css, and rfs, respectively, for the pooled meta - analysis and all omitted meta - analyses (table 2). Both the true nature of an association between bt and cancer recurrence and the biologic mechanism to explain this association are still very much unanswered research questions . The most commonly cited and investigated mechanism is the one that involves immune suppression or trim . However, it has been speculated that the infusion of growth factors (vascular endothelial growth factor and transforming growth factor - b) and an enhanced inflammatory response as a result of the exposure of the recipient immune system to donor microparticles could also stimulate spread and proliferation of cancer cells [24, 25]. The present meta - analysis was not designed to investigate these possibilities; however, our results support the hypothesis that the perioperative administration of prbcs is an independent risk factor for reduced rfs, css, and os after rc for bladder cancer similar to what has been reported for other cancers such as colon, lung, and esophagus [12, 2628]. Although a recent meta - analysis conducted by wang and colleagues showed similar results to ours, we consider the findings of the present study clinically relevant because first we included data from two recently published cohort studies with relatively large sample size . Therefore, a larger number of transfused and not transfused patients were part of the pooled analysis of the present meta - analysis . And second we conducted a different analysis (random effects and fixed effect models) in comparison to that published by wang and colleagues who used a fixed model paradigm . We believe that a random effects model strengthens the analysis and adds significant information to the current evidence because this model assumes that the pooled studies are not functionally equivalent as they were conducted by researchers operating independently . Sources of variation among the studies used in the meta - analysis are, for instance, time of transfusion (intra- versus post- versus intra- and postoperative) and trigger of transfusions . Therefore, our analysis can be generalized to different clinical scenarios of bladder cancer surgery [29, 30]. It is worth mentioning that two studies did try to evaluate the impact of time of transfusion on outcomes and found that intraoperative bts are an independent risk factor for poor survival while postoperative bts do not show an association with worse outcomes [10, 17]. Our meta - analysis shows significant heterogeneity or high degree of dissimilarity among studies for css and os but not for rfs . Although the high level of heterogeneity between studies for css and os tempers the strength of any conclusions that can be made about the effect of bt on these two survival outcomes, the low heterogeneity and identical estimated hrs for rfs using both random effects and fixed effect models suggest a strong association between perioperative bt and bc recurrence after rc . In this meta - analysis, patients who received a perioperative bt had a 21% higher risk of bc recurrence than patients who did not receive bt . The present study has the limitations inherent to any study level meta - analysis of cohort studies . Although we used the ottawa - newcastle score to grade study quality, all of the included studies were retrospective; the possibility exists that confounding variables (i.e., staging and tumor volume) may have influenced the individual study results and by extension the findings of this meta - analysis . Furthermore, the results of the present study cannot be extrapolated to the use of autologous blood transfusion since it is assumed that all studies included in the meta - analysis reported outcomes in patients transfused with mainly allogeneic blood . In conclusion, perioperative bt may be associated with reduced rfs, css, and os in patients undergoing rc for bc . A well - designed prospective rct is needed in this population to provide the high level evidence necessary for answering this question.
Chronic rhinosinusitis (crs) is characterized by severe inflammation of the sinus mucosa leading to blockage of the nasal passageway and the accumulation of mucus and pathogens in the nose and paranasal sinuses [1, 2]. Crs affects around 1.9 million australians and puts a large financial burden on health care systems . Crs is subdivided in crs with nasal polyps (crswnp) and crs without nasal polyps (crssnp) based on the presence or absence of polyps in the sinonasal cavities . Crswnp patients typically display a t helper 2 (th2) polarization, whereas patients without nasal polyps (crssnp) are often characterized by a th1 polarization with high levels of interferon- . Cytokines regulate innate and acquired immunity and can disrupt mucosal barrier function by altering tight junction (tj) composition and structure . This occurs through signalling pathways independent of cell death and the effect is cell type specific, pleiotropic, and time and dose - dependent . Relatively few studies have demonstrated cytokine effects on nasal epithelial tissue or barrier function [5, 6, 9, 10]. Th1 cytokines such as interleukin-2 (il-2), interferon- (ifn-), and tumour necrosis factor alpha (tnf-) are the primary source for proinflammatory th1 responses, in which they are effective in controlling infection with intracellular pathogens and for perpetuating autoimmune responses . In contrast, th2 immune responses are characterized by the production of the interleukins il-4, il-5, and il-13 that are associated with the promotion of eosinophil recruitment and activation, and inhibition of several macrophage functions, thus providing phagocyte - independent protective responses . Th17 cells are a subset of activated cd4 t cells and are characterized by the production of the interleukins il-17a, il-17f, il-22, and il-26 . Th17 cells act as a bridge between adaptive and innate immunity where they play crucial roles in the development of autoimmunity, inflammation, and allergic reactions . Here, we tested the effect of interferon proteins and of th1, th2, and th17 cytokines on the mucosal barrier structure and function of primary nasal epithelial cells harvested from nasal polyps of crs patients . Ethical approval for nasal brushing from crs patients was granted from the queen elizabeth hospital human ethics committee and only consented patients were included in the study . Exclusion criteria included active smoking, age less than 18 years, and systemic disease . Primary human nasal epithelial cells (hnecs) were harvested from nasal polyps by gentle brushing in a method as described in . Extracted cells were suspended in bronchial epithelial growth media (begm, cc-3170, lonza, walkersvill, md, usa), supplemented with 2% ultroser g (pall corporation, port washington, ny, usa). The cell suspension was depleted of monocytes using anti - cd68 (dako, glostrup, denmark) coated culture dishes, and hnecs expanded in routine cell culture conditions of 37c humidified air with 5% co2 in collagen coated flasks (thermo scientific, walthman, ma, usa). Hnecs were tested at passage two and confirmed to be of epithelial lineage via reactivity to pan - cytokeratin and cd45 antibodies (both from abcam, cambridge, ma, usa), and a diff - quick staining method used in the assessment of cell morphology by professional cytologists (imvs, the queen elizabeth hospital, woodville, australia). Hnec were maintained at an air liquid interface (ali) medium, following the lonza ali culture method (lonza, walkersville, usa). Briefly, transwells (bd biosciences, san jose, california, usa) were treated with collagen (stemcell technologies, australia). 70,000 cells were seeded in a volume of 100 l b - ali medium into the apical chamber of the transwell plate and 500 l of b - ali growth medium was added to the basal chamber in all wells containing the inserts . Cells were incubated at 37c . On day 3 after seeding, b - ali growth medium was removed from the apical and basal chambers and 500 l b - ali differentiation medium was added to the basal chamber only, exposing the apical cell surface to the atmosphere . Cytokines were added to the basal transwell chamber at the following final concentrations: recombinant human interferon- (500 ng / ml, sigma, saint louis, usa), interferon 1a (50 ng / ml, sigma, saint louis, usa), interferon- (500 ng / ml, sigma, saint louis, usa), tumour necrosis factor- (500 ng / ml, sigma, saint louis, usa), il-1b (500 ng / ml, sigma, saint louis, usa), il-4 (50 ng / ml, gibco, life technology, usa), il-5 (50 ng / ml, gibco, life technology, usa), il-13 (50 ng / ml, gibco, life technology, usa), il-17a (50 ng / ml, gibco, life technology, usa), recombinant human il-22 (50 ng / ml, sigma, saint louis, usa), and recombinant human il-26 (50 ng / ml, abnova taiwan corp). Paraffin - embedded tissue samples were cut in 4 m thick sections on a microtome (thermo scientific hm 325 rotary microtome). Slides were then stained with routine hematoxylin and eosin (h&e) staining using mayer's hematoxylin and eosin (lillie's modification, dako, thermo fisher scientific, waltham, ma, usa). All slides were then scanned using digital whole - slide imaging technology (wsi) on the nanozoomer digital pathology system (hamamatsu photonics, hamamatsu city, japan) under high resolution (40x objective magnification power). Transepithelial electrical resistance (teer) was measured by using an evom volt - ohmmeter (world precision instruments, sarasota, fl, usa). Briefly, 100 l of b - ali medium was added to the apical chamber of ali cultures to form an electrical circuit across the cell monolayer and into the basal chamber . Cultures were maintained at 37c during the measurement period using a heating platform . Only wells displaying baseline resistance readings greater than 500 /cm were used for the experiments . Cytokines and control (b - ali medium for the negative control and 2% triton 100 for the positive control) were added to the bottom chamber of each well, and teer measurements were obtained at time 0, 4 h, and 24 h. paracellular permeability was studied by measuring the apical - to - basolateral flux of fitc dextran 4 kda (sigma, saint louis, usa). Briefly, after treating the cells for 24 h, the upper chambers were filled with 3 mg / ml of fitc - dextran and incubated for 2 h at 37c . 40 l samples were recovered from the bottom chamber and serially diluted on a 96-well plate (corning costar cell culture plates (96 wells)), and the fluorescence was measured with a microplate fluorometer (fluostar optima, bmg labtech, ortenberg, germany). The amount of lactate dehydrogenase (ldh) in the medium was measured at 24 hours using the cytotox homogeneous membrane integrity assay (promega, australia). Briefly, 50 ml of the media from each well was transferred to a new plate, and 50 ml of ldh reagent was added to the supernatant and incubated for 30 minutes in the dark at room temperature . The od was measured at 490 nm on a fluostar optima plate reader (bmg labtech, ortenberg, germany). After cell incubation with the cytokines, the culture medium was discarded and washed with pbs, and 100 l of trypsin was added to each well . The plate was incubated for 5 minutes, and then 250 l of supplemented culture medium was added . The contents of each well were aspirated, placed into labeled microtubes, and centrifuged at 1000 rpm for 5 minutes . The supernatant was discarded, and the cells were suspended again in 100 l of culture medium . An aliquot of 10 l of cell suspension was removed and mixed with 10 l of trypan blue . After homogenization, the live and dead cells were counted and the percentage of viable cells was calculated . Cells were fixed with 2.5% formalin in phosphate - buffered saline (pbs) for 10 min, and then the cells were rinsed with tris - buffered saline-0.5% tween (tbst) four times, permeabilized with 1% sds in pbs, and blocked with serum free blocker (sfb; dako, glostrup, denmark) for 60 minutes, at room temperature . Mouse monoclonal anti - human zo-1 (invitrogen, carlsbad, ca, usa), diluted to 5 g / ml in tbst-10% sfb, was added to the excised culture support membranes and allowed to incubate for 1 hour at room temperature . Excess primary antibody was removed with tbst, and 2 g / ml anti - mouse alexa-594 conjugated secondary antibody (jackson immunoresearch labs inc ., west grove, pa, usa) the filters were rinsed in tbst, and after the third wash 200 ng / ml of 4,6-diamidino-2-phenylindole (dapi; sigma, aldrich) was added to resolve nuclei . Membranes were rinsed once with ultrapure water, and 95% ice cold ethanol was added for 1 hour at 4c . Membranes were transferred to a glass slide and a drop of anti - fade mounting medium (dako, glostrup, denmark) was added before cover - slipping . Samples were visualized by using a lsm700 confocal laser scanning microscope (zeiss microscopy, germany). The teer experiment was performed using three replicates from four crswnp patients with values normalised against the mean value from the patient at time 0 . Statistical analyses of all data were carried out using anova, followed by tukey hsd post hoc test . Ali cultures were established from 4 independent crs patient donors (2 males and 2 females, aged 4560 years). Two patients were diagnosed with grass - pollen allergy, one had aspirin - exacerbated respiratory disease (aerd), and two were asthmatic . Eosinophil and neutrophil counts [11.1 (4.621.3) and 0.8 (02.4)] per high power field (hpf) were not different between the different patients (p> 0.05). The effect of interferons and of th1, th2, and th17 cytokines was examined by measuring the teer across hnec monolayers from crs patients at different time points . All th17 cytokines tested (il-17, il-22 and il-26) caused a significant reduction in teer (average of 1.9 times; 1.7 times; 1.61 times for il-17, il-22, and il-26 resp .) After 24 h of incubation . Th1 and th2 cytokines or interferon,, or did not show any significant effect on teer (figure 1). All il-17 family cytokines (il-17, il-22, and il-26) led to a significant enhancement of paracellular permeability (p <0.05) (figure 2). Il-17 had the strongest effect, with 89.33% of the fluorescent dextran crossing the hnec monolayer whereas il-22 and il-26 increased paracellular permeability with 49.85% and 53.92%, respectively . Th1 and th2 cytokines and interferons,, and did not show any significant effect on the paracellular permeability in crs patients (figure 2). The effect of interferons and of th1, th2, and th17 cytokines on cellular toxicity was examined by measuring ldh release from hnecs . There was no statistically significant increase in ldh release after 24 h incubation with any of the cytokines (figure 3). In addition, the cell density estimated by the trypan blue assay did not show any significant differences in cell density or cell viability in cytokine treated cells compared to control cells (results not shown). The effect of interferon proteins and of th1, th2, and th17 cytokines on the localization of zona occludens-1 (zo-1) was examined by using immunofluorescence staining and confocal laser scanning microscopy, 24 hours after application of the cytokines . In untreated cells, zo-1 was located at the periphery of the apical side of the monolayer, as expected . Similarly, interferons,, and and th1 and th2 cytokines, which had no effect on either teer or paracellular permeability, led to no alterations in the localization of zo-1 . In contrast, application of th17 cytokines, which significantly altered epithelial barrier function, resulted in profound disruption of zo-1 immunolocalisation evidenced by faint or discontinuous regions of fluorescence (figure 4). Cytokine mediated insult on mucosal membranes, causing disruption of tight junctions and increased paracellular permeability, contributes to a multitude of pathologic conditions in inflammatory diseases of the upper airways [1820]. In this study, we compared the effect of interferons and of signature th1, th2, and th17 cytokines on the barrier function of primary nasal epithelial cells harvested from crs patients with nasal polyps . Immunolocalisation of the tight junction protein zo-1 was used to analyse tight junction integrity to gain insights into mechanisms of cytokines dependent disruption of the airway epithelial barrier . Our study indicates that, in crswnp patients, il-17 family cytokines (il-17a, il-22, and il-26) can significantly disrupt epithelial barrier function in association with a disruption of tight junction integrity and without causing cellular toxicity . In contrast, th1 and th2 cytokines or interferons showed no significant difference on either teer or paracellular permeability of hnecs . It has been well established that different cytokines cause different, often opposing effects on epithelial barrier function depending on the cell type used and that any observed effect is dose and time - dependent (reviewed in). Our results indicate that application of th1 cytokines such as ifn- and tnf- does not have detrimental effects on epithelial barrier function . Rather, application of these cytokines appeared to slightly enhance the teer of human nasal epithelial monolayers derived from some of the crswnp patients 24 hours after application . Whereas ifn- and tnf- generally decrease barrier function in different cell lines [21, 22], in airway epithelial cells, ifn- has been reported to decrease barrier function by soyka et al . And promote epithelial barrier function by ahdieh et al . . These differences in the response to ifn- could be attributed to many factors including interindividual variability in response to cytokines, different origin of cells (mucosa or polyps), and differences in experimental techniques . In the experiments by soyka et al ., for example, teer changes in response to ifn- treatment from crswnp, crssnp and controls were pooled while in our studies, only cells from crswnp were used . Given the small number of samples used in most studies, further experiments using a larger number of donors will need to address the cause of these discrepancies . Tnf- can increase teer in mammalian uterine cell monolayers in a dose - dependent manner . Interestingly, a recent study revealed that patients had developed a recurrence of crs after the start of tnf- inhibitor administration with a remission of the disease only after cessation of tnf- inhibitor treatment . Moreover, we demonstrated no significant reduction of teer by th2 family cytokines (il-4, il-5, and il-13) after 4 h and 24 h in our experiments . Using airway epithelial cells, saatian et al . Showed that il-4 and il-13 caused a reduction in teer 72 h after challenge but not after 24 h . Also used airway epithelial cells from crswnp patients (n = 2) and controls (n = 2) and similarly showed significantly decreased teer after il-4 challenge; however, this effect was already evident after 12 and 24 hours . The reason for these discrepancies is not clear and can be dependent on numerous factors . While physiologically relevant, it is well known that experiments using primary cells have limitations due to inherent interindividual differences of age, genetic make - up and medical history this is particularly important in crs, a multifactorial disease that can be associated with th1 or th2 responses . Also ethnicity may play a role as caucasian crswnp patients are often characterised by a predominant th2 type eosinophilic inflammation with high level of il-5, whereas asian crswnp patients preferentially have a th1/th17 polarization signature . Th17 cytokines also affect the gut mucosal barrier function by promoting the amplification of the host response to secrete neutrophil chemoattractants and antimicrobial peptides such as lipocalin-2 and calprotectin . In addition, th17 cells can expand within mucous layers in association with the presence of pathogens that are resistant to some of the induced antimicrobial responses . The expression level of il-17 was also shown to be significantly higher in recalcitrant crswnp compared to controls . In the present study, we found that il-17 induced barrier dysfunction as assessed by reduction in teer and enhanced macromolecular permeability, whereas soyka et al . We also demonstrated significant reductions in teer and enhanced macromolecular permeability of il-22 and il-26 which is the first such analysis using human nasal epithelial cells . In tight junction formation, zo-1 plays an essential role, by linking the transmembrane proteins occludin, claudin, and junction adhesion molecule (jam) cytoplasmic components of the tight junctions to the actin cytoskeleton . Disruption of the actin - myosin structure has been understood to modulate paracellular permeability . We observed a loss of normal zo-1 immunolocalisation in hnec monolayers of crswnp patients secondary to challenge with th17 (il-17, il-22, and il-26) cytokines, in association with disruption of barrier function . We also observed that il-17, il-22, and il-26 treated cells appeared in higher cell densities than cells treated with other th1 or th2 family cytokines . It is known that tjs regulate epithelial proliferation by different molecular mechanisms, which generally suppress proliferation as cell density (and hence tj assembly) increases (reviewed in). Changes in expression of zo-1 and zo-1-associated nucleic acid binding protein (zonab), a y - box transcription factor, affect cell proliferation; however, these effects take place at least 48 hours after changes in gene expression . Given that the duration of exposure to the treatments in our experiments was only 24 hours and that cell counts did not show significant differences, we believe that tj disruption secondary to il-17, il-22, and il-26 exposure might render the pseudostratified layer of cells into a monolayer which might appear relatively overcrowded . In summary, in patients with crswnp, th1 and th2 in contrast, the th17 cytokines family (il-17, il-22, and il-26) showed significant disruption of the epithelial barrier, leading to increased paracellular permeability associated with reduced tight junctionintegrity . In future studies, it will be important to determine the cellular mechanism of the effect of th17 cytokines on the mucosal barrier in crs patients to provide an opportunity for therapeutic modulation in inflammatory stress.
Splenic pseudoaneurysms (spa) are a serious sequela of blunt traumatic injury to the spleen . They are very rare with fewer than 200 cases reported in english literature in 2006 and their incidence is virtually unknown . Spa have a high risk of rupture and have been reported to be fatal if left untreated in adults . In the setting of trauma, spa can form 1 to 8 days after the blunt splenic injury and could be missed on the initial imaging . However, the management in pediatric patients is poorly defined . With regards to spa, although the american pediatric surgical association does not recommend routine follow - up imaging in pediatric trauma patients regardless of grade of injury, several reviews have described the potential benefits of follow - up ct imaging for detection of pseudoaneurysm . Here, we share our experience at an urban level 1 trauma center of managing a hemodynamically stable pediatric patient who sustained a grade iii / iv splenic laceration following blunt trauma with follow - up imaging demonstrative of splenic pseudoaneurysm and proposed algorithm to manage such a case . A 15 year - old male with no significant past medical history presented to the emergency room complaining of severe abdominal pain of acute onset after sustaining injury to his left side while playing football . His vital signs were within normal limits on arrival to the emergency room with a gcs of 15 . The patient s physical exam was significant for mild tenderness to palpation of his abdomen without peritonitis . Primary survey included a focused assessment with sonography for trauma (fast), which was positive for free fluid in the abdomen . A ct abdomen / pelvis was obtained, demonstrating a grade iii / iv left splenic laceration with moderate to large hemoperitoneum . He was admitted to the surgical intensive care unit for further monitoring . Over the next 24 h serial labs were drawn with a hematocrit as follows: 34.5%> 33.2%> 30.1% . Serial abdominal exams and hemodynamic status remained stable throughout his admission . On hospital - day 3, a follow - up cta abdomen / pelvis was obtained which demonstrated an approximately 6 mm ovoid hypervascular structure within the cleft of the splenic laceration suspicious for a small pseudoaneurysm (fig . Interventional radiology was consulted and on hospital - day 5; they performed a splenic arteriogram which demonstrated multiple pseudoaneurysms arising from the second order splenic artery branches . Proximal coil embolization with 10 mm 20 cm boston scientific interlock coil was performed without complications . He was observed for another 24 h post - procedure without event and was discharged the next day after receiving the pneumococcal and meningococcal vaccines . The patient was seen for follow up in our trauma clinic 5 days post - discharge without complaints . Splenic artery pseudoaneurysms (spa) remain a rare but potentially fatal pathology following blunt trauma which is managed non - operatively . Studies have demonstrated the benefits of repeated follow - up imaging and early intervention upon diagnosis of spa . Management of spa can be operative entailing splenectomy with or without pancreatectomy or non - operative, using endovascular techniques . In recent studies, utilization of angioembolization has been demonstrated to decrease non - operative management failure rate in adults with blunt splenic injuries . However in children, there are no strong evidence - based guidelines that exist to help guide management of spa . Pediatric splenic trauma may be more suitable for non - operative management than that of adult because the differences in pathophysiology of the injury . The pediatric spleen has a fibrous capsule that is thicker than that of an adult, and lacerations in pediatric patients tend to occur in avascular planes . One major controversy in management of pediatric blunt trauma patient is the utilization of predischarge or postdischarge follow - up imaging, usually 37 days after initial presentation [68]. The recommendation from the american pediatric surgical association trauma committee against this practice is based on the fact that less than half of pediatric patient received follow - up imaging without reported complications . Due to small cases presented in literature, . Showed in a retrospective study that follow - up doppler ultrasound found spa in 7.7% of grade iii splenic injuries and 17% of grade iv . The study showed that follow - up imaging may be useful in identifying spa in grade iii and iv splenic injury . However, the study is limited by the size of the sample population . In 2008, mayglothling et al . Described their experience of managing 97 adolescent patients with blunt splenic injury . In the study, mayglothling presented their algorithm with aggressive angiography and embolization as a useful adjunct to non - operative management of adolescents with blunt splenic injuries . The trauma center performed splenic artery angiography screening for patients with grade iii, iv or v splenic injury, contrast blush or pseudoaneurysm on ct . The authors also favored follow - up imaging 4896 h post - injury in their treatment algorithm . When compared to the direct catheter angiography, our patient's follow - up ct angiography of the abdomen demonstrated an underestimation of the size and severity of the pseudoaneurysms . Also, dsa may be better at showing the smaller pseudoaneurysms that cannot be visualized on the ct scan . Direct catheter angiography of the splenic artery has been assumed to be the gold standard to diagnose splenic artery pseudoaneurysm . With the advancing technology of interventional radiology, advanced blood vessel fluoroscopy techniques such as digital subtraction angiography (dsa) can provide high spatial resolution that allows the imaging of small vessels . The use of doppler sonography has also been described in the diagnosis of splenic aneurysms . However, this mode of imaging may be limited by its operators and its low spatial resolution . The use of magnetic resonance imaging (mri) has been reported . Multiple detector computer tomography (mdct) technology is the most utilized tool in critical care . Ct can obtain high resolution images of the whole body in a short amount of time when compared to dsa and to mri . Studies have shown that mdct is favorably comparable to catheter angiography in detecting stenosis and small blood vessels . Cta has also been shown to be safe and effective alternative to dsa for making decisions in cerebral aneurysm management ., brenner et al . Of columbia university have estimated that the lifetime risk of fatal malignancy from both abdominal and head ct examination is roughly 500 out of 600,000 . Although the risk is present, benefit of ct scan may outweigh the small increase in cancer risk in acute situations . Given that many of above sample populations received ct scans over a decade ago, the advancement of ct technology may have significantly decreased the radiation burden to children . Even though there may be an association between incidence of spa and high grade splenic lacerations (i.e., grade iii and iv splenic laceration), there is no strong clinical evidence to show benefits of aggressive angiography of these patients . We concur with safavi et al . In the necessity of follow - up imaging before planned discharge because there is a potential risk of delayed splenic rupture in this population . The limited existing literature leaves us with a still unclear algorithm for the management of non - operative blunt splenic trauma in pediatric patients . Questions still remain regarding the timing of repeat imaging for diagnosis of spa following non - operative blunt splenic trauma, which patients should be imaged, and how to manage spa upon diagnosis . More clinical study and basic science research is warranted to study the disease process of spa in pediatric patient . We believe that our proposed management algorithm timely detect formation of delayed spa formation and addresses the possible fatal disease course of pediatric spa . Roger chen zhu review of literature, preparation of case report, and writing of the paper.
Transitions in care are arguably one of the most important and challenging safety issues in healthcare today . Transitions in care involve the transfer of duties and accountabilities from one person, or group of people, to others . Transitions are a complex business that require a high degree of context - specific coordination and communication among different people with different backgrounds and skills . Two years ago, as the time was approaching for my father to be discharged after emergency hip reconstruction surgery, and three months of non - weight bearing rehabilitation, my mother became increasingly concerned about his impending discharge . In the weeks leading up to his discharge, i had several meetings with his social worker to discuss my mother's reluctance to take my father home and her concern about his ability to once again take care of his colostomy . The social worker arranged for a number of things to happen, prior to his discharge, which enabled a smooth transition to home . A home safety assessment was conducted and bath bars and toilet seat supports were installed a week in advance of his expected date of discharge . Arrangements were made for him to attend a day hospital three times a week, including the provision of specialized transportation services . A personal care worker was arranged to bath dad three days a week and the nurses provided him with up - dated colostomy products to facilitate self - management . My mother had time to adjust to his return home and my dad thrived when he got there . Just last week, my mother was discharged from the same hospital after spending three weeks in rehabilitation for a minor stroke . The day before her discharge i was still trying to meet with her social worker (i.e., a different social worker than the one who cared for my dad); several attempts to reach her by telephone and pager were unsuccessful . On one of my daily visits, i met mom's physiotherapist, who told me to adjust my dad's old walker for mom when she got home . I never did talk with the social worker, or the occupational therapist, or the family physician . I saw nurses, who never seemed to know anything about an expected date of discharge . I was surprised when the social worker called me the day before mom's discharge to ask me what time i was planning on picking her up the next day . We were given a discharge summary and mom was told to take it to her family doctor within a month . Only when i inquired about homecare was i told that a home safety assessment had been requested, but they were not sure if one would be provided . The transition to home was very difficult for us, and a week later we are still waiting for a home safety assessment and for someone to assist her with activities of daily living . Two years ago, as the time was approaching for my father to be discharged after emergency hip reconstruction surgery, and three months of non - weight bearing rehabilitation, my mother became increasingly concerned about his impending discharge . In the weeks leading up to his discharge, i had several meetings with his social worker to discuss my mother's reluctance to take my father home and her concern about his ability to once again take care of his colostomy . The social worker arranged for a number of things to happen, prior to his discharge, which enabled a smooth transition to home . A home safety assessment was conducted and bath bars and toilet seat supports were installed a week in advance of his expected date of discharge . Arrangements were made for him to attend a day hospital three times a week, including the provision of specialized transportation services . A personal care worker was arranged to bath dad three days a week and the nurses provided him with up - dated colostomy products to facilitate self - management . My mother had time to adjust to his return home and my dad thrived when he got there . Just last week, my mother was discharged from the same hospital after spending three weeks in rehabilitation for a minor stroke . The day before her discharge i was still trying to meet with her social worker (i.e., a different social worker than the one who cared for my dad); several attempts to reach her by telephone and pager were unsuccessful . On one of my daily visits, i met mom's physiotherapist, who told me to adjust my dad's old walker for mom when she got home . I never did talk with the social worker, or the occupational therapist, or the family physician . I saw nurses, who never seemed to know anything about an expected date of discharge . I was surprised when the social worker called me the day before mom's discharge to ask me what time i was planning on picking her up the next day . We were given a discharge summary and mom was told to take it to her family doctor within a month . Only when i inquired about homecare was i told that a home safety assessment had been requested, but they were not sure if one would be provided . The transition to home was very difficult for us, and a week later we are still waiting for a home safety assessment and for someone to assist her with activities of daily living . Can we attribute them to differences in the way healthcare providers responded to each client's needs? Do the existing regulatory standards of practice for nurses adequately address safety critical issues in relation to transitions in care? In this paper, we report the findings of a study undertaken with one of canada's provincial regulatory colleges to develop a greater understanding of the kinds of regulatory requirements needed to support safe care transitions . Three assumptions guided this work: (1) the fundamental aim of any transition in care is the transfer of high - quality clinical information between health care providers and a clear understanding by each healthcare provider about who is responsible for which aspects of the client's care; (2) regulation is one element in the web of complex interprofessional relationships and safe handoff communication; and (3) professional regulation sets the standards for skills, knowledge, and behavior for their members . We begin the paper with a review of the literature to gain a better understanding of the current knowledge related to safe transitions in care . We then consider the way in which a traditional and complexity lens helps us develop a fuller understanding of the way in which clients and families can have such dramatically different healthcare encounters, such as that illustrated in the story told above . Next, we describe the complexity informed method used to generate a rich account of client transitions from nurses, physicians, and pharmacists currently engaged in direct client care in a variety of settings in the canadian province . Following this, we examine regulatory requirements for safe client handovers, illuminating the tension between traditional and complexity informed approaches to professional standards of practice . Handoffs, transfer of accountability, and handover are terms used to describe the transfer of information and the accountability for care . Terms such as sign - over, sign - off, and sign - out are used to describe handoffs between on - call physicians . Specific to nursing, terms such as shift change, change - over, and report are used to mark the end of a time period and to transfer the accountability for care to the next . Regardless of the nomenclature, a client handoff involves the transfer of rights, duties, and obligations from one person, or group of people, to another . Communication at the time of handover should result in a clear understanding about who is responsible for which aspects of the client's care . The evidence tells us that major gaps in care occur during these critical exchanges [4, 5]. Frequent misunderstandings, errors, and lost information are related to higher rates of unexpected readmission, failure to adhere to care plans, delays or duplication of tests / treatments, delays in obtaining consents and authorizations, inappropriate use of medication, and potentially negative clinical outcomes [69]. In addition, several operational challenges, such as frequent interruptions, too much noise, and lack of space, have a negative impact on care transitions . Increased client acuity results in stress related to insufficient staff time to complete the client handover [2, 1013]. Despite the reality that many clients are discharged from health care settings quicker and sicker, requiring complex treatment protocols from multiple providers across multiple settings, until recently, little attention has been paid to the role professional standards of practice and safe outcomes . Overall, there is a dearth of empirical evidence that considers the link between regulatory standards of practice and safe transitions in care . Yet, to have a positive impact on the quality of care, regulatory standards of practice have to be contextually relevant, evidence based, and keep pace with the rapidly changing health care environment . A complexity lens helps nurses and other health professionals to develop a fuller understanding of the ways in which people and processes of care are interconnected, emergent and how relationship - rich networks and shared sense - making enable them to manage uncertainty more effectively . Newcomers to complexity science may imagine a picture of utter confusion when they hear someone refer to healthcare as chaotic or complex . For students of complexity science, however, chaos is not about utter confusion, but rather about the existence of patterns where we thought there were none . Unlike traditional models of science, which are cast in the form of linear and proportional relationships between cause and effect, complexity models are nonlinear, and events are related within degrees of proportionality . In other words, in linear models, there is one cause for one effect . In nonlinear models, there is more than one cause for an effect and more than one effect with a cause . From a traditional, linear perspective, the roles and responsibilities for each healthcare provider involved in care transitions are viewed as centrally structured through each discipline's professional standards of practice . In contrast, from a complexity perspective, healthcare providers respond to their own particular local context, producing diversity of agent behaviours and client and family experiences . Our example illustrates how healthcare providers interact with others according to their own evolved principles of local interaction . Complexity theory reminds us that healthcare providers enable and constrain each other in local interactions, and these patterns of interaction constitute control and order . And because complex systems interact with other complex systems, tension and paradox are a common theme seemingly opposing forces of competition and cooperation can work together in ways that can never be fully predicted or resolved . Whereas traditional scientific thinking assumes all issues can be resolved, complexity theory is comfortable with and values inherent tension between different parts of the system . The integration of complexity and traditional approaches to understanding processes of care during transition points will help nurses develop more comprehensive understanding of their work environment and client and family needs . The appreciative inquiry (ai) approach was taken to gain insight into current practices associated with care transitions and to develop insight into whether existing regulatory standards of practice for care transitions provide a sufficient underpinning to clinical practice . What is working well around here and how do we build on what is working well? Ai is based on the assumption that in every group or organization, something works well . Developed by david cooperrider and colleagues at case western university, ai involves the art of asking questions in ways that strengthen a system's capacity to apprehend, anticipate, and exploit procedures and processes that are working exceptionally well . Four generic ai processes were incorporated into design and execution of this study, including (1) using the positive as the focus of inquiry; (2) inquiring into exceptionally positive moments; (3) sharing the stories and identifying life - giving forces; and (4) creating shared images of a preferred future . Relationships are vital in ai and help make sense of emerging patterns across multiple settings and roles, and so we structured the project in a way that all members of the project committee (i.e., ten representatives from the three regulatory agencies that partnered to conduct the inquiry) were directly involved in collecting and analyzing research data . At the outset of the project, we held a one - day workshop focused on ai philosophical approach and data collection methods . Subsequently, the process for data collection required each member of the project team to contact between 8 and 12 individuals from various practice settings and to invite these individuals to be interviewed either in person or on the phone . The sample was opportunistic, and a standardized ai guide was used for the interviews . Interviews lasted between thirty and sixty minutes and took place between the months of february and may 2009 . Regularly scheduled teleconferences were conducted with the project team during data collection phase to address any questions and/or concerns . Each member of the project team summarized their interviews and transcribed their notes using a standardized format (approximately three pages per interview). We began our analysis by reading through the interview notes several times to comprehend their essential features . Preliminary analysis of the data involved open coding to generate a range of key themes that emerged from the data . The initial codes were then organized into provisional categories to build a coding framework divided into major themes and subthemes . The author and a research assistant conducted all aspects of the coding, first independently and then we compared and discussed categories until consensus was reached . Subsequently, we engaged the project team in a one - day workshop to review and validate preliminary findings and explore implications for each profession's regulatory framework . Sixty - six interviews were conducted with a wide range of health care providers (e.g., nurses, physicians, pharmacists, social workers, physio- and occupational therapy, and pastoral care) from a variety of practice settings (e.g., acute care, long - term care, palliative care, mental health, and corrections). The number of participants from each jurisdiction is provided in table 1 . The study protocol received approval by toronto's york university office of research ethics . Overall, study participants emphasized the fact that clients and families are most vulnerable when they transition between health care providers and between organizations, and we heard that nurses and other healthcare providers need to know three things to ensure safe care transitions, including (1) know your client; (2) know your team on both sides of the transfer; and (3) know the resources your client needs and how to get them . Transitions need to be timely, provide comprehensive client and family information, be customized to client needs, involve in - person exchange of information, and provide opportunities for ongoing dialogue as required . Clients are placed at even more risk when there are many interruptions during the exchange of information, when there are insufficient human resources to conduct the transfer of information, when the transfer is unplanned and unprepared, when there is a lack of respect between providers, and when there is a lack of understanding about issues of client privacy and confidentiality . Three key themes emerged from the interviews in response to our questions regarding successful client transfers, including (1) flexible structures; (2) independence and teamwork; and (3) client- and provider - focused care . We describe each theme here with examples from the best transfer experiences . This theme speaks of the process of client transitions, highlighting tension between the need for well - defined structures for successful transitions and the need to be flexible in order to meet unique client needs . At their best, client transitions were described as a structured process comprising several stages, including prehandoff planning, movement of the client, and posthandoff followup . They involve in - person reciprocal exchange of client information, including comprehensive data on the client's history, medications, and diagnostic testing results . Verbal report prior to physically receiving the client helps the team prepare for the client's arrival, and written documentation helps to decrease reliance on memory, thus enhancing the accuracy of the client's data . Advanced notice of a transition is especially important when clients have complex clinical needs and are being discharged between acute and homecare settings.during a home visit, the community nurse determined that the client needed to be assessed in the emergency department (ed). First, the community nurse provided a verbal report to the ed triage nurse telling her why she was sending the client to the ed . She also sent written documentation to the ed in the event that to a different nurse than the one to whom she provided the verbal report to ended up assessing the client . The written documentation also provided a detailed list of the correct dosages of the client's medication . (community) during a home visit, the community nurse determined that the client needed to be assessed in the emergency department (ed). First, the community nurse provided a verbal report to the ed triage nurse telling her why she was sending the client to the ed . She also sent written documentation to the ed in the event that to a different nurse than the one to whom she provided the verbal report to ended up assessing the client . The written documentation also provided a detailed list of the correct dosages of the client's medication . (community) participants also told us that successful transitions involve a high degree of flexibility, or professional latitude . Specifically, several participants spoke about how they stretch standard operating procedures in order to assist clients, a situation which happens frequently with handoffs between institutional and home settings . Participants spoke of situations in which the hospital provided supplies and equipment for discharged clients until arrangements could be made with community providers . This flexibility contributes to successful handoffs while building a sense of good will between health care professionals working in hospital and community . This palliative care client was being discharged home and was prescribed iv mixture to be administered by an epidural which needed laminar flow compounding and several days to order ingredients . There was insufficient time to arrange for this medication from a community pharmacy, so the client was sent home with sufficient meds to cover the gap . This was not official hospital policy; the communication and arrangements were made through the hospital pharmacist . (pediatrics) this palliative care client was being discharged home and was prescribed iv mixture to be administered by an epidural which needed laminar flow compounding and several days to order ingredients . There was insufficient time to arrange for this medication from a community pharmacy, so the client was sent home with sufficient meds to cover the gap . This was not official hospital policy; the communication and arrangements were made through the hospital pharmacist . (pediatrics) this theme speaks of independent and collaborative roles health care practitioners play in relation to successful handoffs . Specifically, participants discussed the importance of knowing their independent roles and responsibilities in any given situation, while respecting the skills and knowledge of teams of providers from different health care sectors and organizations . Participants stressed the importance of mutual respect when negotiating across multiple organizational and team boundaries while remaining focused on complex client needs . My role [as a nurse] in transferring accountability for the client is to make sure that the doctor's orders are correct, the charting is up to date, the time and date is set for transfer and to ensure that the receiving institution knows that the client is being transferred . They made it [the handoff] a great experience because the team was very fluid and every member contributed to the efficiency of the team (mental health and corrections)this transfer would have been even better if there was a greater understanding of the role of health workers and parish nurses within the community, and more willingness to share health information with client consent . (long - term care) my role [as a nurse] in transferring accountability for the client is to make sure that the doctor's orders are correct, the charting is up to date, the time and date is set for transfer and to ensure that the receiving institution knows that the client is being transferred . (acute care) everyone knew their role really well, especially the forensic team . They made it [the handoff] a great experience because the team was very fluid and every member contributed to the efficiency of the team (mental health and corrections) this transfer would have been even better if there was a greater understanding of the role of health workers and parish nurses within the community, and more willingness to share health information with client consent . (long - term care) participants also identified the importance of collaboration between multiple healthcare teams and organizations . Collaboration is particularly important in situations of conflicting client goals, changing condition, and complex needs . In addition, several participants suggested that transfers need to be seen as a seamless continuum of care rather than independent points at which one kind of care is concluded and another starts . The following describes a situation in which multiple health and social services providers collaborated to improve the situation for one client who was living in substandard conditions: the individual had mental health issues and the family found it difficult to care for the individual, to the extent that they were neglectful . Efforts to improve care his care involved partnership of various organizations, including coordinated police and emergency medical services who removed the client from the unsafe environment . When presented to emergency room, the link between the his community and acute care case manager ensured sharing of his history and current situation, which facilitated an expedited review of his case by the capacity board . While the change in his care took a month of planning, it was an extremely positive experience for all involved due to teamwork and in the end, the client was in a (mental health and corrections) the individual had mental health issues and the family found it difficult to care for the individual, to the extent that they were neglectful . Efforts to improve care his care involved partnership of various organizations, including coordinated police and emergency medical services who removed the client from the unsafe environment . When presented to emergency room, the link between the his community and acute care case manager ensured sharing of his history and current situation, which facilitated an expedited review of his case by the capacity board . While the change in his care took a month of planning, it was an extremely positive experience for all involved due to teamwork and in the end, the client was in a better place . (mental health and corrections) the third theme speaks of the information needs of clients, families, and members of the healthcare team . Clients are especially vulnerable during times of transfer and a successful handoff is measured by the degree to which the client is informed, and comfortable, the extent to which the family is informed and the degree to which staff are confident they have met client and family care needs . Specifically, study participants spoke to the importance of making sure that the client and their family members understand the reasons for the transfer and what they can expect from healthcare providers in the new healthcare setting . As illustrated by this participant, family members play a critical role in ensuring that client information has been shared with and between health care providers, including between nurses at shift change.nurse going off shift brought the oncoming nurse to the bedside, introduced her to us and included us in a portion of her report as i d band and fluid levels checked . As a spouse, i had an opportunity to provide input regarding my husband's limitations and needs . I felt much more confident / comfortable with care provided by on - coming nurse, and my husbands' needs were better met on that shift (acute care) nurse going off shift brought the oncoming nurse to the bedside, introduced her to us and included us in a portion of her report as i d band and fluid levels checked . As a spouse, i had an opportunity to provide input regarding my husband's limitations and needs . I felt much more confident / comfortable with care provided by on - coming nurse, and my husbands' needs were better met on that shift (acute care) in addition, participants told us that transfers from institutional to home care are more successful when family members are involved in planning and when they have time to obtain needed supplies and equipment . This is especially true in the case of pediatric care when family members need to learn new ways of caring for their child, or when family members are entering long - term care . I want to share my experience of the transfer of my husband from medical floor to long - term care . Found out preferences / needs / wants plan of care had been shared ahead of time . Found out areas of chief concern, including the need to give rescue meds quickly when shortness of breath started . Decision to transfer was made based on bed availability and palliative care teams identification that my husband's needs were not being met on the medical floor . Nurses gave me a tour and showed me what was available and opportunities for me to discuss my role as a contributor to my husband's care . Were allowed to go out on pre - planned pass and when we returned, even though it was a different team, they knew our story and our preferences . (long - term care) i want to share my experience of the transfer of my husband from medical floor to long - term care . Found out preferences / needs / wants plan of care had been shared ahead of time . Found out areas of chief concern, including the need to give rescue meds quickly when shortness of breath started . Decision to transfer was made based on bed availability and palliative care teams identification that my husband's needs were not being met on the medical floor . Nurses gave me a tour and showed me what was available and opportunities for me to discuss my role as a contributor to my husband's care . Were allowed to go out on pre - planned pass and when we returned, even though it was a different team, they knew our story and our preferences . (long - term care) study participants also spoke of the importance of transferring complete and accurate information between all members of the sending and receiving healthcare team . As stated above, successful transfer involves the exchange of comprehensive client information, including the client's medical history, a summary of the current situation, medications, test results, and those pending . Participants told us that they were more confident about their ability to provide safe care when they receive accurate, complete, and appropriate information from other team members . In addition, they spoke of the importance of trusting the providers from whom they are receiving the information . The positive part of the experience was the verbal report received as well as the phone call prior to transferring the client to the critical care unit that seemed to help everyone be comfortable with the admission . The rn who took the report was able to ask questions once the client arrived because they already had that head start in the information about the client before they arrived which allowed time for the rn to use critical thinking and come up with appropriate probing questions . (acute care) the positive part of the experience was the verbal report received as well as the phone call prior to transferring the client to the critical care unit that seemed to help everyone be comfortable with the admission . The rn who took the report was able to ask questions once the client arrived because they already had that head start in the information about the client before they arrived which allowed time for the rn to use critical thinking and come up with appropriate probing questions . To goal of this project was twofold: to gain insight into the practice setting realities for nurses and other health care providers as they manage increasingly complex care transitions across multiple settings and with a wide variety of care providers and to determine if current regulations reflect practice setting realities . Our findings demonstrate that healthcare providers responded to client and family needs in different ways, and how the outcomes of care can be dramatically different, depending on that responsiveness . Here first, participants seem to be contradicting themselves as they reinforce the importance of well - defined structures for successful transitions and the need to be flexible in order to meet unique client needs . From a traditional perspective, we heard how rules, structures, and boundaries are important elements of establishing minimum expectations of performance . We believe that when treated from a traditional mechanistic framework, structures might inadvertently impede safe transitions in care by inhibiting our ability to adapt in the dynamic, ever - changing healthcare environment . From a complexity perspective, study findings alert us to the fact that successful care transitions, and safe client and family outcomes, depend on the ways in which individual healthcare providers learn from each other and respond effectively to novel situations as they arise . We heard of situations that, when facing exceptional situations, individual healthcare providers worked hard to overcome difficult dynamics and organizational policies that affect safe care transitions . In reality, the need for structures and flexibility coexists . Standard operating procedures are critically important to establish general goals and boundaries, while at the same time, the ability to explore, act on experience, and interact and respond are key to the delivery of safe and effective care in the event of ambiguous objectives and divergent problems . Second, while demonstrating the importance of discipline - specific knowledge and roles, our findings also demonstrate the ways in which successful transitions in care require a high degree of collaboration and teamwork with all stakeholders, and with those who have the most to gain (or lose): clients and families . Complexity science reminds us that successful care transitions involve multiple agents who are constantly interacting and changing in response to each other, and the strength of the partnership is a key factor in achieving desired outcomes . Exemplar transitions in care illustrate how relationships between individual care providers and with clients, and family members, also known as clinical microsystems, are the essential building block of larger systems of care . Clinical microsystems are the place where clients, families, and care teams meet, and as our findings demonstrate, are the place where interactions determine the outcome of care transitions . Consistent with what stacey refers to as complex responsive processes of relating, our finding that, at times, nurses and other healthcare workers make extraordinary responses and engage in superb teamwork to ensure safe transitions, reinforces the social nature of healthcare processes and of how safe and effective outcomes of care emerge in the social act of call and response . We suggest that nurses and other healthcare professionals take different paths, depending on whether they use traditional and/or complexity informed approaches, to manage care transitions . Do they use a for - granted approach when they interact with clients and with each other, seeing themselves as one person in a web of constrained and constraining relationships, not fully responsible for outcomes of care? Or do they fully engage with each client, making sense of their need and the opportunities to collaborate with other team members to become agents of possibility and transformation? Third, with respect to regulatory implication, our findings demonstrated that while professional standards of practice provide the underpinning for safe care transitions, the use of good clinical judgment in the context of each client situation is a key to successful outcomes for clients, families, and care providers . In addition, findings suggest that it is easier to transfer clients within systems you know and to people you have worked with before, and that increased flexibility is required in a less familiar context . Cook and rasmussen provide a complexity informed model of system dynamics that helps us understand the adequacy of existing standards of nursing practice and of how nurses and other health professionals uphold professional accountability while coming up with innovative responses and strategies to ensure safe transitions in care . In the going solid model of system dynamics, cook and rasmussen differentiate between safe and unsafe operating space; safe operating space is conceptualize as an envelope created by three boundaries: economic, workload, and performance . There is room within the safe operating space to analyze risk and respond to unique client needs with creative adaptation while still upholding practice standards . However, nurses and other health care providers often operate at the margins of acceptable performance, and as our findings demonstrate, flexibility with regulation and standards is often required in complex sociotechnical work to make the system more efficient and adaptive to changing circumstances . The dynamic modeling of care transitions raises the fundamental question of how tightly regulated nurses and other healthcare professionals should be so as not to cause a trap of overregulation, which inevitably leads to more safety violations because of the tendency of standards to restrict behaviours . Given the complex and context - specific nature of transitions, and the importance of clinical judgment for client safety outcomes, we concluded that a regulatory standard of practice, designed specifically for care transitions is not necessary, nor would it likely be meaningful . The present study was limited to one canadian province and the results may, to some extent, reflect contextual factors that are not shared by other national and international jurisdictions . In addition, because this study is unique in its use of appreciative inquiry as a method of research, findings may be biased toward an optimistic view of care transitions . However, we adopted this approach because we were most interested in hearing about how nurses and other health providers contribute to safe care transitions and learning about ways to support exemplary care . Members of the study team who were new to ai expressed concerns about the positive approach at the outset of the project; however, by the end of the project, these same individuals endorsed its use because the approach allowed us to build on current strengths and avoid the trap of only focusing on deficiencies . Moreover, the project provided an excellent and unique opportunity for nurses and other health professionals to gain insight into each others' practice environments and explore ways to uphold professional accountability in exceptional situations where innovations are required to ensure safe transitions in care . Examples of positive and successful transitions demonstrated universal features across settings and healthcare providers, including the involvement of the client and family in decision making and planning, comprehensive and concise client information, opportunity for questions and followup by client and family as well as health care providers, time for planning and availability of staff to execute the transfer, and interprofessional and interorganizational collaboration . Our findings demonstrated that exemplar transitions in care are laden with paradoxes, or the coexistence of apparent opposites, including flexible structures; independence and teamwork; and client and provider focus . To ensure safe and effective care transitions, nurses and other healthcare professionals need to accept the paradoxes as a normal part of their practice environment and they need to engage in relationship - rich networks which foster creativity and responsiveness . All regulatory bodies establish expectations through policies, position statements, practice standards, guidelines and/or other documents, for how members do what they do in an effective, safe, and ethical manner . Viewed through a complexity lens, we see how the social act of call and response can lead to improved client and family outcomes, and that flexibility with regulation and standards is often required in complex sociotechnical work to make the care more efficient and adaptive to changing circumstances . We conclude that the development and introduction of new regulations holds the risk of constraining professionals' responsiveness at the point of care, thereby threatening client safety outcomes . Rather than focusing on new standards of practice, priority needs to be given to creating conditions where nurses and other healthcare providers are free to creatively engage and respond in ways that will optimize safe care transitions.
Positron emission tomography - computed tomography is useful for diagnosing primary and metastatic lesions of esophageal cancer . The sensitivity and specificity of a pet / ct are also relatively preferable to other diagnostic imaging tools . Although the number of cases is few, false positives exist in which an inflammatory tumor mass and a nonspecific tumor mass are difficult to differentiate between, even when combined with other image findings . In cases of esophageal cancer, the types of treatment and their prognosis are greatly affected if there is a metastasis or recurrence . Positron emission tomography - computed tomographic false positives cannot simply be ignored even if they appear to be due to inflammation or some other factors . This case report discusses the reasons to conduct a pet / ct test and its limitations in diagnosing esophageal cancer . The subjects of this investigation had suspected metastasis from a pet / ct which occurred before surgery of the 129 resected cases of esophageal cancer at our department from june 2010 to march 2015 . However, after surgery, biopsy and/or follow - up observation, only the cases that were confirmed as metastasis negative were considered to be pet / ct false positives for this assessment . There were 3 cases of preoperative pet / ct false positives (table 1). Two cases in which bone metastasis was suspected revealed almost normal range of squamous cell carcinoma (scc)associated antigen, alkaline phosphatase, and serum calcium levels . Abbreviations: ln, lymph node; m, metastasis; mt, middle thoracic lesion of esophagus; n, node; t, tumor; ut, upper thoracic lesion of esophagus . In case 1, a 59-year - old man, without particular medical history, had a diagnosis of esophageal carcinoma by endoscopy . Multiple bone metastases were suspected by a pet / ct in the spine, sternum, ribs, and pelvic bone (figure 1). After conducting a ct - guided biopsy of the right iliac bone, the patient is alive with no recurrence 4 years and 10 months after surgery, although the false - positive lesions in pelvic bone are still detected by pet / ct . In the case 1, multiple bone metastases were suspected by a positron emission tomography - computed tomography in the pelvic bone (suvmax = 2.94). In case 2, a 71 year - old man, without particular medical history, abnormal accumulations of fluorodeoxyglucose (fdg) in the manubrium sterni, vertebra th5, and the pedicle of vertebral arch were found by pet / ct . Therefore, after the sufficient informed consent, the patient underwent a laryngopharyngeal esophagectomy, a transhiatal esophagectomy without thoracotomy, and reconstruction with the gastric tube through the posterior mediastinal route . Pathological finding showed poorly differentiated scc of esophagus and moderately differentiated scc of hypopharynx with a cervical lymph node metastasis . The patient is alive with no recurrence 3 years and 11 months after the surgery . In case 3, a 76-year - old man, without particular medical history, had a diagnosis of a superficial esophageal cancer by endoscopy . He was suspected of lymph node metastasis of right recurrent nerve lymph node (figure 2) by preoperative pet / ct . . Scattered silicotic nodules and inflammatory lymphadenopathy might be caused by pneumoconiosis (figure 3). Case 3 was suspected of right recurrent nerve lymph node metastasis by preoperative positron emission tomography - computed tomography (suvmax = 3.00). Pathological finding of case 3 revealed scattered silicotic nodules in resected lymph nodes with infiltration of histiocytosis to sinusoid . In esophageal cancer treatment, fdg - pet / ct is also used to diagnose metastases and to judge the effectiveness of treatment . Especially, pretherapeutic there are cases when the surgery may not apply to a patient if remote metastasis or high - grade lymph node metastasis is found . Many cases have already been reported claiming that fdg - pet is a useful modality for a pretherapeutic staging . Esophageal cancer causes 30% to 40% of lymph node metastases among cancers that spread to the submucosa, and the prognosis of esophageal cancer is known to be defined by the presence of lymph node metastasis, how many metastases are present, and how much the metastasis spreads . Therefore, evaluating lymph node metastasis is a very important factor in deciding the course of treatment . The diagnosability of lymph node metastasis using a fdg - pet for esophageal cancer is generally reported to have a sensitivity of 32% to 51.9%, a specificity of 94.2% to 100%, and an accuracy of 48% to 93% . In addition, fdg - pet / ct, which is a combination of a fdg - pet and a ct, improves the diagnosability even more . Yuan et al have reported that they evaluated the diagnosability of lymph node metastasis using fdg - pet / ct for esophageal squamous cancer and found a sensitivity of 94%, a specificity of 92%, and an accuracy of 92% . However, the size of a lymph node that can be detected by pet / ct is said to be only 6 to 8 mm, thus it is difficult to detect micrometastasis . Also, detecting metastasis to neighboring lymph nodes could be difficult in cases with a lot of fdg accumulation in the primary lesion, or accumulation in the intestinal tract such as in the stomach, or in the heart . The fdg accumulates in inflamed lymph nodes as well, so false positives often appear in hilar lymph nodes or in patients who have chronic lung disease . Lymph node metastasis was suspected after a preoperative pet / ct for the case 3, as described above, but it was actually an inflammatory lymphadenopathy caused by pneumoconiosis . Dual - time - point fdg - pet has been reported to be useful in reducing this kind of false positive . It uses the time differences of peak fdg accumulation for inflammation versus for tumors . After injecting the fdg, pet has revealed occult distant metastases at nodal and non - nodal sites in 5% to 40% of patients . Distant metastases of esophageal squamous cancer are mainly found in the lungs, liver, and bones . Kato et al reported the diagnosability of fdg - pet for esophageal cancer as having a sensitivity of 92%, a specificity of 94%, and an accuracy of 93% . When comparing those numbers to bone scintigraphy, whose diagnosabilities are 77%, 84%, and 82%, respectively, the diagnosability of fdg - pet is superior, especially for osteolytic lesions . But 2 false positives out of 44 cases came from pet, showing that fdg - pet is not perfect . The suvmax of metastic tumor might be helpful to increase the accuracy of pet / ct . Our previous study demonstrated that the suvmax of the primary tumor was positively correlated with tumor size and vessel invasion and was positively related to the suvmax of metastic tumor . In the report, the diagnostic accuracy of pet / ct (87.3%) was higher than that of conventional ct scans . When diagnosing metastatic bone tumors, the positive predictive value of bone metastasis is known to be quite high if the findings for both pet and ct match . Therefore, even when bone metastasis is suspected due to abnormal fdg accumulation after a fdg - pet, yet no bone destruction image is found in the same site by ct, then interventional radiology or a surgical biopsy should be conducted to obtain a pathological diagnosis . This case report was on 3 patients with suspected metastasis due to a false - positive pet / ct who were later confirmed to not have metastasis . The metastases were originally suspected after conducting a pet / ct before surgery to treat esophageal cancer or during the postoperative course . Conducting a pet / ct is useful when diagnosing esophageal cancer metastasis, but there should be an awareness of the possibility of false positives . Therapeutic decisions should be made based on appropriate and accurate diagnoses, and a pathological diagnosis should be actively introduced if necessary.
Class ii malocclusion can be appraised as skeletal and dental class ii.1 the treatment of skeletal class ii malocclusion in non - growing individuals involves either surgical correction of the jaw abnormality, or orthodontic camouflage which usually requires the extraction of premolars or distal movement of the maxillary molars.23 for several years, extraoral appliances such as headgear were the most widely used distalizing appliances, but they are not esthetically desirable or socially acceptable, especially for adults . They are also removable and require patient compliance, which can compromise the results.4 several intraoral noncompliance devices for maxillary molar distalization have been recommended since the 1980s.5 these include but are not limited to the hilgers pendulum appliance, jones jig distalization apparatus, open nickel - titanium (niti) push coils, distal jet, repelling magnets, and molar slider.67891011 numerous side effects have been reported in association with these tooth - borne distalizing appliances, including anchorage loss of the maxillary premolars and flaring of the incisors, and a significant amount of relapse can occur . Also, the distalized molars must be used for anchorage during retraction of the premolars and anterior teeth,5 resulting in a bite - opening effect that is not tolerable for most patients . Temporary anchorage devices (tads) have been investigated, in an effort to overcome some of the side effects associated with tooth - borne distalizing appliances.1 dental implants, miniscrews, and miniplates have been used for skeletal anchorage.11112131415 tads have several advantages . They are relatively easy to place, inflict less trauma on the oral tissues, are stable under normal degrees of force, and can bear force immediately after placement . Miniscrews, in particular, are relatively inexpensive and patient compliance is limited to maintaining good oral hygiene . Given their small size, they can be inserted in a variety of sites on the alveolar and basal bones.316171819 however, they have been associated with damage to anatomical structures such as dental roots, nerves, and blood vessels, and there are the possibilities of screw breakage on placement and removal, and screw failure with peri - implant inflammation . The reported success rates of miniscrews range from 80% to 95%.20 the placement of miniscrews in the buccal interradicular bone is one of the most common approaches used to provide skeletal anchorage . The interradicular space is a potentially advantageous region for insertion, because there is less potential for complications related to soft tissue irritation, particularly if they are placed through the attached gingiva.20 although adjacent teeth may limit mesiodistal tooth movement, buccal interdental miniscrews are very useful for molar distalization, due to their ease of placement, and simple application during treatment . With a properly positioned tad, 3 mm of distal movement per side can be achieved.21 on the palatal side, a tad may be placed paramedian (near the midline) or in the interdental space . While paramedian appliances usually require more expensive and sophisticated attachments, the risk of root damage associated with them is minimal.161222 tads placed on the palatal interdental area can apply distalization forces directly to the molar . Moreover, these tads can also control the mesiodistal axis of the molar via manipulation of the line of action.22 we have introduced a new appliance using palatal miniscrews " mimi - implant - aided (mia) in the interdental area between the 1st molar and 2nd premolar root and a trans - palatal arch (tpa) to overcome some of the mechanical shortcomings of previous appliances, in contexts including anterior protrusion, extrusion of posterior teeth, and tipping of molar teeth (mia - tpa). Traction between tads in the palatal interdental area and a tpa can produce direct distalization force that travels through the center of resistance of molars (figure 1). The purpose of the present study was to measure the three - dimensional (3d) movement of 1st molars after using this newly designed distalizing appliance . The sample group consisted of 26 patients (22 females, 4 males) with an average age of 19.8 6.3 years (range 12 - 36 years) at the beginning of treatment (table 1). The main inclusion criterion was angle class ii malocclusion of no more than one cusp on each side . Patients with missing teeth, substantial restorations, or any craniofacial malformation were excluded from the study . The study was approved by the university research and ethics committee, and registered as a clinical trial at iranian registry of clinical trials (no . Irct201302269085n3). All patients (and their parents in the case of minors) were informed about the treatment procedures involved, and informed consent was provided . After preparing the initial diagnostic records including dental casts, a dentist placed two miniscrews, each with a range of 8 - 10 mm in length and 1.4, 1.6, or 2.0 mm in diameter in the palatal interdental area under local anesthesia . Miniscrews were inserted between maxillary 2nd premolars and 1st molars with a self - tapping technique, approximately 5 - 6 mm from the gingival crest at an angle of 60 - 80 relative to the long axis of adjacent teeth . The heads of the screws were approximately 2 mm above the mucosal surface, to facilitate the attachment of elastic modules . A tpa was made on a cast with stainless steel wire of 0.8 mm in diameter, with two helices or hooks soldered into the lateral incisors area . Miniscrews were loaded with an average force of 150 - 200 gram - force / side via elastic modules, and ligature wire was used to connect the tpa, almost parallel to the occlusal plane of the miniscrew . All of the other teeth were bonded from 2nd molar to 2nd molar with standard edgewise brackets and tubes (dentaurum, ispringen, germany), and leveling and aligning were performed before the termination of distalization to reduce any possible anchorage loss after distalization . The final arch wire was 0.018-in stainless steel . A cone - beam computed tomography (cbct) scan (newtom 3 g; quantitative radiology, verona, italy) was performed to evaluate the precise position of mini screws relative to adjacent teeth and the direction of the force relative to molar teeth . After maxillary 1st molars were driven to super class i relationship, distalization was terminated and a final impression was taken for final evaluation . Digital model analysis of molars was used to evaluate changes in the 3d position of the maxillary molars . Digital scans of models before and after distalization were prepared via a maestro 3d dental scanner (age solutions s.r.l ., maestro ortho studio software version 2.5 (age solutions s.r.l .) Was used to analyze the digital models . The palatal rugae possesses unique characteristics, and as it tends to exhibit reasonable stability during growth, it may serve as a suitable reference point for longitudinal cast analysis.23 as the patients in the current study were not in an active growth period and no potential incisor movement was anticipated during the distalization period, as in nalcaci et al.,1 the distance from the central point of the incisive papilla to the mesiopalatal cusp of the 1st maxillary molars was used to measure distalization . The distances between the mesiopalatal cusps of the left and right molars were measured to evaluate intermolar width . The angles between the incisive papilla and the distopalatal and mesiobuccal cusps of the 1st maxillary molars were measured to evaluate the rotation of maxillary molars1 (figure 2). Interocclusal space at the mesiobuccal, distobuccal, and mesiopalatal cusps of the 1st maxillary molars was evaluated by occlusion inspection, and maestro 3d scanner and software was used to evaluate vertical movement . After putting the casts in the maximum intercuspal position, the spaces between the maxillary 1st molar cusps and the antagonist tooth at t1 and t2 were measured via a 0.5-mm interval scale with a color coded tool (figure 3). For cbct analysis, digital imaging and communications in medicine (dicom) files were imported to a dolphin 3d 11 (dolphin imaging, chatsworth, ca, usa). The angle between this line and a line drawn parallel to the occlusal plane was measured on right and left 3d sagittal view . In 3d axial view, lines between the helix and the miniscrews on the right and left sides were drawn, and the angle between those lines (axial angle) was measured (figure 4). Measurements were repeated one month after the initial measurements, and the reliability of the measurements was calculated via the intraclass correlation test . Reliability was between 79.7 and 97.0 for linear measurements, and between 93.8 and 96.6 for angular measurements . The paired t - test was used to compare measurements acquired before and after distalization in a digital cast . A probability of p <0.05 was deemed to indicate statistical significance . Pearson's correlation was used to evaluate the relationships between cbct parameters and digital cast parameters . Statistical package for the social sciences ver . 13.0 (spss inc ., the palatal rugae possesses unique characteristics, and as it tends to exhibit reasonable stability during growth, it may serve as a suitable reference point for longitudinal cast analysis.23 as the patients in the current study were not in an active growth period and no potential incisor movement was anticipated during the distalization period, as in nalcaci et al.,1 the distance from the central point of the incisive papilla to the mesiopalatal cusp of the 1st maxillary molars was used to measure distalization . The distances between the mesiopalatal cusps of the left and right molars were measured to evaluate intermolar width . The angles between the incisive papilla and the distopalatal and mesiobuccal cusps of the 1st maxillary molars were measured to evaluate the rotation of maxillary molars1 (figure 2). Interocclusal space at the mesiobuccal, distobuccal, and mesiopalatal cusps of the 1st maxillary molars was evaluated by occlusion inspection, and maestro 3d scanner and software was used to evaluate vertical movement . After putting the casts in the maximum intercuspal position, the spaces between the maxillary 1st molar cusps and the antagonist tooth at t1 and t2 were measured via a 0.5-mm interval scale with a color coded tool (figure 3). For cbct analysis, digital imaging and communications in medicine (dicom) files were imported to a dolphin 3d 11 (dolphin imaging, chatsworth, ca, usa). The angle between this line and a line drawn parallel to the occlusal plane was measured on right and left 3d sagittal view . In 3d axial view, lines between the helix and the miniscrews on the right and left sides were drawn, and the angle between those lines (axial angle) was measured (figure 4). Measurements were repeated one month after the initial measurements, and the reliability of the measurements was calculated via the intraclass correlation test . Reliability was between 79.7 and 97.0 for linear measurements, and between 93.8 and 96.6 for angular measurements . The paired t - test was used to compare measurements acquired before and after distalization in a digital cast . A probability of p <0.05 was deemed to indicate statistical significance . Pearson's correlation was used to evaluate the relationships between cbct parameters and digital cast parameters . Statistical package for the social sciences ver . 13.0 (spss inc ., maxillary 1st molars were moved distally an average of 2.3 1.1 mm, and the difference between the right and left sides was not statistically significant (p = 0.63). The mean duration of distalization was 6.8 2.8 months (range 2 - 14 months), thus the average rate of distalization achieved via the method utilized in the current study was 0.4 0.2 mm / month . Maxillary 1st molars were slightly rotated mesially around the palatal root after distalization, though the amount of rotation was not statistically significant (this rotation is shown with a minus sign in table 2). The mean intermolar width increase was 2.9 1.8 mm (range 0.6 - 6.8 mm). The examination of vertical movement of three cusps of the maxillary molars showed relative molar intrusion that was statistically significant, except in the left mesiopalatal cusp (table 2). Orthodontists are frequently faced with mild to moderate class ii molar and canine relationships that they prefer not to treat via the extraction of premolars, and for which treatment via auxiliary methods is often unsatisfactory due to insufficient patient compliance . Many studies on intraoral maxillary molar distalization without patient cooperation have been performed, in order to solve the patient compliance problem associated with extraoral distalization appliances.110111220222425262728 in the present study, maxillary 1st molars were distalized via mini - implant - aided tpa . In mia - tpa, direct traction from elastic modules spanning from a miniscrew to a tpa helix is used to achieve molar distalization . Miniscrews were inserted into the palate in this study, wherein attached gingiva is not a major concern, in contrast to the buccal side . Moreover, there is more interdental space in the palate relative to the buccal side, and larger miniscrews (2 mm in diameter) can be inserted . In the present study, the mesiodistal axis of molar movement can be controlled by adjusting the vertical position of miniscrews and/or the direction of the line of action relative to furcation . Thus, bodily tooth movement is promoted, rather than tipping movement . In the current study, a removable tpa was used which made possible both proper adjustment of the line of action, and the control of molar rotation . The appliance is of a reduced size relative to other similar devices, and was tolerated by all of our patients without any concerns . There were also no problems reported with regard to the maintenance of oral hygiene . In the treatment protocol used in the current study, the elastic properties of the modules were lost relatively quickly in the oral cavity however, so the force applied was intermittent in nature, and they needed to be changed at least every 3 weeks . While niti pull coils allow for the application of more continuous force for distalization, they require a distance of at least 20 mm between the miniscrew and helix, to produce an appropriate level of force . In this study, 3d models were used for the 3d analysis of maxillary molar movements . Two - dimensional (2d) analysis by superimposing pretreatment and posttreatment cephalometric tracings has commonly been used in orthodontic studies.26 there are some disadvantages of cephalometric radiographs and superimpositions . The superimposition of lateral cephalograms is not easily accomplished, because the relevant radiographic landmarks and structures are frequently difficult to accurately trace . Also, the process of superimposing can be technique - sensitive.29 cephalometric superimposition can reveal positional changes in the maxillary and mandibular dentition in both vertical and sagittal dimensions, but not in the buccopalatal direction . In contrast to 2d analysis, 3d analysis of serial dental models can provide further information on tooth movements, particularly in the buccolingual or transverse direction . The projection of bilateral teeth on the midsagittal plane also causes greater tracing errors because of the difficulty in identifying the bilateral teeth . Consequently, the separate investigation of the tooth movements on each side, which can be achieved relatively easily via 3d models, is seldom attempted via cephalometric analysis.26 in this study, the distal movement of molars was measured along the line connecting the central point of the incisive papilla and the mesiolingual cusp of the 1st molar . While this measurement does not reflect true distal movement, measuring the intermolar change can provide a clear determination of both the distal and buccal movement of maxillary molars according to best available anatomical reference points . Applying a distal force to maxillary molars moved them in three dimensions . With the device used in the current study, maxillary 1st molars were distalized 2.3 1.1 mm . In other studies based on intraosseous anchorage appliances, distalization was between 1.3 and 6.4 mm.110202224262728 distalization was higher in studies that used tooth - borne appliances such as pendulums or dual force distalizers supported by mini - implants for molar distalization.222527 the main reasons for the reduced distal movement of maxillary molars in our study were related to characteristics of our sample . The majority of patients in our sample required less than one cusp of distal movement . Nalcaci et al.1 distalized molars via miniscrews with open coil springs by an average 3.95 1.35 mm . Gelgr et al.10 distalized molars by an average 3.9 1.6 mm via screws supported by tpas and niti open coil springs . Yamada et al.20 used miniscrews and elastomeric chain or niti closed coils for distalization, and average molar movement was 2.8 1.6 mm . The mean duration of distalization in our study was 6.8 2.8 months, thus the rate of distalization was 0.4 0.2 mm / month . In other studies, the duration of distalization was between 4.5 and 10.2 months.11020222425262728 nalcaci et al.1 reported an average duration of 9.61 2.1 months, and a rate of 0.41 mm / month . Yamada et al.20 reported a duration of 8.4 months and a rate of 0.33 mm / month . In this study, mean maxillary 1st molar rotation was -0.86 4.6 and -0.35 4.1 in the right and left sides respectively . Nalcaci et al.1 reported 0.54 and 0.74 rotation in the right and left sides respectively . Buccal movement of molars was evident in the current study, with a mean increase of 2.9 1.8 mm in intermolar width . Many studies1112224 reported buccal movement of molars, while yamada et al.,20 and lai et al.,26 reported palatal movement of molars . Tpas made up of 0.8 mm round stainless steel wire do not have enough strength to resist expansion due to distal traction force . Intrusion was the hallmark of this appliance, and numerous authors reported intrusion of molars in their studies.1222425 in a study similar to the current study, maxillary molars were intruded by an average of 0.3 0.8 mm.1 on the other hand, yamada et al.20 and lai et al.26 reported extrusion of molars . In the majority of our cases, the line of action of force passed through the furcation and the center of resistance of molars in an apical direction, concomitant intrusion of 2nd molars and premolars could be masked by mandibular overclosure, if present at all . So, we did not measure absolute intrusion via this type of measurement, instead interocclusal discrepancy was measured . The absolute measurement of intrusion is more accurately achieved via cbct before and after distalization . However, a second cbct was not approved by the relevant research ethics committee . Two miniscrews of 1.4 8.0 mm inserted in the same patient failed in the current study . They were found to be loose at the first visit after fitting . Larger miniscrews (2.0 10.0 mm) were then inserted in this patient, but these also became loose so the patient was removed from the study . Among the remaining patients, 3 screws (5.7%) became loose . In these cases, excessive contact between the tpa and the miniscrew was the probable cause of screw failure in these cases . The mia - tpa is an appliance that can achieve absolute anchorage, and successfully drive maxillary 1st molars distally.
Acute liver failure (alf) is a rare but life - threatening illness which occurs mostly in young adults without any known underlying liver disease [1, 2]. In the united states, the most common cause of alf is acetaminophen overdose (39%) but in 13% of cases, the causes are indeterminate . Epstein - barr virus (ebv) is a dna virus associated with infectious mononucleosis (i m) in children and young adults . It may cause moderate and transitory increase of liver enzymes; however, in rare cases, severe liver injury and even fatal alf may occur . We report a case of successful orthotopic liver transplantation (olt) in a 67-year - old female who presented with ebv - associated alf . A 67-year - old woman with bilateral osteoarthritis of the knee, hypothyroidism and recurrent deep vein thrombosis presented with 3 weeks of fatigue and a 1-week history of jaundice . Her medications include diclofenac, rivaroxaban, desiccated thyroid, alprazolam, selenium and glucosamine . Four weeks prior to admission, the patient was found to have mildly elevated serum aminotransferases during a routine visit . One week prior to admission, she reported to become jaundiced and her aminotransferases were found to be elevated to about 1,000 one day prior to admission, her serum alanine aminotransferase (alt) was 3,542 u / l, aspartate aminotransferase (ast) was 3,610 u / l and her inr was 2.7 . On admission, her vital signs were normal, and a physical exam was positive only for jaundice . Laboratory findings included a white blood cell count of 13,200/l (neutrophils 84.9%, lymphocytes 12.4%, monocytes 3.5% and eosinophils 0.2%). U / l, ast was 1,236 u / l, alp was 215 u / l and inr was 2.9 . Hepatic artery, hepatic veins, and portal vein were patent, with a normal direction of flow by doppler study . Serum acetaminophen level was 7 g / ml . Hav antibody, hbsag, hcv and hev antibody were all negative . Blood tests for human immunodeficiency virus (hiv, hiv1/2 antibody and p24 antigen), cytomegalovirus (cmv), rubella and rubeola were negative . Antinuclear antibody, anti - mitochondrial antibody and anti - smooth muscle antibody were negative . The patient was initially diagnosed with alf possibly, secondary to diclofenac use, because she had begun to take diclofenac potassium 50 mg twice daily since 5 months ago . She was admitted to the icu and received multiple transfusions of fresh - frozen plasma (ffp) to correct her coagulopathy . She was given midodrine and octreotide and underwent hemodialysis for hepatorenal syndrome . Despite multiple transfusions of ffp, her coagulopathy worsened, and her inr was 3.7 the following day . Her serum alt and ast decreased rapidly in the following 2 days and the patient became more lethargic and somnolent, and difficult to arouse . Four days after admission, she underwent a successful liver transplantation from a cmv - negative, ebv (igg) positive cadaveric donor . The explanted liver was significantly smaller than normal indicating acute failure of the liver (fig . After transplantation, she received thymoglobulin, methylprednisolone taper and was later switched to prednisone taper, and she also started tacrolimus 4 days after transplantation . (a) small explanted liver weighing 720 g. (b, c) histologically, the liver showing 75% of parenchyma extinction and massive necrosis with sinusoidal lymphocytosis and atypical lymphocytes (h&e, b: 100; c: 400). (d) ebv in situ hybridization (left, 600) and cd20 immunostain (right, 600) showing numerous ebv - positive atypical b lymphocytes . Her histological report of the explanted liver showed that there was 75% massive hepatic parenchyma necrosis (fig . There were also acidophilic bodies, microvesicular steatosis and mild macrovesicular steatosis present in the residual hepatic parenchyma . There was a mixed population of inflammatory cells with predominant lymphocytes with sinusoidal lymphocytosis (fig . 1d, left) and positive for cd20 immunostain (1:100 dilution, horse radish peroxidase technique; dako, carpenteria, ca) (fig . Hsv staining of the liver tissue was negative although the serum antibodies were slightly higher than normal . A review of the liver biopsy 5 days prior to admission showed about 25% hepatic parenchyma necrosis with bridging necrosis (fig . A whole blood assay for ebv dna using pcr of edta anticoagulated peripheral blood showed 462,000 copies / ml . She was started on intravenous acyclovir immediately after the diagnosis of ebv infection, and later she was switched to oral acyclovir which was administered for 3 months . Two weeks after transplantation, ebv viral capsid antigen (vca)-igg test was positive and vca - igm was negative, ebv d early antigen (ea - d) and epstein - barr nuclear antigen (ebna) were negative . One month later, the transplanted liver underwent biopsy and showed negative cd20 and ebv rna (data not shown). The patient is presently doing well 6 months after liver transplantation with no evidence of clinical ebv infection or lymphoproliferative disease . (a, b) liver showing 25% parenchyma necrosis with bridging necrosis, acidophilic bodies, mixed inflammatory infiltrate and sinusoidal lymphocytosis (a: 100; b: 400). (c) ebv in situ hybridization showing occasional ebv - positive b lymphocytes (400, arrows). Lt: liver transplant; acv: acyclovir; alp: alkaline phosphatase; ast: aspartate aminotransferase; alt: alanine aminotransferase . We describe a case of alf - associated with ebv infection in a previously healthy elderly woman that underwent successful emergent liver transplantation . Fulminant liver failure, is a sudden and severe hepatic injury in patients without previous liver diseases . The clinical manifestation usually includes loss of hepatic function, coagulopathy, hepatic encephalopathy and in many cases, progressive multi - organ failure [1, 2]. Despite recent advances in intensive care management, it has been reported that 5-year survival rate is about 80% in patients who underwent emergency liver transplantation for alf . The etiologies of alf vary greatly by country and have evolved over time . In the unites states, drug - induced alf, mainly acetaminophen, accounts for about 58% of cases, and hepatitis a and b accounts for 14% of cases according to the us acute liver failure study group data . These cases often follow a relatively insidious presentation before patients quickly develop liver failure, and rates of survival are poor without transplantation . Recent studies showed that the cause may be related to ebv infection . In our case, the cause of alf was initially thought to be diclofenac, because the patient had been taking diclofenac for about 5 months prior to admission, and other causes such as acetaminophen, infectious agents and metabolic diseases were excluded through serological tests; however, the biopsy and blood ebv dna pcr results confirmed that ebv is the major cause of alf, and diclofenac may have played no or a minor role in this process . Ebv - associated alf is very rare, accounting for only 0.21% of adult alf cases . It usually occurs in adults younger than 40 years of age, which is consistent with the fact that young adult patients tend to have symptomatic infectious mononucleosis [10, 11]. It has been reported that patients with neoplasm, cmv and hiv and patients on immunosuppressant drugs are at high risk for ebv - associated alf, although recent studies also showed that immunocompetent patients can also develop alf, indicating that other unknown risk factors may also be involved in the development of ebv - associated alf [12 - 14]. In adult aged 60 years or older, there were only two cases of ebv - associated alf in the literature; however, both of them had impaired immune system: one had just started steroids and the other had recent heart surgery [15 - 17]. In our case, the patient does not have any of the above mentioned problems and did not take any immunosuppressant medications . This is the first case of ebv reactivation - associated alf in a healthy elderly patient . This indicates that in older immunocompetent patients presenting with alf, a primary ebv infection or ebv reactivation should be considered when other causes have been excluded . Early diagnosis of ebv - associated hepatitis was difficult as reliable diagnostic criteria are not clearly defined . Molecular approaches, such as ebv - dna pcr or eber - rish, are frequently used to diagnose ebv - related hepatitis [9, 18]. Serological ebv tests such as vca - igm, vca - igg, ea - d and ebna can also help to detect a recent infection or a prior infection, or a reactivated ebv infection, although they are not reliable, especially in the elderly, as they have significantly higher titers of the serum tests than young adults [9, 18]. In our case, the liver biopsy 1 week before admission showed 25% hepatic necrosis, some atypical b lymphocytes with positive ebv rna . In explanted liver, there was over 75% necrosis, significantly increased atypical b cells and strongly positive expression of ebv rna, which is consistent with the progression of the liver failure . We also tested the blood ebv level by pcr 4 days after liver transplantation and it was extremely high, indicating a severe active ebv infection . The ebv serology tests 10 days after liver transplantation showed very high vca - igg but normal vca - igm, indicating that this is an ebv reactivation . Moreover, the liver enzyme showed cholestatic enzyme changes after transplantation with continuously increased alp . We think that the increased alp was due to damage caused by ebv infection to the implanted liver . Treatment with acyclovir should be strongly considered after liver transplantation for alf from ebv to prevent infection of the liver graft during the period of high level immunosuppression . First, we were not able to diagnose ebv hepatitis until after evaluation of the explanted liver . Liver transplantation could be potentially avoided if an early diagnosis of ebv hepatitis could have been made . Second, the ebv pcr test was done after transplantation, when patient had already been on steroids . The extremely high ebv pcr value may not reflect the severity of ebv infection, as steroids can suppress the immune system and ebv could replicate faster than expected . Third, the serological test was done 10 days after we started acyclovir, which may not show the initial changes of ebv infection . In addition, the use of diclofenac in this patient since a few months ago might have caused some initial injury to the liver . Diclofenac is reported to cause liver injuries by two main mechanisms, hypersensitivity and metabolic aberration [19, 20]. Acute liver injury caused by diclofenac is thought to be due to hypersensitivity and usually presents within days to a few weeks after starting diclofenac, which can result in fatal liver failure . Metabolic aberration can cause persistent mild liver injury and once diclofenac is stopped, the liver can recover quickly . In our patient, diclofenac might have caused mild liver injury initially; however, the reactivation of ebv plays a major role in developing alf . We are confident that ebv is the cause of alf in the patient based on liver enzyme changes, characteristic histopathological changes, and most importantly strong ebv rna expression in the explanted liver . In conclusion, our case demonstrates that ebv infection or reactivation may be under - recognized as a cause of alf . Early recognition and diagnosis is extremely important, however difficult . In both healthy young and elderly adults with alf liver biopsy should be strongly considered in patients with unexplained alf with hepatitis pattern and ebv rna by in situ hybridization should be mandatory if sinusoidal lymphocytosis is present . Emergent liver transplantation is the only definitive treatment option, with very favorable prognosis for such patients . Treatment with acyclovir should be strongly considered after liver transplantation for alf from ebv to prevent infection of the liver graft during the period of high level immunosuppression.
Cranial nerves were composed of two different histological portions, glial and non - glial portion . The transitional zone refers to the junction between glial portion and the non - glial peripheral portion . The most outer layer of brain cortex is coated by pia glial membrane called glial limiting membrane or glial limitans10). Skinner12) reported the extent of glial out growth into the various cranial nerves and glial out spread of facial nerve was extended as far as 2.6 mm in length from the origin of brain stem . In general, root exit zone or root entry zone (rez) of cranial nerves means the area nerves come from the brainstem . Recently, some authors proposed to replace the term of rez by root exit point (rexp), root detach point (rdp), and root exit zone (rexz) in detail13). The purposes of this study were to measure the length of the central myelin, especially from root detach point to transitional zone and determine histological structures of central myelin of facial nerve . Twenty brain stems were obtained from formalin embedded cadavers . The 12 men and 8 women of cadavers ranged in age from 32 to 78 years (mean, 58 years). During the brain stems were served, care should be taken to preserve the cisternal segment of facial nerve . We obtained 40 facial nerves but 17 facial nerves were put out of shape during preparation for histological study . Twenty three facial nerves could be examined properly . To make a facial nerve - brainstem tissue block, we cut the midline of brain stem into halves and slice them along the facial nerve . First, one slice was stained with hematoxylin and eosin (h&e) for screening the shape of facial nerve and brain stem . And then, other slices were stained with periodic acid - schiff (pas) for peripheral myelin and glial fibrillary acid protein (gfap) for central myelin and glial membrane . After staining, photomicrographs of each section were taken and measure the distance of medial rez, from detach point of facial nerve at the brain stem to the most distal part of central myelin, and thickness of glial membrane of central myelin of rez . The thickness of glial membrane was obtained at two different points, the thickest point of proximal and distal area of central myelin . Usually to correct for the shrinkage error during fixation, occulomotor nerve of same specimen was used as a standard . But we did not calculate the percentage of facial nerve shrinkage because of being used pre - fixed specimens with formalin . With the h&e staining, the shape of facial nerve was easily determined (fig . Special staining methods, such as pas and gfap, could be clearly distinguished the facial nerve into peripheral and central myelin . Peripheral myelin was stained dark blue with pas staining and central myelin was stained dark brown with gfap (fig . 2). Glial outgrowth from the brain stem, central myelin, ends in dome or cone formation . The outermost membrane surrounding the brain stem, glial limiting membrane, continues to the central myelin could be visible . The mean thickness of glial membrane of central myelin was 66.5 um (40 - 110 um) at proximal and 7.4 um (range of 5 - 10 um) at distal (table 1). The length of medial rez was mean 2.6 mm (range of 1.6 - 3.5 mm) (table 2). There is no clear description of the relationship between the rez and the transitional zone . In the strict sense, rez means the area nerve comes from the brainstem and transitional zone refers to glia - schwann cell border . The term of rez was defined by jannetta as a junctional area between central and peripheral myelin6). Many authors used the terms of rez and transitional zone interchangeably1,3,11). In a recent study, tomii et al.13) described the transitional zone as the region where the myelin sheath is composed of both central glial and schwann cell myelin . They have proposed the use of terms root exit point (rexp), rdp (medial detach point) and transitional zone instead of rez . And they stressed the rdp appears to be a good landmark for the microvascular decompression surgery . In 1915, henschen5) has been descried that cranial nerve was composed of two different segments, such as glial and non - glial portion . Skinner12) also reported several characteristics of various cranial nerves as follows . From the brain there is an outgrowth of glia cells, astrocyte and oligodendroglia, along the cranial nerve trunks and the extent of glial outgrowth varies in different nerves . Many investigators named the glial outgrowth segment of cranial nerve as cns segment of cranial nerve, central myelin, or central glial myelin2,10,13). To measure the exact length of central myelin of facial nerve is difficult because of its irregular distal margin . Skinner12) reported that the glial outspread in the facial nerve extended as far as 2.6 mm from the plane of superficial origin . Adams1) described that the transitional zone is only 1 to 3 mm in length . Jannetta6) also reported the distance to the peripheral boundary of the rez was 0.5 to 1 cm in the fifth, seventh, and ninth cranial nerves . In 2003, he measured the length of the lateral and medial transitional zone were mean 1.9 mm and 0.58 mm, respectively . We measured the distance from medial detach point of brain stem to the most distal glial segment of facial nerve . Glial portion of the cranial nerve, central myelin, is composed of fibrillary astrocyte and oligodendroglia cells12). And it has a structure similar to that of white matter of the cns, consisting of parallel traveling nerve fibers, and lacks funicular structure . Nerve fibers are embedded in supporting tissue, but endo-, peri, and epineurium are absent . De ridder et al.2) stated the layer of pia mater surrounds the central myelin . Glial limiting membrane, pia glial membrane, or glial limitans, is a thin barrier of astrocyte foot process associated with the parenchymal basal lamina surrounding the brain10). The main function of glial limiting membrane is to act as a physical barrier of the cns . We observed that the glial limiting membrane extending from the outermost layer of brain stem, strong stained with gfap, surrounds the central myelin . We called the layer closing the central myelin as glial sheath like epineural sheath of peripheral nerve . We also measured the thickness of glial sheath of facial nerve and it becomes thin at distal portion than proximal . The glial sheath of central myelin may act as a barrier of the nerve fiber from vascular compression . The cause of hemifacial spams is generally known as that facial nerve compressed by blood vessels located at the rez . The mechanism of hemifacial spasm is thought that thinning or defect of neural sheath of facial nerve because of vascular compression may result in developing cross talking of neural transmission4,9). Jannetta et al.7) reported that myelin defect exist in the rez in patient of hemifacial spasm . There is no clear description of the relationship between the rez and the transitional zone . In the strict sense, rez means the area nerve comes from the brainstem and transitional zone refers to glia - schwann cell border . The term of rez was defined by jannetta as a junctional area between central and peripheral myelin6). Many authors used the terms of rez and transitional zone interchangeably1,3,11). In a recent study, tomii et al.13) described the transitional zone as the region where the myelin sheath is composed of both central glial and schwann cell myelin . They have proposed the use of terms root exit point (rexp), rdp (medial detach point) and transitional zone instead of rez . And they stressed the rdp appears to be a good landmark for the microvascular decompression surgery . In 1915, henschen5) has been descried that cranial nerve was composed of two different segments, such as glial and non - glial portion . Skinner12) also reported several characteristics of various cranial nerves as follows . From the brain there is an outgrowth of glia cells, astrocyte and oligodendroglia, along the cranial nerve trunks and the extent of glial outgrowth varies in different nerves . Many investigators named the glial outgrowth segment of cranial nerve as cns segment of cranial nerve, central myelin, or central glial myelin2,10,13). To measure the exact length of central myelin of facial nerve is difficult because of its irregular distal margin . Skinner12) reported that the glial outspread in the facial nerve extended as far as 2.6 mm from the plane of superficial origin . Adams1) described that the transitional zone is only 1 to 3 mm in length . Jannetta6) also reported the distance to the peripheral boundary of the rez was 0.5 to 1 cm in the fifth, seventh, and ninth cranial nerves . In 2003, he measured the length of the lateral and medial transitional zone were mean 1.9 mm and 0.58 mm, respectively . We measured the distance from medial detach point of brain stem to the most distal glial segment of facial nerve . Glial portion of the cranial nerve, central myelin, is composed of fibrillary astrocyte and oligodendroglia cells12). And it has a structure similar to that of white matter of the cns, consisting of parallel traveling nerve fibers, and lacks funicular structure . Nerve fibers are embedded in supporting tissue, but endo-, peri, and epineurium are absent . De ridder et al.2) stated the layer of pia mater surrounds the central myelin . Glial limiting membrane, pia glial membrane, or glial limitans, is a thin barrier of astrocyte foot process associated with the parenchymal basal lamina surrounding the brain10). The main function of glial limiting membrane is to act as a physical barrier of the cns . We observed that the glial limiting membrane extending from the outermost layer of brain stem, strong stained with gfap, surrounds the central myelin . We called the layer closing the central myelin as glial sheath like epineural sheath of peripheral nerve . We also measured the thickness of glial sheath of facial nerve and it becomes thin at distal portion than proximal . The glial sheath of central myelin may act as a barrier of the nerve fiber from vascular compression . The cause of hemifacial spams is generally known as that facial nerve compressed by blood vessels located at the rez . The mechanism of hemifacial spasm is thought that thinning or defect of neural sheath of facial nerve because of vascular compression may result in developing cross talking of neural transmission4,9). Jannetta et al.7) reported that myelin defect exist in the rez in patient of hemifacial spasm . Our study shows the length of medial rez is mean 2.6 mm (range of 1.6 - 3.5 mm) and the rez is covered by glial sheath continued from glial limiting membrane of brain stem.
Falls are both common and debilitating in patients with parkinson's disease (pd). They have devastating consequences for affected individuals, often leading to injuries, secondary immobility, and reduced quality of life . Survival is reduced once falls have occurred, although a recent report failed to identify a relationship between falls and mortality risk in pd . Falls are also important for the public health system, as the costs associated with falls and fall - related injuries are enormous . A prospective 20-year follow - up of 136 patients with newly diagnosed pd confirmed the high prevalence of falls (87%) and resulting fractures (35%). For the management of environmental factors such as slippery floors, loose rugs, poor lighting, or inadequate footwear may also contribute . There is an increasing awareness that freezing of gait - a sudden and episodically occurring inability to generate effective forward stepping movements - is one of the leading causes of falls, presumably because patients are caught by surprise due to the often unexpected nature of freezing events . Recent work has underscored the additional importance of cognitive impairment as a key factor contributing to both falls and freezing; falls are an issue in demented patients in particular . Possible explanations for this link include more prominent dopaminergic denervation of the caudate nucleus or more generalized cholinergic dysfunction . Preventing falls is generally perceived as being difficult, but is not impossible . Given the complex multifactorial nature of falls and the experience in elderly non - parkinsonian populations, a multidisciplinary approach appears preferable, but for patients with pd this strategy is not yet backed by scientific evidence . Crucial elements in the therapeutic approach include optimizing pharmacotherapy - increasing pd medication for dopa - sensitive signs, and stopping sedative drugs - and tailored physiotherapy, based on evidence - based practice guidelines . Here, we highlight a few important new developments in this field . The quest should not stop when one causative factor has been found because falls are typically multifactorial in origin . A recent prospective study in 101 patients with early - stage pd assessed the best prediction was reached by combining disease - specific measures (such as pd severity and freezing of gait severity) with balance measures (such as the occurrence of symptomatic orthostatic hypotension, the tinetti total score, and the extent of postural anterior - posterior sway). However, even this combination attained a sensitivity and specificity for predicting falls of only around 80% . Including cognitive measures that are more specific may further improve these predictive scores, but this remains to be examined . Particular emphasis should be placed on testing for freezing of gait - using a series of provoking tests, including rapid 360 degree turns on the spot - and for frontal executive dysfunction . Specific attention should be paid to fear of renewed falling as this is common in pd . Fear of falling is not only a risk factor for renewed falls, it may also lead to secondary immobilization with all its related adverse consequences . Most balance deficits are resistant to dopaminergic medication . However, gait problems - including freezing of gait - can improve with dopaminergic medication, although doses that are higher or more frequent than those typically needed to increase hand functioning may be required . Recent work points to a possible role for cholinesterase inhibitors in the treatment of gait and balance deficits, both in pd and in alzheimer's disease, given that many axial motor deficits may result from misbalance between central cholinergic and dopaminergic systems . It has become clear that stereotactic deep brain surgery should be reserved for patients whose gait and balance deficits are still levodopa - responsive pre - operatively . Several studies are comparing bilateral stimulation of two different targets, namely the subthalamic nucleus (stn) and the internal globus pallidus . The initial results indicate that both approaches are effective in providing short - term relief of motor symptoms, but one study suggested internal globus pallidus stimulation may offer better long - term outcomes for gait and balance deficits . This difference was not evident in the largest study, although falls after a 2-year follow - up tended to be more common after stn stimulation, but this requires more study . In particular, stn stimulation has been associated with a worsening of gait and balance deficits, not only in the immediate postoperative phase, but also several years after follow - up . Adjusting the stimulation parameters (e.g., markedly lowering the stimulation frequency) may be helpful in such patients . The pedunculopontine nucleus has been studied as a promising new target, specifically for gait and balance deficits, but so far the results have not been very impressive . The evidence - based guidelines on physiotherapy for pd were recently updated, providing a menu of treatment modalities to improve mobility and reduce falls . Examples of evidence - based physiotherapy strategies include cueing techniques, cognitive movement strategies, and the use of exercise . Rhythmic auditory or visual cues can improve gait in pd, including freezing of gait . New inventive cueing approaches include walking glasses with different patterns of visual and auditory stimulation and mental singing while walking . Another promising approach, especially for pd patients with freezing of gait, is the use of cycling, the skill for which can be remarkably preserved in some patients . Evidence based mainly on the effects of cueing on laboratory measures of gait and balance remains; the challenge is to ascertain an enduring clinical improvement in daily life, including a reduction of falls . Fears that cues might worsen the tendency to fall - for example, by increasing overall mobility - have not been substantiated . A systematic review concluded that exercise in patients with pd results in improvement in postural stability and balance task performance . However, power and quality of exercise studies have hitherto been insufficient to make definitive recommendations . Future randomized controlled trials will look into the (cost) effectiveness of exercise to reduce falls . There is a growing body of literature on the use of treadmill training for gait rehabilitation in patients with neurologic disorders in general and for patients with pd in particular . A recent cochrane review concluded that treadmill training may help to improve gait akinesia in pd, but the effect on falls remains unclear . Another interesting new development is the use of robotics, which can assist pd patients in making medio - lateral anticipatory weight shifts in preparation for taking a step . The initial results are promising, and such new techniques now need to be implemented in situations that are more realistic to evaluate the clinical merits of these techniques in relation to everyday gait performance and falls . Delivering such specific physiotherapy interventions to patients with pd a large cluster - randomized trial showed that a community - based professional network with trained expert physiotherapists improved the quality of physiotherapy care and reduced health care costs, but without health benefits for patients . The quest should not stop when one causative factor has been found because falls are typically multifactorial in origin . A recent prospective study in 101 patients with early - stage pd assessed the best prediction was reached by combining disease - specific measures (such as pd severity and freezing of gait severity) with balance measures (such as the occurrence of symptomatic orthostatic hypotension, the tinetti total score, and the extent of postural anterior - posterior sway). However, even this combination attained a sensitivity and specificity for predicting falls of only around 80% . Including cognitive measures that are more specific may further improve these predictive scores, but this remains to be examined . Particular emphasis should be placed on testing for freezing of gait - using a series of provoking tests, including rapid 360 degree turns on the spot - and for frontal executive dysfunction . Specific attention should be paid to fear of renewed falling as this is common in pd . Fear of falling is not only a risk factor for renewed falls, it may also lead to secondary immobilization with all its related adverse consequences . Most balance deficits are resistant to dopaminergic medication . However, gait problems - including freezing of gait - can improve with dopaminergic medication, although doses that are higher or more frequent than those typically needed to increase hand functioning may be required . Recent work points to a possible role for cholinesterase inhibitors in the treatment of gait and balance deficits, both in pd and in alzheimer's disease, given that many axial motor deficits may result from misbalance between central cholinergic and dopaminergic systems . It has become clear that stereotactic deep brain surgery should be reserved for patients whose gait and balance deficits are still levodopa - responsive pre - operatively . Several studies are comparing bilateral stimulation of two different targets, namely the subthalamic nucleus (stn) and the internal globus pallidus . The initial results indicate that both approaches are effective in providing short - term relief of motor symptoms, but one study suggested internal globus pallidus stimulation may offer better long - term outcomes for gait and balance deficits . This difference was not evident in the largest study, although falls after a 2-year follow - up tended to be more common after stn stimulation, but this requires more study . In particular, stn stimulation has been associated with a worsening of gait and balance deficits, not only in the immediate postoperative phase, but also several years after follow - up . Adjusting the stimulation parameters (e.g., markedly lowering the stimulation frequency) may be helpful in such patients . The pedunculopontine nucleus has been studied as a promising new target, specifically for gait and balance deficits, but so far the results have not been very impressive . The evidence - based guidelines on physiotherapy for pd were recently updated, providing a menu of treatment modalities to improve mobility and reduce falls . Examples of evidence - based physiotherapy strategies include cueing techniques, cognitive movement strategies, and the use of exercise . Rhythmic auditory or visual cues can improve gait in pd, including freezing of gait . New inventive cueing approaches include walking glasses with different patterns of visual and auditory stimulation and mental singing while walking . Another promising approach, especially for pd patients with freezing of gait, is the use of cycling, the skill for which can be remarkably preserved in some patients . Evidence based mainly on the effects of cueing on laboratory measures of gait and balance remains; the challenge is to ascertain an enduring clinical improvement in daily life, including a reduction of falls . Fears that cues might worsen the tendency to fall - for example, by increasing overall mobility - have not been substantiated . A systematic review concluded that exercise in patients with pd results in improvement in postural stability and balance task performance . However, power and quality of exercise studies have hitherto been insufficient to make definitive recommendations . Future randomized controlled trials will look into the (cost) effectiveness of exercise to reduce falls . There is a growing body of literature on the use of treadmill training for gait rehabilitation in patients with neurologic disorders in general and for patients with pd in particular . A recent cochrane review concluded that treadmill training may help to improve gait akinesia in pd, but the effect on falls remains unclear . Another interesting new development is the use of robotics, which can assist pd patients in making medio - lateral anticipatory weight shifts in preparation for taking a step . The initial results are promising, and such new techniques now need to be implemented in situations that are more realistic to evaluate the clinical merits of these techniques in relation to everyday gait performance and falls . Delivering such specific physiotherapy interventions to patients with pd a large cluster - randomized trial showed that a community - based professional network with trained expert physiotherapists improved the quality of physiotherapy care and reduced health care costs, but without health benefits for patients . Asking about falls and their impact on daily functioning should be a standard part of the evaluation of patients with pd . While awaiting further evidence, neurologists should consider installing a multidisciplinary team approach to tackle the vexing problem of falls in patients with pd . Management involves a systematic search for risk factors for falling, and a subsequent multifactorial approach aimed at eliminating or alleviating all patient - related and environmental risk factors for falling . Apart from optimizing dopaminergic medication, cholinergic therapies are now beginning to enter the field of play as well . Fear of falling must be addressed, and immobilization must be avoided as long as independent movements can still be made reasonably safely . The multidisciplinary team should ideally consist of trained and experienced professionals who treat large numbers of patients . Using this integrated approach, the goal should be to at least reduce falls or perhaps prevent them altogether, restore mobility and independence, and thereby help to maintain the quality of life for patients with pd.
Prostate cancer (pca) is one of the major causes of cancer death in men worldwide . The molecular basis of the disease involves an irregular behavior of the functions mediated by the androgen receptor (ar). Human ar belongs to the nuclear receptor (nr) superfamily of transcription factors, which regulate gene transcription upon ligand binding . The structure of nrs is extensively documented in the literature, and in general, nrs share the following common organization: a variable amino - terminal activation function domain (af-1), a highly conserved dna - binding domain (dbd), a hinge region that contains the nuclear localization signal, a conserved c - terminal ligand - binding domain (lbd) comprising a 12 helical structure that encloses a central ligand binding pocket (lbp), and a second activation function domain (af-2) that is located at the carboxy - terminal end of the lbd and mediates ligand - dependent transactivation . Ar is activated by the endogenous hormone testosterone (tes) and its more potent metabolite dihydrotestosterone (dht), both of which bind in the lbp . The binding of these endogenous modulators induces a reorganization of helix 12 to the so - called agonist conformation, generating a structured hydrophobic surface (af-2) suitable for the recruitment of tissue - specific nr coactivators . Such nr coactivators can be thought of as master switches, directing and amplifying the subsequent transcriptional activity of the target nr . In a recent work, an additional secondary function site called binding function 3 (bf-3) has been reported on the surface of the ar that could also play a relevant role in the allosteric modulation of the af-2 . Nr drug development has traditionally focused on advancing full or partial agonists / antagonists interacting within the lbp of the lbd . Pca has been treated by intervention at the early stages through utility of classical antiandrogens, which act by displacing the natural hormones from the pocket and inducing a conformational change of the helix 12 so that coactivators cannot be recruited . Tissue specificity, detrimental side effects, and a loss of the pharmacological effect (acquired drug resistance) over time are major and ongoing concerns with such lbp targeting treatment regimes . It has been demonstrated that it is possible to inhibit the transcriptional activity of the nrs by directly blocking the critical receptor: coactivator interaction . This alternative approach to traditional nr modulation may furnish greater pharmacological insight and afford opportunities to modulate not only under tissue specific circumstances but without adversely affecting natural ligand binding and so preserving the beneficial / nondisease linked functions of the receptors . Specifically, the steroid receptor coactivator (src) family has been postulated as a feasible target for pharmacological intervention . The viability of targeting ar coactivator interaction using small molecules has been recently demonstrated . Moreover, it has been postulated that circumventing the lbp will overcome the problem of drug resistance in pca . Here we describe the discovery and characterization of a novel class of selective non - lbp true antiandrogens, characterized by full ar antagonism in inhibiting the recruitment of coactivators and lacking intrinsic partial agonistic properties . Mechanistically, these compounds are totally differentiated from the recent description of true lbp antiandrogens like mdv3100 and rd162, while their selectivity and druglike nature underpin the potential of a non - lbp intervention strategy in advanced prostate cancer resistant to classical therapy, first described for the true non - lbp targeting antiandrogens pyrvinium pamoate (pp) and harmol hydrochloride (hh). The biological data obtained both on target with time - resolved fluorescence resonance energy transfer (tr - fret)/fluorescence polarization (fp) assays and in cellular pca models demonstrate the non - lbp antagonist activity of the series and an alternative mechanism of inhibition, furnishing a new class of nonpeptidic, small molecule ar: coactivator selective disruptors as leads for the development of novel treatments for prostate cancer . A virtual (computational) screen of six vendor compound databases (see experimental section) was performed through a combination of 3d pharmacophore generation and docking . Seven x - ray structures of coactivator peptide bound ar were used to define key ligand - derived pharmacophoric features of the most represented motifs occurring in known ar coactivators . Initially, common key interaction motifs within the peptide of the form fxxlf, lxxll, or fxxlw were considered to generate a consensus af-2 pharmacophore . Subsequently, a second site - derived pharmacophore model was advanced based on the specific characteristics of the androgen receptor af-2 region, which demonstrates known selectivity toward the fxxlf coactivator motif (figure 1b). The cocrystallization of the ar lbd bound with dht in the presence of the fxxlf peptide (pdb i d 1t7r) provided the structural basis of the af-2 interaction for docking studies . Virtual screening and identification of diarylhydrazide scaffolds . (a) a series of coactivator peptides cocrystallized in the af-2 groove was employed; for illustrative purposes we present the fxxlf coactivator motif from pdb entry 1t7r . The af-2 groove is represented in dark gray . For clarity reasons, only lys720 and glu897 are shown and dht is not illustrated; (b) a 3d pharmacophore model was derived containing the common features between ar coactivators and the two aromatic features of the fxxlf motif . Pharmacophores were used to screen vendor compound databases and to guide the docking of putative hits into the af-2 site . (c, d) two first round actives 1 (mdg173) and 2 (mdg15) docked poses in the af-2 site, with the surface rendered and only key amino acids shown . Partial mapping of initial hits to the pharmacophore suggested additional virtual screening to identify more potent family members . Images were generated with molecular operating environment (moe) and pymol . From the virtual screen, a first series of compounds with predicted target affinity was selected from commercially available databases (see experimental section) and evaluated for biological activity using tr - fret and fp techniques . This initial screen (figures 1c, d and 2) identified two small molecules, 1 and 2, both diarylhydrazides, as possible non - lbp ar antagonists . Non - lbp modulatory activity was evidenced by demonstration of an ic50 in the range of 50100 m in ar tr - fret coactivator displacement assay and their inability to displace bound fluorescently labeled ligand from the lbp through an fp assay . These first round hit molecules map only partially to the screening pharmacophore (figure 1c, d). Accordingly, an optimization round of screening was initiated to explore the utility of the scaffold for more effective disruption of ar: coactivator interaction . From these initial data, a simple molecular similarity search was performed (tanimoto coefficient> 70%) to furnish a new screening series of 37 compounds bearing the desired diarylhydrazide scaffold . This second round screen identified four small molecules (figure 2), 3 (mdg 483), 4 (mdg 292), 5 (mdg 506), and 6 (mdg 508), with improved activity (ic50 <50 m in an ar tr - fret assay). These ligands were taken forward for additional investigation and characterization . The series of diaryl - substituted hydrazides identified through the vs process (figure 2) inhibited the recruitment of the fluorescent labeled d11-fxxlf coactivator peptide in the presence of an agonist (dht) concentration equal to ec80 using time - resolved fret assays . D11-fxxlf is a peptide developed from random phage display technology that resembles the src family of coactivator proteins in its flanking sequence but that also has an ar n - terminal interaction domain . Thus, it is a biological mimic of the n - terminal and the src coactivator interactions with the lbd . A 12-point dose response curve was determined for those compounds that inhibited coactivator binding in the micromolar range, acting as full ar antagonists, 36 (figure 3a and table 1). The background signal, representing diffusion - enhanced fret in the absence of ar, was subtracted from the fret value of each compound and from the maximal signal, representing fxxlf - bound ar in presence of dht . Diarylhydrazides inhibit the ar recruitment of a fluorescent - labeled d11-fxxlf peptide but do not displace a potent fluorescent ligand from the ar - lbp . (a and b) compounds were tested in a tr - fret assay across a concentration range from 100 m to 45 nm in the presence of a concentration of dht = ec80 in ar - lbd wt (a) and ar - lbd t877a (b). Error bars represent the standard error of the mean (sem) for n = 6 values . Data was fitted using log antagonist concentration vs response (variable slope) with graphpad prism 5 (see experimental section for details). (c) fluorescence polarization data is plotted as percent maximal activity represented by ar - lbd and fluorophore complex (0% inhibition). The minimum control value represents free fluorophore (free f) in solution (100% inhibition). The tr - fret assay cannot differentiate between direct coactivator antagonists acting on the lbd surface and classical ar antagonists, which also functionally disrupt coactivator recruitment by displacing dht from the ligand binding pocket . To characterize the nature of the antagonist effect, compounds were tested for their ability to displace a potent fluorescent ligand (fluorophore) from the ar lbp through a fluorescence polarization (fp) assay at a single point concentration (50 m), using cyproterone acetate (cpa) at the same concentration as a reference, a known ar lbp - mediated antagonist . All compounds tested showed 0% inhibition of the ar - lbd and fluorophore complex, indicating a non - lbp - mediated mechanism of ar transactivation inhibition (figure 3c). Compound 3 gave an unusually high value of millipolarization units (mp), 20% higher than the maximal control (figure 3c). This could be indicative of solubility issues in the assay buffer and therefore could generate a false negative result . It is known that fp assay outcomes can be influenced by intrinsic fluorescence of the test compounds and/or light scattering phenomena due to poor solubility and precipitation . To minimize the possibility of such false negative or positive reporting, none of the compounds tested showed competing autofluorescence in the assay conditions or was shown to be a false negative . Results are shown in the supporting information (supplementary figure 2). To further validate the utility of these ligands in pca, on - target binding experiments were also performed using the recombinant t877a ar mutant characteristic of advanced stage androgen - independent pca . In tr - fret, the compounds demonstrated similar activity to that observed in the wild type assays, indicating their potential in advanced phases of prostate cancer (figure 3b). Activity data are in agreement for 4 and 5 in both ar wt and art877a . The higher confidence mp values and experimental reproducibility obtained for 4 and 5 in coactivator studies were used as the basis to advance these compounds to cellular characterization and receptor subtype selectivity evaluations . We undertook to profile the selectivity of these compounds for ar over other members of the same phylogenetic branch of the steroidal nuclear receptor subfamily . Compound binding affinities for progesterone receptor (pr), glucocorticoid receptor (gr), estrogen receptor (er-), and estrogen receptor (er-) were determined using tr - fret (table 2). Er- and estradiol er- complex and do not displace fluorescent - labeled coactivator src1 - 4 from dexamethasone gr complex at concentrations up to 100 m (supporting information, supplementary figures 35). Compound 5 binds pr with comparable affinity to that observed for ar, while 4 demonstrates approximately 2-fold binding selectivity for the ar over pr . In functional evaluation we determined that the diarylhydrazide compounds are full ar antagonists, with a partial antagonistic profile demonstrated in pr, displacing src14 from progesterone the non - lbp nature of this interaction was confirmed by an fp assay (supporting information, supplementary figure 6). Compounds were tested at a concentration range from 100 to 1 m in the presence of a concentration of progesterone = ec80 . Error bars represent the standard error of the mean (sem) for n = 6 values . Data was fitted using log antagonist concentration vs response (variable slope) with graphpad prism 5 (see experimental section for details). Ic50 values are shown in table 2 . To ascertain the translational (clinical) potential of these ligands, compounds were evaluated in cellular models of prostate cancer (lncap, an androgen - dependent cell line and pc-3, an androgen - independent cell line) and in normal prostatic epithelia cell line pwr-1e . Cell viability was assessed after 24 h of incubation with the test compounds at three different concentrations (figure 5). The classical antiandrogen cpa was used as a reference, showing a minor effect at 50 m in the androgen independent cell line pc-3 . At 50 m 4 reduces cell viability to a 5060%, whereas 5 acts consistently across the three cell lines, retaining cell viability at around 80% . Compounds were tested at 5 10, 1 10, and 5 10 m. error bars represent the sem of two independent experiments done in triplicate (n = 6). The diarylhydrazides were evaluated for their effects on the ar signaling pathway and on hormone - dependent cellular growth of lncap cells . Compound 5 was well - tolerated after 5 days of treatment at 10 and 20 m concentrations and enabled observation of a specific reduction in dht - treated cell count (figure 6a). Prostate specific antigen (psa) is a serine protease normally secreted by the prostate epithelia . Psa is widely used as a marker for pca, as its serum levels are increased in this condition . Compound 5 was shown to reduce dht - induced psa secretion in a dose response fashion as quantified by an elisa experiment in lncap cells (figure 6b). It is well - documented that classical antiandrogens (i.e., those binding within the lbp / competing with endogenous ligands) have partial agonistic properties, which make them less useful in the management of advanced prostate cancer . Arising from this inherent agonism, in an androgen - deprived lncap cell line, antiandrogens such as cpa can actually activate the ar pathway and stimulate cell growth . In direct contrast to the behavior of traditional antagonists, 5 shows no detectable agonist or partial agonist activity at tested concentrations, consistent with an alternative mechanism to that of the classical antiandrogens . Finally, treatment with 5 at 10 m was found to antagonize cpa partial agonist activity (measured as secreted psa levels in the cellular media in an elisa experiment), suggesting its potential benefit in combination therapy for advanced stages of prostate cancer (figure 6c). To further challenge this hypothesis, the compounds also demonstrated similar effects in this alternate system, supporting the hypothesis of their functioning as true antiandrogens (supporting information, supplementary figure 7). (a and b) 5 reduces androgen - stimulated cell growth and dht - dependent ar signaling measured as psa levels secreted in the cellular media in a dose - dependent fashion . (c) 5 at 10 m reduces cpa - induced ar signaling (in absence of androgens) measured as psa levels secreted in the cellular media in a dose - dependent fashion . Data are presented as the mean of two independent experiments, and bars show sem for n = 6 values (a). Secreted psa (ng / ml) was measured considering the optical density at 450 nm minus the optical density at 540 nm and interpolating the values from the standard curve . Data are presented as mean of two independent experiments, and bars show sem for n = 4 values (b and c). Classical antiandrogen therapy is known to have limited beneficial effects in hormone - insensitive pca . Alternative ar inhibitors are therefore needed in the treatment of pca . In this study, we demonstrate the successful implementation of a virtual screening approach in the identification of small molecule ar modulators, where the structural motif of ar coactivators was included in a 3d pharmacophore . We report the discovery, identification, and characterization of a novel series of diarylhydrazide non - lbp - binding antiandrogen compounds, with demonstrated ability to displace ar coactivators and with established potency in ar - dependent prostate cancer cell lines . Activity was measured with a tr - fret assay and a non - lbp - mediated mechanism of inhibition was confirmed by fp assay . These compounds are shown to function without any demonstrated intrinsic or partial agonist activity in ar and therefore can be classified as true non - lbp antiandrogens . The nature of nr coactivators and the high homology of nr coactivator binding sites are such that, to more fully profile the potential utility of these ligands, their selectivity was evaluated across members of the subclass of steroid receptors, including er- and er-, gr, ar, and pr . The selectivity of the diarylhydrazide scaffold for the ar was demonstrated through tr - fret evaluation in the estrogen and glucocorticoid receptors, where agonist bound receptor recruitment of coactivator was unimpaired at screening concentrations up to 100 m . We additionally investigated the potential cytotoxicity of the diarylhydrazides in three different cell lines, selecting 5 for its favorable cytotoxic profile (cell viability was retained at around 80% in different prostatic cellular models). Unmodified diarylhydrazide screening hits were also shown to have 2-fold selectivity for ar over pr, with partial antagonist activity demonstrated for the scaffolds in a pr functional assay, remarkable given the high (> 60%) homology of these nr family members . Futhermore, given the established utility of mifepristone (a pr modulator which also has antiandrogenic activity) in the treatment of castration resistant prostate cancer, the narrower selectivity window observed for these ar ligands in pr over the other nr s assessed is not a significant concern in the context of the therapeutic area under consideration . Classical antiandrogens can be also distinguished for their different behaviors at a cellular level . Save for two recent examples, all lbp antiandrogens described to date have also intrinsic partial agonist activity, demonstrated by induction of psa in the absence of hormone stimulation in lncap cells . In this study, the novel non - lbp diarylhydrazide antiandrogen 5 did not induce psa expression in absence of hormone stimulation when compared to cpa . In androgen - deprived lncap cells, 5 reduces psa expression in combination with cpa, antagonizing its partial agonist activity in a dose responsive fashion . This result supports the hypothesis of a nonclassical mechanism of ar inhibition for these diarylhydrazide ligands and it also demonstrates the potential application of these and other non - lbp antiandrogen small molecules targeting alternative ar sites in combination with existing prostate cancer therapy . Through application of virtual screening methodologies, we present and characterize novel diarylhydrazide scaffolds as true antiandrogens displacing ar coactivator interaction and having a full antagonistic profile on ar (both wt and t877a), partial antagonistic profile for pr, and selectivity for the other members of the nr-3 family (gr, er-, and er-). The initial small molecule non - lbp true ar modulators provided by this study will be used to further characterize the ar coactivator interface, to understand the basis of selectivity, and to further guide rational drug design in the search of other novel scaffolds directed at this interface . Black, low volume, 384-well assay plates (corning, ny, cat . No . 3676) were used to perform the assay (total volume 20 l), and tr - fret signal was measured with pherastar equipment (bmg labtech) using a lanthascreen optic module (excitation, 335 nm; emission, 520 nm channel a and 495 nm channel b). Tr - fret values were calculated at 10 flashes per well, using a delay time of 100 s and integration time 200 s as recommended by the invitrogen assay guidelines . A serial dilution of compounds was first prepared in 100 dmso (sigma - aldrich) starting from the maximum desired concentration to achieve a 12 point range concentration using 96-well polypropylene plates (nalgene nunc, rochester, ny). Each 100 solution was diluted to 2 concentration with tr - fret coregulator buffer a (invitrogen proprietary buffer), yielding a final concentration of 1% dmso in each well . Ten microliters of 2 solution was then added to the 384-well plate, following addition of 5 l of 4 ar - lbd and 5 l of d11-fxxlf / tb anti - gst antibody in agonist mode and 5 l of d11-fxxlf / tb anti - gst antibody / dht (included at a concentration equal to ec80 as determined by running the assay in agonist mode first).d11-fxxlf and tb antibody were premixed in light protecting vials prior to use . A final concentration of 5 mm dtt was used in the assay buffer in order to prevent protein degradation . All plates (agonist and antagonist mode) were incubated between 2 and 4 h at room temperature protected from light prior to tr - fret measurement . Ic50 values were determined by testing each ligand at concentrations ranging from 100 m to 45 nm using 2- and 3-fold dilutions to generate a 12 point dose data was fitted using the sigmoidal dose response (variable slope) available from graphpad prism 5.the z factor for these assays was> 0.5, as calculated by the equation provided by zhang et al . In line with the assay protocol, a known agonist, dihydrotestosterone (dht, cat no . A8380, sigma), and a known antagonist, cyproterone acetate (cat no . A control with no ar - lbd present was included to account for diffusion - enhanced fret or ligand - independent coactivator recruitment . A negative control with 2 dmso was present to account for any solvent vehicle effects . The assay was adapted to exclude possible nonspecific aggregation mechanism of inhibition by adding very low concentration of detergent triton x-100 (0.01%) to the assay buffer following the shoichet review guidelines (supporting information, supplementary figure 1). P3018) was used to investigate the binding of the test compound to the lbp site, occupied by a high - affinity fluorophore ligand (fluormone). The 100 test compound solutions in dmso were diluted in ar green buffer (invitrogen) to achieve 2 concentrations and placed in a 384-well plate (corning, cat no . Ar - lbd and fluormone (2) mix were prepared separately and then added to each compound dilution to achieve a final concentration lbd - fluormone of 50 and 2 nm, respectively . Plates were incubated protected from light for at least 4 h. controls included a maximum mp positive control, which consists of the ar - lbd and fluormone mix (2), and a minimum mp control, containing only fluormone (2). A vehicle control was added to account for dmso effect, and a blank control containing buffer only . Fluorescence polarization was measured with pherastar equipment (bmg labtech) using an optic module with excitation at 485 nm and emission at 530 nm . Lncap cells (androgen - dependent), pc-3 (androgen - independent), and pwr-1e (normal prostatic epithelia) were cultured in rpmi-1640 glutamax (invitrogen), f12k (invitrogen), and k - sfm media (invitrogen). The first two were supplemented with 10% fetal bovine serum (fbs), penicillin (100 units / ml), and streptomycin (100 g / ml). K - sfm was supplemented with 5 ng / ml epidermal growth factor (egf) and 0.05 mg / ml bovine pituitary extract (bpe). Cells were propagated at 1:3 or 1:6 dilutions at 37 c in 5% co2 . For cell viability (end point) assays lncap, pc-3, and pwr-1e cells were seeded at 2.5 10/ml density in 200 l volume of a 96-well plate in triplicate and incubated for 24 h prior testing . Test compounds were included at different concentrations to achieve a final concentration of 0.5% dmso in each well . Cell viability was assessed after 24 h of treatment using 10% alamarblue reagent (invitrogen) for each well . Cell viability was monitored by the reduction of resazurin, a blue, cell - permeable, nontoxic compound, to resorufin, a red and highly fluorescent product . Viable cells continuously convert resazurin to resorufin, increasing the overall color and fluorescence of the media surrounding cells . Fluorescence intensity can be quantitatively determined with a fluorescence microplate reader at excitation / emission 544 nm/590 nm (spectramax gemini). For hormone - dependent cell proliferation assays in androgen - deprived lncap cells, cells were seeded at 2 10 cells / ml in a 24-well plate in triplicate . Cells were plated in phenol red free rpmi glutamax (invitrogen) supplemented with 10% charcoal - stripped fbs to deplete endogenous steroids 48 h prior to the assay, as described in previous reports . The optimal condition for the treatment was found to be 5 days and the concentration of dht included to stimulate the cells was 0.1 nm . Cells were treated with different concentrations of test compounds with or without 0.1 nm dht to achieve a final concentration of 0.1% dmso in each well . A control for the vehicle was included to ensure that no effect on viability could be detected . Media and treatments were replaced every second day, after washing the cells twice with 1 pbs . Supernatants were collected after 5 days for evaluation of secreted psa levels, and cell proliferation was assessed for the same plate using alamarblue in order to exclude nonspecific effects due to toxicity issues . Secreted levels of prostate specific antigen were evaluated with a commercially available kit (quantikine human kallikrein 3/psa immunoassay, r&d systems)., 50 l of standards and cell culture samples were added to precoated wells containing assay diluent rd1w (r&d systems) and incubated for 2 h at room temperature . Unbound material was washed several times and 200 l of horseradish peroxidase (hrp) labeled psa conjugate antibody was added to each well and further incubated for 2 h at room temperature . Wells were washed and treated with colored substrate (tetramethylbenzidine) for an additional 30 min, after which 50 l of stop solution (2 n sulfuric acid) was added per well and optical density (450 nm with correction at 540 nm) was read with a plate reader within 30 min (versamax). A virtual screen was designed to select compounds mapping onto the peptide binding surface (af2) of the ar receptor, based on an ensemble of documented x - ray crystal structures (pdb i d 1t73, 1t74, 1t76, 1t79, 1t7f, 1t7 m, 1t7r, and 1t7 t). Molecular operating environment (moe) software was employed to preprocess the proteins and to remove the coactivator peptides from the complexes . An initial pharmacophore was generated using the moe pharmacophore elucidator and considering the most significant features, which involved hydrophobic, donor, and acceptor features . A second pharmacophore was developed including two additional hydrophobic / aromatic features to represent the phe side chains present in the fxxlf coactivator motif (1t7r), so as to increase the selectivity for ar over other families of nuclear receptor . These pharmacophore models were then applied for in silico screens of small - molecule commercial libraries to identify compounds that resemble the active principle of the starting peptides . A number of vendor databases were selected for screening of ligands, including amsterdam (5389 compounds), peakdale (8188), asinex platinum collection (75 258), specs (175 800), maybridge (56 870), and zinc (4.6 million) compounds . A bayesian analysis was performed on the peptide structures to estimate parameters of an underlying distribution based on the observed distribution . The above databases were then filtered for those compounds with properties similar to the peptides, thus focusing the search on the ar ligand chemical space . All molecules were standardized for stereochemistry and charges and ionized at a ph of 7.4 and all calculable tautomers were enumerated . At this stage the conformational flexibility of the screening compounds was explored using the omega software (openeye scientific package). A maximum of 50 conformations were generated for each molecule in the data set . The virtual molecules were overlaid on and compared to the generated pharmacophore of the active ligands, and those molecules that compared favorably were advanced for additional virtual screening and scoring . The fast rigid exhaustive docking (fred) software as implemented in openeye scientific s package was used to exhaustively examine all possible poses within the protein site, filtering for shape complementarity and scoring . The smaller databases (amsterdam and peakdale) were screened on all 13 crystal structures and only ligands scoring well on more than one crystal structure were considered . The larger databases specs, asinex, maybridge, and zinc were screened on the 1t7r crystal structure . A structural similarity search was conducted on 1 and 2 using a tanimoto coefficient of> 70% on the specs compound database . Thirty - seven compounds were purchased and four small molecules were selected for optimization and characterization studies based on their improved on - target activity determined by tr - fret . All screening compounds described in this work were purchased as commercial samples from specs nv . Compound purity in all instances was greater than 95% as determined by lcms and nmr.
During the last years tyrosine kinase inhibitors (tkis) have changed the natural history of metastatic non - small - cell lung cancer (nsclc) harboring epidermal growth factor receptor (egfr) mutations . Eight important studies were conducted to evaluate the efficacy and tolerability of tkis on advanced nsclc in comparison with standard platinum - based chemotherapy . Not surprisingly, the use of tkis was correlated with a higher response rate, a longer progression - free survival and a better quality of life in patients with advanced nsclc activating egfr mutation . The iressa pan - asia study (ipass), which enrolled 1,217 patients, was the largest trial in which patients were randomized to receive gefitinib or standard chemotherapy, and in the group of tkis therapy the primary endpoints were reached obtaining a statistically significantly higher response rate, a longer progression - free survival and better symptom control . Similar results were reported by first - signal and by west japan thoracic oncology group (wjtog 3405) studies . The north - east japan study group (nej002) trial was stopped early because gefitinib showed a significantly higher progression - free survival in comparison with standard chemotherapy in patients with advanced lung adenocarcinoma activating egfr mutation . Impressive results were also reported with the use of other tkis such as erlotinib or afatinib versus chemotherapy in patients carrying the same egfr mutations . Better responses were observed in patients with mutations in exons 1821 of the tyrosine kinase domain of egfr . However, egfr gene mutations were also identified in small - cell lung cancer (sclc) [3, 4] and in large - cell neuroendocrine carcinoma (lcnec) of the lung . Lcnec is a high - grade carcinoma (> 10 mitoses/2 mm) belonging to the neuroendocrine tumors of the lung . It represents about 3% of all pulmonary malignancies and is characterized by neuroendocrine cytologic features (formation of rosettes, trabeculae and perilobular palisading pattern) and markers (neuron - specific enolase, cd56, synaptophysin, chromogranin and leu7). In fact, the cytologic and biologic features of lcnec are different from those of large - cell carcinoma . The molecular alterations that are commonly found in lcnec are p53, bcl-2 overexpression and rb mutation . To our knowledge, few cases of lcnec with egfr gene mutation have been described up to now, and only one case was treated with gefitinib, with a good response [7, 8]. A 47-year - old caucasian woman with no family history of neoplastic diseases and no comorbidities was examined by a general practitioner after the appearance of back pain unresponsive to usual non - steroidal anti - inflammatory drugs . Standard chest x - ray showed a left lung perihilar lesion, probably suggesting pneumonia . As a consequence, thus, after 2 weeks, chest x - ray was repeated and showed persistence and stability of the left lung lesion . About 1 month later, the patient came for the first time to our attention for appearance of vomiting, dyspnea, fatigue and abdominal pain (visual analog scale 7). Abdominal physical examination revealed a painful hepatomegaly . She underwent a total body computed tomography (ct) scan that showed multiple focal liver lesions, solid left lung tissue and multiple secondary brain lesions (two left frontal cerebral lesions, one right parietal lesion and two cerebellar lesions) (fig . 1). As a result, a liver biopsy was performed . Since all investigated tumor markers (carcinoembryonic antigen, carbohydrate antigen 19 - 9, carbohydrate antigen 125, neuron - specific enolase, glycoprotein hormones alpha polypeptide) were increased, it was not possible to identify the primary site of localization of the tumor and to reach a definitive diagnosis . Given the rapidly progressive impairment of her clinical conditions and performance status, we administered an empirically not targeted chemotherapy with gemcitabine 1,000 mg / m die 1 and oxaliplatin 100 mg / m die 2 q 2 weeks although we did not yet have definitive histopathological results . Although the sample was poor, the diagnosis was evocative of lung adenocarcinoma (ttf-1 positive, cytokeratin 7 positive). However, since a further deterioration of her clinical condition was observed, a biopsy was repeated in order to have an additional sample for molecular analysis . Therefore, we started tki therapy and gefitinib was administered at 250 mg p.o . Once a day . The patient reports a good quality of life and no relevant side effects (skin toxicity grade 1) have been registered . A restaging total body ct scan showed a significant improvement of disease, with a partial response> 50% . Particularly, the brain ct scan showed a significantly reduced volume of the lesions (valuable only 2/5 lesions) and complete resolution of edema (fig . Lcnec is a rare tumor which is usually treated with cisplatin - based chemotherapy as sclc as it shares many characteristics with it . Usually, the administration of tkis is considered only for advanced lung adenocarcinoma because the most important studies demonstrating higher activity of tki, in comparison with standard platinum - based chemotherapy, were conducted in patients affected by nsclc with egfr mutations . Some features are associated with a higher possibility that egfr mutations are present in nsclc . The research of egfr mutations is not performed routinely on lcnec since this mutation is not commonly present in this subtype . The prognosis of this tumor with standard chemotherapy is poor, thus we need to evaluate alternative diagnostic strategies such as the investigation of egfr mutational status in order to explore new effective treatments . We report the case of a patient affected by lcnec carrying all of the predictive characteristics associated with egfr mutation (caucasian, female, never - smoker). When the patient came to our attention she was very symptomatic, so in an attempt to reach a better and faster response, we decided to test egfr mutational status . Strikingly, the mutation was present, and we were allowed to administered gefitinib, obtaining an impressive and objective response . Therefore, we suggest the research of egfr mutations also in patients with lcnec to offer them one more therapeutic option.
Tetralogy of fallot (tof) accounts for approximately 5% to 7% of all cases of congenital heart disease . Complications related to surgical repair of tof are clinically relevant and include pulmonary regurgitation, residual obstruction of the right ventricular (rv) outflow tract, rv systolic dysfunction, and arrhythmias . Among these, residual rv outflow tract obstruction with accompanying pulmonary regurgitation is the most frequently reported sequela after initial surgical correction of tof . Therefore, a systolic murmur that is audible over the base of the heart is frequently interpreted as a residual rv outflow tract lesion . However, this is not always true . We herein report a rare case of subaortic stenosis in association with a previous tof surgical repair that was not considered to be a differential diagnosis . Discrete subaortic stenosis is generally thought of as a disease of children or young adults (<25 years of age) and is thus rarely reported in subjects older than 25 years, particularly in association with tof . A 29-year - old woman presented for a routine check - up of surgically corrected congenital heart disease . At 7 years of age, she had undergone surgical repair of tof with excision of the infundibular stenosis and closure of a 10-mm ventricular septal defect . She had been asymptomatic until her first decade of life, and only a 5-year clinical follow - up had been performed after the operation . Approximately 6 months prior to presentation, she began to feel exertional dyspnea that was progressively aggravated . On physical examination, she was alert and well - oriented . Her blood pressure was 100/60 mmhg, pulse 60 beats / min, respiration rate 18/min, and body temperature 36.6. the left ventricular (lv) impulse was almost normal in location . On auscultation, a grade 3 of 6 mid- to late - systolic murmur was audible over the base of the heart and heard clearly on both sides of the sternum . Routine blood examinations, including a complete blood count, chemistry panel, coagulation panel, and c - reactive protein level, were all within normal ranges . Transthoracic and transesophageal echocardiograms both demonstrated severe subaortic stenosis due to a subvalvular membrane, on which a mobile mass - like structure that was suspicious of a vegetation was noted (fig . There was a mild degree of tricuspid regurgitation, but no clinically significant obstruction of the rv outflow tract . The maximum velocity measured across the tricuspid valve was 2.8 m / sec, and thus the maximal pressure gradient was estimated to be 31.4 mmhg using the simplified bernoulli equation . Due to the up to 20 mmhg pressure gradient across the pulmonary valve her rv was not dilated, and its systolic function was good . Due to the suspicion of infective endocarditis despite the absence of clinical evidence, blood cultures were performed three times; however, no pathogen was cultured . The patient underwent resection of the subaortic membrane and myomectomy of the lv outflow tract along with aortic valve commissurotomy . The membrane, with the exception of the noncoronary cusp area, was located beneath the aortic annulus (i.e., inverted u shape). The mobile mass - like structure described on transesophageal echocardiography was proven to be loose tissue attached to the subaortic membrane . Although subaortic stenosis is the second most - common form of lv outflow tract obstruction, it is predominantly found in children or adolescents [4 - 8]. Subaortic stenosis is strongly associated with other congenital heart defects; however, subaortic stenosis in association with tof, especially in women, is extremely rare . According to a medline database review of the last 30 years furthermore, all were diagnosed in childhood or adolescence (<20 years of age) with two exceptions . The present case is peculiar in that subaortic stenosis manifested more than 20 years after the initial tof surgical correction . Although we could not definitively say that the subaortic stenosis in the present case was acquired, we consider it an acquired or progressive form given the long interval (> 20 years) before echocardiographic confirmation . Thomas and foster suggested that acquired subaortic stenosis can develop gradually due to hemodynamic disturbances derived from coexisting lesions or after surgical correction . Moreover, the mass - like mobile structure observed on transesophageal echocardiography could be regarded as a degenerative byproduct of flow convergence induced by the subaortic membrane, further supporting the concept of an acquired or progressive form of the disease . Discrete subaortic stenosis is well - known to be linked to important complications, such as bacterial endocarditis and significant aortic regurgitation . Flow turbulence distal to the obstructing subaortic membrane plays a crucial role in progressive damage to the aortic valve with resultant thickening of its leaflets and regurgitation . The secondary jet lesion also predisposes the patient to bacterial endocarditis . In this context, in particular, patients with surgically corrected tof tend to be misinterpreted as having only residual rv outflow obstruction without consideration of the lv outflow tract lesion . Transthoracic and transesophageal echocardiography can assist differentiation of the rv from the lv outflow tract obstruction . Therefore, periodic performance of echocardiography should be kept in mind during follow - up of patients with surgically corrected tof.
Pregnancy - associated plasma protein - a (papp - a) is a metzincin metalloproteinase primarily produced by the placental syncytiotrophoblast during pregnancy . Papp - a is also synthesized by fibroblasts, osteoblasts, vascular smooth muscle cells (vsmcs), and endothelial cells (ecs). In vitro, papp - a functions to cleave insulin - like growth factor - binding protein 4 (igfbp-4), an inhibitory igfbp, consequently increasing igf bioavailability for receptor activation [14]. In vivo, several studies have shown a similar role for papp - a in modulating site- and event - specific igf signaling during injury repair processes . Recent studies have indicated that papp - a is a novel biomarker for plaque instability and inflammation useful in early diagnosis, risk stratification, and prognostic prediction in patients with acute coronary syndrome (acs) [5, 6]. Papp - a was found abundantly expressed in ruptured and eroded human atherosclerotic plaques, colocalized with activated smooth muscle cells and macrophages [7, 8]. Since plaque - derived papp - a is being considered as a new biomarker that may potentially play a role in the development of atherosclerotic lesions [9, 10]. A better understanding of its cellular source and regulation is important to the future development and implementation of therapeutics utilizing this biomarker . Previous studies have indicated that proinflammatory cytokines, interleukin- (il-) 1, and tumor necrosis factor- (tnf-) were potent stimulators of papp - a expression in cultured human fibroblasts, osteoblasts, coronary artery smooth muscle cells, and endothelial cells (ecs) [9, 11]. Despite these significant findings, little is known about the effect of c - reactive protein (crp) and tnf- on papp - a expression in human peripheral blood monocytes (pbmcs). The current study investigates the ability of crp and tnf- to induce papp - a expression in the pbmcs of healthy volunteers . Furthermore, inhibitor experiments have been designed to explore the underlying intracellular signaling pathways involved in papp - a expression . The focus of these studies is nuclear factor- (nf-) b pathways, a major pathway associated with cytokine stimulation in various cell types . Peripheral blood was collected from the forearm vein of healthy volunteers enrolled in the current study . For each experiment, 30 ml samples were freshly collected from 6 healthy subjects for use in pbmc preparations . Each of these 6 subjects provided 4 blood donations over the course of the study . The study was approved by the ethics committee of the beijing friendship hospital and conforms to the principles outlined in the declaration of helsinki . Human pbmcs were isolated from 30 ml fresh blood samples obtained from healthy volunteers by ficoll - paque (amersham bioscience, uppsala, sweden) centrifugation . Resultant cells were washed 3 times with pbs and subsequently resuspended in rpmi 1640 (gibcobrl, grand island, ny, usa) supplemented with 10% fetal calf serum (gibcobrl, grand island, ny, usa), penicillin (100 u / ml), and streptomycin (100 g / ml). Cells were then cultured for 24 hours in plastic dishes at 37c in a humidified atmosphere of 5% co2 . Upon observation of subconfluent growth, the medium was replaced with fresh medium . All nonadherent lymphocytes were discarded during the medium change, reserving only healthy adherent monocytes . After 24 hours, trypan blue exclusion indicated that 95% of cultured pbmcs were living . The pharmacological agents recombinant human tnf- (perprotech, rocky hill, ct, usa), crp, actinomycin d, and bay11 - 7082 (sigma chemicals, deisenhofen, germany) were dissolved into solution according to the manufacturer's instructions . Resultant solutions were added to cultured pmbcs at defined time intervals (2, 8, 16, 24 hours) and concentrations (crp: 5, 10, or 20 mg / l; tnf-: 25, 50, or 100 ng / ml; bay11 - 7082: 20 m) in the absence or presence of actinomycin d (1 g / ml), as further described in the following sections . Individual controls were determined for each experiment, as described in figures 14 . Briefly, total rna was isolated using trizol (invitrogen, calsbad, ca, usa) according to the manufacturer's instructions . Reverse transcription - generating cdna was performed using the superscript iii first - strand synthesis system (invitrogen, calsbad, ca, usa). Papp - a cdna was amplified using forward (5-ata tct cac gtg acc gag ga-3) and reverse (5-aga tga tgg tgc tgg aag tc-3) primers, which produce a 529 bp product . Amplification was performed at 94c for 2 min for preheating, followed by 30 cycles of 94c for 45 s, 65c for 45 s, 72c for 60 s, and a final extension of 72c for 10 min . -actin was amplified using forward (5-gca tgg agt cct gtg gca t-3) and reverse (5-cta gaa gca ttt gcg gtg g-3) primers, which produce a 320 bp product . Amplification was performed at 94c for 2 min for preheating, followed by 28 cycles of 94c for 30 s, 60c for 30 s, 72c for 30 s, and a final extension of 72c for 20 min . The resulting bands were photographed under ultraviolet light and analyzed using a gel imaging system (gel doc2000, bio - rad, hercules, ca, usa). The relative intensity of bands of interest was expressed as the ratio to -actin mrna bands . For cell lysates, cells were washed twice with ice - cold pbs and lysed in ripa buffer . Total protein was quantified using the bca assay (pierce, rockford, il, usa). Equal amounts of protein (40 g) were separated by sds - page in 14% tris - glycine gels (tefco, tokyo, japan). After electrophoresis, the proteins were blotted onto a nitrocellulose membrane and blocked with 5% skim milk powder diluted in tris - buffered saline (tbs) with 0.05% tween 20 . Rabbit polyclonal antibodies against human papp - a (1: 1000, abcam systems, cambridge, usa) were used as the primary antibody . Membranes were incubated with diluted antibody preparations overnight at 4c . After washing the next day, membranes were incubated with horseradish peroxidase- (hrp-) conjugated affinity - purified goat anti - rabbit igg antibody (1: 3000, santa cruz . Papp - a levels were determined using the ultrasensitive elisa kit (diagnostic systems laboratories, webster, tx). The assay was calibrated using recombinant papp - a calibrated against the world health organization's international reference preparation 78/610 for pregnancy - associated proteins, by definition containing a papp - a concentration of 100 minimum sensitivity was 0.24 miu / l with intra- and interassay coefficients of variation of 4.7% and 4.2%, respectively . All statistical analyses were carried out using the spss statistical package, version 13.0 (spss inc ., p - values less than 0.05 were considered statistically significant (p <0.05). The time course of papp - a mrna expression in pbmcs under basal and cytokine - stimulated conditions is presented in figure 1(a). Little papp - a expression was observed in pbmc cultures under basal conditions after 24 hours . Treatment with crp (20 mg / l) or tnf- (100 ng / ml) significantly increased paap - a mrna expression at all time points (2, 8, 16, 24 hours). Papp - a mrna levels increased 2 hours after stimulation with crp (20 mg / l) and remained elevated by approximately 3.7-fold up to 24 hours . Pappp - a mrna expression, however, rapidly increased and peaked at approximately 6.8-fold 2 hours after tnf- (100 ng / ml) stimulation . A subsequent decrease was then observed, though levels remained elevated at approximately 4.5-fold up to 24 hours . Maximal papp - a protein expression in pbmcs and concentrations in culture supernatants were achieved with crp stimulation by 24 hours and tnf- stimulation by 8 hours (figures 1(b) and 1(c)), reflecting the changes in papp - a mrna expression . As shown in figure 2, dose - response experiments confirmed crp or tnf- treatment elicited dose - dependent increases in papp - a mrna expression, protein expression in pbmcs, and secretion in the supernatant after 24 hours . Crp showed half - maximal effectiveness at approximately 5 mg / l, with maximal effectiveness at approximately 20 mg / l (figure 2). Tnf- showed half - maximal effectiveness at approximately 25 ng / ml, with maximal effectiveness at approximately 100 ng / ml . The dependence of papp - a expression on mrna synthesis was explored in the following three experiments . Figure 3 showed that the effects of these proinflammatory cytokines appeared to be at the level of transcription, as the dna - directed rna polymerase inhibitor, actinomycin d, completely prevented crp or tnf- induction of papp - a mrna expression, protein expression, and concentrations in culture supernatants . These results showed that crp or tnf- was responsible for new protein synthesis of the papp - a protein . Furthermore, papp - a protein was actively secreted into the supernatant . As indicated in our previous experiments, treatment of human pbmcs with crp (20 mg / l) or tnf- (100 ng / ml) significantly increased papp - a mrna expression, protein expression, and concentrations in culture supernatants . To confirm the role of nfb activation, bay11 - 7082, which inhibits inducible phosphorylation of ib, was shown to effectively inhibited crp and tnf--stimulated papp - a expression (figures 4(a), 4(b), and 4(c)). It is well known that the nfb pathway is the critical mediator of prooxidant stimuli, such as inflammatory cytokines . The basic mechanism by which nfb is activated is through phosphorylation of intrinsic inhibitors, with the ib, subsequently freeing nfb to translocate into the nucleus where it regulates gene expression . Novel markers of coronary artery disease progression have been confirmed in recent years, with circulating levels of papp - a standing out as one of the most prominent indicators of this profile . The current study indicates that papp - a expression in human pbmcs may be regulated by crp and tnf- through the nf-b pathway, a mechanism that may play a critical role in increases in serum papp - a levels during acute coronary syndrome (acs). These findings are consistent with previous reports indicating that papp - a is a marker of atheromatous plaque instability as well as extent and prognosis of cardiovascular disease [1315]. Serum papp - a levels increase in patients with acs, indicating that papp - a may also be a marker of adverse events [1618]. Moreover, in chronic stable angina (csa) patients, papp - a is an independent predictor for the extent of vessel stenosis, where it has been shown to correlate with the presence of vulnerable coronary artery stenosis [19, 20]. Thus, papp - a levels have demonstrated a firm association with angiographic plaque complexity in csa patients . The stimulation of papp - a expression by tnf- has been observed previously in human fibroblasts, osteoblasts, vsmcs, and ecs [4, 11, 22]. Using specific monoclonal antibodies, bayes - genis et al . Reported that papp - a was abundantly expressed in both eroded and ruptured plaques, but was only minimally expressed in stable plaques . Moreover, in plaques with large lipid cores and cap rupture, staining for papp - a occurred mostly in the inflammatory shoulder region, in areas surrounding the lipid core, and in areas with localized cd68-positive cells . Thus, papp - a levels have been associated with inflammation in regions of atherosclerotic plaques, potentially contributing to progression and poor outcomes in patients . Most evidence of monocyte involvement of papp - a expression has been completed through analysis of atherosclerotic plaques, where monocytes are the predominant leukocyte contributing to the development, progression, and instability of atherosclerotic lesions, they often contain high levels of papp - a . Because samples of plaques may contain a mixture of leukocytes and circulating compounds, these tests have left the source of papp - a observed in these monocytes a point of debate among researchers . Double immunofluorescence confocal microscopy (icm) has been used to characterize cell types expressing papp - a, suggesting that monocytes are the primary source of papp - a in plaques and the target cells for cytokines . It has also been speculated that papp - a may be produced by activated monocytes or macrophage cells in unstable plaques and released into the extracellular matrix and circulation . Conversely, conover et al . Suggested that in vivo macrophages actually failed to produce papp - a, but instead internalized the compound through their membranes, thus accounting for the accumulation of circulating papp - a produced by other sources in macrophage cells associated with plaques . The current study, however, indicates that the specific mrna expression associated with papp - a production increases in macrophages in vitro, resulting in expression and supernatant secretion of papp - a . These findings indicate that papp - a production occurs in macrophages rather than being internalized through the membrane, as suggested by conover et al . Further in vivo studies will be required to assess the affect of circulating papp - a levels on macrophage papp - a expression and secretion, which may account for the discrepancies between these two studies . Cumulatively, the findings of the current study indicate that macrophages in cultured human pbmcs can synthesize and secrete papp - a . Furthermore, crp and tnf- were indicated to be potent stimulators of papp - a gene expression and protein secretion in human pbmcs, suggesting a link between the increase in local inflammatory cytokine production and papp - a during acs . While further studies of the in vivo effects in acs patients will be required to confirm these results, these findings suggest that serum papp - a, hscrp, and tnf- levels may be significantly higher in acs patients than in patients with stable angina pectoris and that increasing papp - a mrna levels in patients with acs may also have a positive association with serum hscrp and tnf- mrna expression . Exploration of these effects is a topic being explored in our current research based on the initial positive findings of the current research . Furthermore, the time course of stimulation by crp and tnf- differed, with papp - a expression increasing 2 hours after stimulation with crp, peaking at approximately 3.7-fold by 24 hours, whereas a rapid increase to approximately 6.8-fold was seen in only 2 hours with tnf- stimulation . The current study provides significant evidence for papp - a production in pmbcs and stimulation by the cytokines crp and tnf-; however, further in vivo studies will be required to verify these findings and assess the effects of circulating papp - a on pmbc papp - a production . Actinomycin d was observed to complete block the induction of papp - a mrna expression by crp and tnf-, indicating potential regulation at the level of transcription . The rapid increase in papp - a mrna levels after treatment with these cytokines further confirms the hypothesis of transcriptional regulation . As expected, increases in papp - a protein expression and secretion into the supernatants were paralleled by increases in gene expression . Based on this observation, it is possible that the biological consequence of crp- and tnf--induced papp - a expression in human pbmcs was enhanced igf - i bioactivity mediated by papp - a proteolysis of igfbp-4, thus contributing to the progression of both coronary atherosclerosis and restenosis . It is well known that the free fraction of circulating and locally synthesized igf - i stimulates vsmc proliferation, migration, and extracellular matrix synthesis . In macrophages, igf - i also promotes excess ldl cholesterol uptake, production of proinflammatory cytokines, and chemotaxis [26, 27]. Crp and tnf- were also identified as potential regulators of papp - a expression, operating through a mechanism involving the activation of the nfb system in pbmcs of healthy volunteers . Nfb is a ubiquitous transcription factor that is activated by inflammatory cytokines, infection, oxidative stress, and shear stress . Ritchie reported that nfb was activated in peripheral monocytes using an electromobility shift assay in patients suffering from unstable angina . The nfb family of transcription factors plays a critical role in coordinating and regulating the expression of a wide variety of inflammatory genes that have been linked to the pathologies of acs . Some studies have even demonstrated that crp induces nfb activity in various cell types, including peripheral monocytes, saphenous vein endothelial cells, and human aortic endothelial cells [23, 33, 34]. Moreover, resch et al . Reported that tnf- induced ib degradation and papp - a - regulated expression in human fibroblasts by tnf- was mediated by nfb activation . The current study also demonstrated that bay11 - 7082 was a potent inhibitor of both crp- and tnf--stimulated papp - a expression in human pbmcs . The view of nfb as a transcription factor for papp - a gene expression, however, will require further study to identify the promoter region and validate these findings . The current study provides a variety of evidence to support the expression on papp - a by leukocytes in pmbcs, including increased papp - a - associated mrna expression, papp - a expression, and papp - a secretion into the supernatant of fresh in vitro samples collected from healthy subjects . Furthermore, the cytokines crp and tnf- were shown to stimulate papp - a expression in these cells . Based on these findings and the previously observed association between pmbcs and acs, it is likely that activated monocytes or macrophages in pmbcs surrounding developing artherosclerotic plaques may secrete proinflammatory cytokines, thus stimulating the expression and secretion of papp - a . Further studies will, however, be required to assess the effects of elevated papp - a concentrations on autocrine and paracrine mechanisms for the exacerbation of atherosclerosis procession and plaque rupture through igfbp-4 cleavage and enhanced local igf - i bioavailability . The current results, however, provide the fundamental mechanistic groundwork for further understanding of the entire mechanism associated with cytokine regulation of papp - a expression and igf bioavailability . This understanding may lead to the future development of novel therapeutic targets for the treatment of acs.
There is an increasing interest in the study of the consultation process and patients' satisfaction with it . The core activity in primary care is the consultation irrespective of whether patients consult for cure, services, counseling, prevention, or care . A widely accepted model views the consultation as a dialogue involving elements of negotiation to create a common reality to which agenda setting is paramount . In the medical consultation the doctor and patient meet on common grounds with tolerance for each other's rights . This consultation by necessity requires a doctor who is expected to possess the requisite knowledge which will be useful in solving the problems the patient presents with the assumption that the doctor will act in the best interest of the patient . Guided by rules of professional conduct, objectivity, and being emotionally detached the doctor is guaranteed the right to examine the patient physically and to enquire into intimate areas of the patient's physical and emotional life . During the consultation, the reason for attendance is defined and an appropriate action is chosen . This process aims at achieving a shared understanding, involving the patient in management and using time and resources appropriately . Physicians have been noted to have fixed ideas about what is best for a patient, and this inflexibility leaves little room for negotiation . Quite often animosity is expressed when the patient attempts to negotiate . However, as demonstrated by a study in the netherlands, interindividual and intra - individual variability does occur among physicians, who were noted to adjust their styles according to the situation . This study sought to identify the factors contributing to patients' satisfaction, shed more light on the burden of patients' dissatisfaction with the consultation in our environment, and help devise strategies for practicing physicians to strive for an improvement in the overall patient care in our cultural context . The findings would provide some of the information needed to further fill the knowledge gap about our patient needs in our environment and highlight the need to teach the consultation process at both undergraduate and postgraduate medical training . This study comes from a background where patient awareness of their opportunities in the patient doctor encounter is still in its early days, and thus this study brings to the literature a unique perspective of the patients' views from this environment . This study was conducted in the general outpatient clinic (gopc) of the university of calabar teaching hospital, (ucth) calabar . The university of calabar teaching hospital is a tertiary hospital located within calabar metropolis, which lies along latitude 4, 58 north of the equator and longitude 820 east of the greenwich meridian . Margaret's annex, maternity annex, the permanent site, and the comprehensive health centre (chc) okoyong . The general outpatient clinic (gopc) is situated at the permanent site and has fourteen outpatient consulting rooms in which about 1014 doctors (family physicians or resident doctors in family medicine) consult from 8 am4 pm on a daily basis . Three consulting rooms are for consultants, three for doctors dedicated to the hiv clinic, one for consultancy patients, one for patients who are staff, and six consulting rooms are for outpatient consultation by other doctors . The department also had 16 nurses, 6 records' personnel, 5 orderlies, 3 counselors, and 5 administrative staff who usually assisted the doctors during the consultation . All adults between the ages of 18 and 65 years who consented to participate in the study were recruited . Informed consent was obtained from the patients before they were given a questionnaire to complete . The average attendance of patients at the ucth gopc in the three months preceding the study was 74 patients per day . Therefore, the number of patients estimated to attend the clinic was 74 22 = 1628 . From the calculated sample size of 430 subjects, a sampling interval of 4 was used to systematically select subjects who were recruited to participate in the study . The patients' attendance register for each day was used as the sampling frame from which patients were selected . The first subject was chosen randomly from this sampling frame, and subsequently every fourth patient was selected and invited to participate . If a selected subject did not meet the inclusion criteria or refused to participate in the study, the next patient was approached until the recommended sample size was recruited . A self - administered, pretested questionnaire adapted from the general practice assessment questionnaire the general practice assessment questionnaire was developed in the united kingdom and used to study certain components of the consultation . The questionnaire consisted of 21 questions divided into five sections that investigated the proportion of patients that are satisfied with their patient - doctor encounter, which patient - factors are associated with patients' satisfaction or lack of satisfaction with the consultation . Some of the questions had options from which the patient selected the response while others made room for a narrative response . Prior to the commencement of the consultation, all the patients waiting to be consulted were addressed on the possibility of being approached to join an ongoing study . Selected patients on leaving the consulting rooms were approached by the trained assistants and requested to complete the questionnaire . Data generated in the study was analyzed using the epi info software for analyzing medical data from the centre for disease control, atlanta, georgia, usa . Privacy of the patients was maintained during the study, and all information provided by the patients was treated with utmost confidentiality . Patients' consents were sought and formally obtained after a detailed explanation of the intention of the author concerning the research findings . Ethical approval for this study was sought and received from the ethical committee of the university of calabar teaching hospital . The age distribution of the respondents varied with the highest proportion being young adults aged 2640 years (44%), adolescents aged 1825 years (34%), middle - aged persons aged 4160 years (18%), and elderly patients aged 60 + 9 (4%), table 1 . Two hundred and fifty - five (59.3%) were satisfied with their patient - doctor encounter . The average age of the respondents was 29 years while the average age of all the patients who presented to the hospital during the study period was 31 years . The sex ratio was almost equal with males accounting for 201 (47%) and females 229 (53%) of the respondents (table 1). There was a wide variation among the occupational characteristics with patients who had any form of paid employment accounting for 27%, students 32%, retired persons 5%, unemployed 9%, housewives 7%, and others 22% (table 1). Among the others were artisans, self - employed businessmen, and farmers . Sex and occupational distribution of the respondents were shown to be similar to those of all the patients who presented in the clinic during the study period . Majority of the patients 230 (53%) felt the time they spent with the physician was adequate or very adequate (table 2). Only 26 (6%) respondents assessed the time they spent with the physician as inadequate (table 2). Two hundred and twenty - five (52%) respondents felt they understood the illness much more than when they came to visit the doctor (table 2). Seventy - eight percent of the patients who participated in the study and perceived that the encounter had made it possible to cope with the illness were satisfied with their encounter (table 2). A good majority of the patients perceived that their ability to cope with the illness after the visit influenced the patients' satisfaction with the encounter (p <0.001). Three hundred and fifty (81%) of patients who found an improvement in their ability to maintain their health were satisfied (table 2) with their encounter (p <0.001). Table 3 shows that the frequency of visits did not statistically influence the patients' satisfaction with the consultation (p> 0.25). The patient's assessment of time spent in the consultation was shown to have a statistically significant influence on the patients satisfaction with the consultation (p <0.001). This table also shows that the patients preference for a particular physician did not statistically influence the patients satisfaction with the consultation (p> 0.05). None of the sociodemographic variables studied were found to have any statistically significant relationship with a patient satisfaction in a consultation . This study could not demonstrate any statistical significance between a patient age and their satisfaction with the consultation . This agrees with some studies which demonstrated similar findings [8, 9] but differs from other studies which demonstrated that patients' satisfaction rates usually improve with advancing age [10, 11]. The elderly patients included in this study were only 18, and perhaps with a larger elderly population the study could have demonstrated an age - related effect on satisfaction in the consultation . Many elderly patients did not agree to complete their questionnaires, and this was probably due to the influence of the accompanying persons who often insisted they had to return to work as soon as possible . The living arrangements in our society possibly make us share similar illness perceptions of what is good or bad accompanied by a shared cultural understanding of wellness or illness . This study could not demonstrate any statistically significant influence of a patient's sex on his / her satisfaction with a medical encounter (table 1). The patients' frequency of visits to the gopc was not found to statistically influence the patient's satisfaction with the consultation (table 3). It was believed that the higher the number of visits, the higher the level of dissatisfaction because this was thought to be related to the higher likelihood of social factors not being addressed in these frequent users of the hospital services . . Patients often request to see particular doctors, but this was not shown to influence their satisfaction in this study (table 3) and is supported by a study among israeli patients . This finding differs from other studies which have shown that continuity and being seen by a particular doctor improve concordance and satisfaction [10, 13, 14]. The difference in this study may be accounted for by the fact that the patients were in a teaching hospital and many were usually referred to other clinics when the need arose . This may explain the fact that 95% of patients in this study did not insist on seeing a particular doctor as many patients often see the clinic as a transit route to other specialist clinics . Only about 32% of patients had been to the clinic on at least three previous visits with the majority (44%) having attended just 1 - 2 times or with no previous visits (25%) in the last 12 months . It is possible they had not cumulatively spent enough time with the doctors to form an opinion . There is also the practice of doctors changing rooms, duties, and postings in between patients' visits . This makes patients wary of requesting for a particular doctor who may not be on duty . Usually in the study area clinic, patients are not given the choice of selecting a doctor and may be rebuked by the nurses who do the sorting if they request for a particular doctor . Also patients probably did not request for a particular doctor because they did not know the doctors, were not aware if a particular doctor was on duty, or how long they needed to wait to see a preferred doctor . The use of time in the consultation has been shown to be crucial to consultation satisfaction ratings . This study demonstrates a statistical significance between patients' perception of time spent in the consultation and satisfaction (table 2), but not all studies agree . Fifty - three percent of the patients rated the time spent in their consultation as adequate or very adequate, with 47% describing the time spent as either fair or inadequate (table 2). Patients' assessment of the adequacy of time is crucial in gauging their satisfaction as it has been linked to satisfaction with psychosocial issues in the consultation . Patients' assessment of time spent in the consultation may be influenced by certain individual traits such as age . In this study, patients often confused the time spent in the consultation with the time spent in the waiting room, and throughout the data collection patients were encouraged to make this distinction as they completed their questionnaires . The patient perception of time is crucial in the consultation, and this influenced whether the patient was satisfied with the consultation or not . Duration of a patient illness has been shown to have an influence on the consultation by a study of chronic illnesses while a patient satisfaction with a consultation can also affect the duration of his illness . However, this study could not demonstrate any statistically significant influence of chronic illness on patients' satisfaction (table 3). Patients with chronic illnesses are expected to know more about their illness than those with acute illness and are thought to require more attention . Chronic illnesses are usually not what doctors expect to manage when they graduate, and their management may be a form of psychological burden to the physician . This study did not demonstrate any effect on satisfaction rates by the presence or absence of a chronic illness (table 3). This finding may be explained by the fact that many patients in our environment are not well informed about their illnesses, so their knowledge of the illnesses does not necessarily increase as the durations of their illnesses increase . This study could not demonstrate any significant influence of occupation on a patient's satisfaction with the consultation (table 1). Students made up 32% of the respondents, employed persons 27%, and housewives 7% . Despite this spread the patient's occupational status is closely linked to the person paying for their medical expenses . This was also found not to have any statistically significant influence on patients' satisfaction with their consultation (table 1). Fifty - two percent of the patients were paying for their medical expenses themselves while families were paying for 30% . Ten percent of the patients including two males were being paid for by their spouses . However, 3% of the patients did not know who would pay and an equal number was being sponsored by their employers . The number being sponsored by their employers was unexpectedly low (2.6%) considering the nigerian government's efforts at promoting a national health insurance scheme (table 1). The effect of managed care in this study cannot be discussed owing to the low number of patients who were using health care insurance, but studies in the united states have demonstrated that managed care affects neither the perception of time used in the consultation nor patients' satisfaction with it . There was also a possibility that the number of students might be lower than the observed figures because some young people in calabar town often claimed to be students when they were not . This finding suggests that people from different occupational backgrounds in our practice environment may not bring their psychological expectations to influence their satisfaction with the consultation . The more a consultation contributes to a patient's understanding of his illness, the higher the likelihood for the patient to be satisfied with the consultation [18, 19] but very often patients get less information than they expect . Fifty - nine percent of the patients had a satisfying consultation, while 52% had some improvement in the understanding of their illness . However, of the 41% of patients with unsatisfying consultations, 28% of them still had an improvement in their illness understanding (table 2). This suggests that, despite the lack of illness understanding there is still some satisfaction with the consultation . However this study clearly demonstrates that a patient understanding of his / her illness has a statistically significant effect on the patient's satisfaction with the consultation (table 2). It seems clear that the more informing a consultation is, the more likely a patient is to be satisfied with the consultation . This finding supports the call by one report for the patient to be more involved in decision making . Many patients were observed in this study to have shown great interest when they found the doctor to be willing to provide some explanation about their illness . A patient's ability to cope with his illness can be helped or marred by a consultation, and this is more evident in chronic illnesses . Patients' abilities to cope with their illness based on the information received have been demonstrated by this study to statistically affect patients' satisfaction with the consultation (table 2). It is increasingly clear that better informed patients have better outcomes, choose less risky procedures, and avoid equivocal treatments . A patient's ability to maintain health after a consultation would be addressing one of the core issues in family medicine by promoting prevention of illnesses . This study demonstrates a statistically significant effect of a patient's ability to cope with his illness on his / her satisfaction with the consultation (table 2). This is vital in our environment considering that the bulk of illnesses we manage is due to preventable diseases . In conclusion, factors influencing the patient - doctor consultation are numerous, and the exact influence of any of these factors is not easily isolated, but together these factors influence the interaction either positively or negatively . However, the study has shown that, despite the various factors that are considered to encourage client satisfaction at primary care consultation, a few of such factors contributed to end of consultation satisfaction in our environment . This calls for a refocusing if improvement in the overall patient care in our cultural context is to be achieved with the aim of meeting patient needs . Teaching of consultation process must take these factors into consideration . To further address effort towards improving patient satisfaction rates in the study centre, it is recommended that the physician - related factors that influence the doctor - patient encounter should be further studied . In particular the effect of sociodemographic variables such as same - sex consultation, cultural / language diversity, and experience should also be further explored.
Acute pancreatitis is an inflammatory intra - abdominal process, which in approximately 1520% of patients presents in a severe form, with a gradual establishment of multiple organ dysfunction or local complications, including necrosis, pseudocyst, and abscess . Severe acute pancreatitis is a condition associated with high mortality, which is characterized by a complex and incompletely understood pathophysiological mechanism [2, 3]. The deficit in our understanding of the mechanism driving the inflammatory process in acute pancreatitis is a reason why our therapeutic strategy has failed to reduce mortality, despite ongoing research . When acute pancreatitis leads to the establishment of acute kidney injury, there is a 5- to 10-fold rise in mortality, which can reach 70% [46]. The prevention of acute kidney injury can be a useful strategy in the prevention of the morbidity and mortality associated with acute pancreatitis . Eugenol (1-allyl-4-hydroxy-3-methoxybenzene) is a naturally occurring substance, found in the essential oil of commonly consumed spices such as clove oil as well as cinnamon, basil, and nutmeg oils . It has many pharmacological properties which are mainly analgesic, anti - inflammatory, antioxidant, and vasodilatory action, while it has been shown to ameliorate kidney injury in a model of gentamycin - induced nephrotoxicity . The aim of this study is to assess the possible reduction in the extent of acute kidney injury after administration of eugenol in an experimental model of acute pancreatitis . 106 male wistar rats, aged 3 - 4 months and weighing 220350 gr, were used in this study . They were housed in cages under standard laboratory conditions (12 hr light - dark cycles, 2225c room temperature, and 5558% humidity), with free access to food and water . The animals were procured from the hellenic pasteur institute (athens, greece). The experiment took place at the elpen experimental research center (pikermi, greece), while the histological analysis was carried out at the lab of histology, embryology, medical school, democritus university of thrace . The experimental surgical procedures and the general handling of the animals conformed to the international guidelines of directive 86/609/eec on the protection of animals used for experimental and other scientific purposes . The animals were randomly assigned in 3 groups: sham (n = 20), control (n = 46), and eugenol (n = 40). The animals were anaesthetized initially by being placed in a glass box containing isoflurane and then through administration of 0.25 ml of butorphanol (dolorex; intervet / schering / plough animal health, boxmeer, holland) by subcutaneous injection . The animals were intubated with a 16 g venous catheter, which was then connected to a ventilator set at 70 breaths / min and a tidal volume of 3 ml . After confirmation of the success of intubation, anaesthesia was maintained by a mixture of 93% o2, 5% co2, and 2% isoflurane . Briefly, after induction of anaesthesia and preparation of the surgical site, the abdomen was entered via a 3 cm midline incision under sterile conditions . The biliopancreatic duct was identified and ligated near the duodenal wall with a 4 - 0 silk sutures (in the control and eugenol groups, but not in the sham group). 1 ml of normal saline and 1 ml of 5% d5w were instilled in the abdominal cavity . The abdomen was closed with vicryl 2 - 0 sutures . In the eugenol group, eugenol was administered by a nasogastric catheter in a dose of 15 mg / kg, while the sham and control groups received corn oil solution without eugenol . Postoperatively, analgesia was maintained through subcutaneous administration of 2 ml / kg butorphanol (dolorex; intervet / schering / plough animal health, boxmeer, holland). Euthanasia was performed at a predetermined time for each animal with the use of ketamine (narcetan; vetoquinol, buckingham, uk) 0.30.6 ml and xylazine (rompun; bayer, uxbridge, uk) 0.10.3 ml, followed by a midline laparotomy and exsanguination of the abdominal aorta . Time points for analysis were 6, 12, 24, 48, and 72 hours postoperatively . Serum samples for measurement of urea and creatinine as well as specimens from both kidneys for histopathological examination were acquired . Louis, mo, usa) was purchased and prepared in an oily solution in the chemical laboratory of elpen pharmaceutical co. inc . This was achieved with the admixture of pure eugenol in a corn oil solution in a concentration of 1.5 mg eugenol / ml . Samples were placed in 10% buffered formalin solution, and 4 m paraffin - embedded sections were stained with hematoxylin / eosin . All specimens were evaluated by a pathologist blinded to the sequence of the biopsy specimens . Slides were evaluated with regard to 5 histopathological parameters and with the use of a semiquantitative scoring system as depicted on table 1 . The scores of each individual parameter for each slide were added and a histopathological score was obtained for each specimen . Immunohistochemical staining was applied to detect the possible expression of inflammatory cytokines like il-6, tnf-, and myeloperoxidase . The following antibodies were used: myeloperoxidase (rabbit polyclonal), dako (a 0398), diluted 1: 400 tnf- (rabbit polyclonal), abnova (pab8016), diluted 1: 1000, il-6 (rabbit polyclonal), and abcam (ab6672), diluted 1: 500 . The buffers, blocking solutions, secondary antibodies, avidin - biotin complex reagents, and chromogen were supplied in a detection kit (envision hrp, mouse / rabbit detection system (k 5007), dako). To inhibit endogenous peroxidase, the specimens were incubated with 3% h2o2 (200 ml h2o and 6 ml h2o2) for 15 min in a dark room . Before the primary antibody was applied, the sections were immersed in 10 mm citrate buffer (ph 6.0), rinsed in tris - buffered saline, and subsequently heated in a microwave oven (650800 w) for three cycles of 5 min . The slides were washed with tris - buffered saline before application of the primary antibody in order to reduce nonspecific binding of antisera . Sections were then briefly counterstained with mayer's hematoxylin, mounted, and examined under a nikon eclipse 50i microscope (nikon instruments inc, ny, usa). The average labeling index was assessed according to the proportion of positive cells, after scanning the entire section of the specimen . The results were graded as negative (0) for <10% of stained cells, low (1) for> 10% and <30% of cells stained, moderate (2) for> 30% and <70% cells stained, and high expression (3) for> 70% cells stained (table 1). The statistical analysis of the results was completed with the use of the 20th version of spss (statistical package for the social sciences, spss inc ., we performed an analysis in which the data were treated as qualitative using fisher's exact test (this test was preferable to x because of the small number of animals in each subcategory / time point). Evaluation of the different variables was performed to determine whether they were normally distributed (kolmogorov - smirnov are shapiro - wilk). The three different groups were then analyzed using the kruskal - wallis one - way analysis of variance test . Finally, the mann - whitney u test was further used to compare the groups in pairs . These tests were applied to the overall sample and for each individual subgroup corresponding to individual time points (6, 12, 24, 48, and 72 hours postoperatively). The difference between the eugenol and control groups is apparent at 48 and 72 hours after induction of pancreatitis (figures 1 and 2). The histological score for these two groups is higher compared to the sham group at 48 and 72 hours and for the whole sample . Eugenol administration lowers hyperemia and dilation of renal parenchyma capillaries and the difference was statistically significant for the 48 and 72 hour time points and for the whole sample . The eugenol group exhibited lower values than the control group and both exhibited higher values than the sham group . The same was true for hyperemia and dilation of renal corpuscles capillaries for the 48- and 72-hour time points, but not for the whole sample . There were no inflammatory infiltrations in any of the animals in our experimental model and measurement of this factor did not produce any results . Edema was reduced through the administration of eugenol and, again, the difference to the control group was significant for the 48- and 72-hour time points and the whole sample . The control group had higher values than the sham group at 48 hours and also higher values than both the sham and eugenol groups at 72 hours . When values of the whole sample were considered, the control group had higher values than the eugenol group, which in turn had higher values than the sham group . Analysis of the whole sample showed only higher values for the eugenol and control groups when compared to the sham group . There was no clear difference regarding il-6 expression between the different groups (figures 3 and 4). On the contrary there was a statistically significant difference between the eugenol and control groups 72 hours after induction of pancreatitis, while both groups exhibit higher tnf- expression than the sham group . There was no statistically significant difference between the eugenol and control groups for mpo expression, although there was a trend toward higher expression for the control group after 72 hours . Eugenol administration resulted in lower serum levels of urea and creatinine especially at the 48- and 72-hour time points, compared to the control group . Urea and creatinine levels were higher for both the eugenol and control groups, when they were compared to the sham group (figure 4). The results of this study suggest that eugenol attenuates the intensity of the histopathological changes and the expression of tnf- and mpo in the renal parenchyma, while lowering the values of serum urea and creatinine when administered in a rat acute pancreatitis experimental model . To evaluate the extent of kidney injury, we decided to evaluate serum urea and creatinine levels and the histopathological changes in the kidney, as well as the expression of tnf-, il-6, and mpo in the renal parenchyma . The role of cytokines, such as tnf- and il-6, in the pathophysiology of acute pancreatitis has been studied extensively and they have been found to contribute to the activation of the systematic inflammatory response process and multiorgan failure, which is a hallmark of severe acute pancreatitis and is, ultimately, correlated with the observed high mortality rates [10, 11]. The role of cytokines in acute kidney injury has been found to be equally important . The cytokine - mediated inflammatory response has a central role in the pathophysiology of acute renal failure irrespective of its cause . Mpo has been used as a marker of neutrophil migration in acute pancreatitis studies and has been correlated to the severity of kidney injury [1214]. The histopathological evaluation showed that the histologic score was lower for the eugenol group in comparison to the control group at 48 and 72 hours from the initiation of the inflammatory process (means: 3.75/6.5 and 4.12/7.62, resp .) And this difference was statistically significant . This difference between the two groups was also present for individual histological changes such as hyperemia and dilation of renal parenchyma and renal corpuscles capillaries and edema . The difference observed in the degree of acute tubular necrosis and inflammatory infiltration was not statistically significant . Regarding the expression of inflammatory mediators, tnf- levels were higher for the control group in comparison to the eugenol group with the difference reaching statistical significance at the 72-hour time point, while there was a trend for higher mpo expression in the control group at 72 hours, which was, however, not statistically significant . In contrast, il-6 levels did not show the same correlation and there were no statistically significant differences between the eugenol and control groups . We chose the bile - pancreatic duct ligation model as it is a well - characterized model of acute pancreatitis, which mimics acute pancreatitis caused by biliary obstruction, which is a frequent clinical scenario and results in multiorgan failure similar to that observed in humans [15, 16]. We have previously used this experimental model and we were able to show that it generates acute pancreatitis with histopathological changes in the pancreatic tissue including hemorrhage and necrosis . Out of a total of 106 animals, 6 died and the fact that they were all in the control group could be seen as further evidence supporting the protective role of eugenol . It is possible that these animals would have exhibited signs of severe kidney injury, if they had survived until the predetermined time of euthanasia . However, since the distal bile - pancreatic duct ligation model is not usually fatal, we cannot directly attribute the death of these animals to the severity of acute pancreatitis . Eugenol has been shown to possess a multitude of pharmacological effects, some of which make it a likely candidate for use in the setting of acute pancreatitis and can explain the results observed in our study . The analgesic action of eugenol has been well documented and doses in the range of 40100 mg / kg have been shown to be effective in rat experimental models [1820]. In addition, eugenol acts as an anti - inflammatory substance inhibiting cyclooxygenase and reducing the release of proinflammatory mediators such as il-1, tnf-, and pge2 [2224]. The antioxidative potential of eugenol has been studied in a number of, mainly in vitro, studies where it has been shown to bind to free oxygen radicals and attenuate the action of oxidative substances [2528], while a recent study of gentamycin - induced nephrotoxicity offers insight into how eugenol can prevent kidney injury by reducing oxidative damage . These combined properties of eugenol can be used to explain the observed reduction in tnf- expression, as well as the reduction of kidney inflammation . Eugenol administration causes a dose - dependent, reversible vasodilation through its effect on the endothelial cells [29, 30], which is comparable to nifedipine . The potential of eugenol to inhibit the vasoconstriction that is associated with kidney injury points to another potential mechanism for its effect in the model of acute pancreatitis . A number of authors have proposed strategies to reduce kidney injury caused by acute pancreatitis . Zhang et al . Have tried dexamethasone administration in an experimental model of retrograde injection of sodium taurocholate in the pancreatic duct . The dexamethasone group exhibited milder congestion of the glomerular capillary, swelling of the renal tubular epithelial cells, and less inflammatory cell infiltration than that of the control group, which was shown by the lower histological score at the 6- and 12-hour time points . The same authors found a significant difference in the serum levels of tnf- in favor of the dexamethasone group, while expression of nf-b in the renal tissue was more pronounced in the dexamethasone group . The same model has been used to study octreotide and baicalin (5,6,7-trihydroxyflavone-7-o - d - glucuronic acid). The administration of these substances had a protective effect on the kidney and both the histological score and renal parenchyma nf-b expression were lower in comparison to the control group . Il-6 were reduced compared to the control group in another study with the same experimental protocol . There have been a number of studies of plant derived substances, used in traditional chinese medicine . Ligustrazine proved to be protective for the kidney as was demonstrated by the lower creatinine levels and the milder histopathological changes in comparison to the control group . In another study, the administration of 3 traditional chinese medicine substances (ligustrazine, kakonein, and panax notoginsenosides) resulted in reduced mortality and milder histopathological changes in the rat kidney . Finally, the model of induction of acute pancreatitis through sodium taurocholate administration was used for the study of poly(adp - ribose) polymerase inhibition, through 3-aminobenzamide (3-ab) administration . The administration of 3-ab resulted in reduced mortality and a reduction in the increase of creatinine, tnf-, il-1b, and il-6, milder histopathological changes, and reduced mpo expression in the kidney . The half life of eugenol in the rat has been determined to be 18,3 hours; therefore, at 72 hours, most of the initial dose would have been cleared from the circulation . It is possible that a repeat administration of eugenol could further increase the therapeutic result . Moreover, the time frame of our protocol reached 72 hours, which was not adequate for the complete evaluation of the effect of eugenol . Indeed, a difference in the extent of kidney injury between the eugenol and control groups is first observed 48 hours after the onset of acute pancreatitis and it is greater at 72 hours . In conclusion, the administration of eugenol in a rat model of acute pancreatitis was protective for the kidneys in our experimental model . Further research is necessary to determine the possible role of eugenol in the management of acute pancreatitis.
Periodontal health can be described as a dynamic state where the activity of proinflammatory / antimicrobial cytokines to control infection is optimally balanced by anti - inflammatory mechanisms to prevent unwarranted inflammation . In subjects susceptible to periodontal disease (pd), an imbalance of the inflammatory response results in excessive production of proinflammatory cytokines and the subsequent loss of periodontal attachment . On the other hand, furthermore, the release of tissue regenerating factors may contribute to periodontal regeneration by regulating the function of periodontal ligament cells, endothelial cells, and cementoblasts . In this setting, neurotrophin brain - derived neurotrophic factor (bdnf) has been reported to enhance periodontal tissue regeneration [4, 5]. Bdnf is a member of the neurotrophin family which is expressed by vascular endothelium and osteoblastic, immune, and neuronal cells . Bdnf is reported to be involved in the joint inflammatory process and its production is increased in response to proinflammatory cytokines . Although a role for bdnf in periodontal regeneration has been proposed, no information is available concerning bdnf and periodontal disease . The aim of this study was to measure the levels of bdnf in periodontal tissues from patients with chronic periodontitis . The presence of polymorphisms rs6265 and rs4923463 of the bdnf gene and its correlation with inflammatory and clinical parameters were also assessed . Twenty - eight patients with cp, treated at the periodontal clinic, school of dentistry, at universidade federal de minas gerais (ufmg, brazil), were enrolled in this study . Patients in this study met the following inclusion criteria: previous history of cp, diagnosed according to previously described criteria: (1) exhibiting more than one tooth with probing depth higher than 5 mm, (2) exhibiting more than two sites with clinical attachment loss deeper than 6 mm, and (3) exhibiting lesions distributed in more than two teeth in each quadrant . Patients who met the following criteria were excluded: (1) having a history of smoking, (2) use of antibiotic, (3) usage of anti - inflammatory and/or immunosuppressive medications during the 6 preceding months, and (4) a history of any systemic diseases (i.e., immunologic and autoimmune disorders, diabetes mellitus). The control group (hc) comprised 29 age and gender matched periodontally healthy patients enrolled for third molar removal surgery . Periodontal examination was performed in both groups of patients, cp and hc, at the initial visit to determine probing depth (pd), clinical attachment loss (cal), and bleeding on probing (bop). Measurements were performed full - mouth at 6 sites per tooth (mesiobuccal, midbuccal, distobuccal, mesiolingual, midlingual, and distolingual). All the measurements were performed by the same examiner . At the time of the examination a peripheral blood sample periodontal tissue samples from periodontal pockets or healthy oral mucosa extracted during surgery of impacted third molars were fixed in 10% buffered formalin, embedded in paraffin wax, and cut longitudinally (3 m). The sections were deparaffinized, rehydrated, and stained with h&e for evaluation of the inflammatory infiltrate . Inflammatory cells were counted in four fields in two independent sections, using a light microscope (axioskop 40 zeiss; carl zeiss, gottingen, germany) at 400x magnification . The concentrations of the il-17a, bdnf, il-10, and tnf- and the chemokine cxcl10 were measured in periodontal tissues by enzyme - linked immunosorbent assay (elisa) using commercially available kits (r&d systems, minneapolis, mn, usa). The lower limit of detection for each cytokine was 15 pg / ml, 3.9 pg / ml, 5.5 pg / ml, 20 pg / ml, and 4.5 pg / ml, respectively, for il-17a, il-10, tnf-, bdnf, and cxcl10 . The data were determined using a standard curve prepared for each assay and expressed as picograms of cytokine / chemokine per 100 mg of tissue . Periodontal tissue samples were also used for determination of myeloperoxidase (mpo) activity, a neutrophil enzyme marker, as described earlier . The mpo activity in homogenized periodontal tissues was evaluated by enzymatic reaction and absorbance was measured at 450 nm . The mpo content was expressed as relative units calculated from standard curves based on mpo activities from 5% casein peritoneal - induced neutrophils assayed in parallel . Total genomic dna was extracted from blood samples using qiaamp dna blood mini kit (qiagen, valencia, ca, usa) according to manufacturer's instructions . Quality, integrity, and quantity of dna were analyzed by nanodrop spectrophotometer (thermo scientific, wilmington, de, usa). All amplifications were carried out in an abi 7900h thermal cycler (applied biosystems, foster city, ca, usa) using taqman genotyping master mix and following manufacturer's recommended amplification conditions . Chi - square test analysis was used to test for deviation of genotype frequencies from hardy - weinberg equilibrium . The levels of cytokines in periodontal tissues and the frequency of gene polymorphisms were compared by the student's t - test and chi - square tests . The sample included in the current study was composed by age and gender matched groups . The clinical features pd, cal, and bop were significantly higher in the cp than in the hc group (p <0.0001) (table 1). The levels of il-17a, cxcl10, il-10, tnf-, and bdnf in periodontal tissues were greater in cp patients than in controls (figure 1). Moreover, the mpo activity and the inflammatory infiltrate in the periodontal tissues, characterized by polymorphonuclear and mononuclear leukocytes, were significantly higher in the cp than in the hc group (figure 2). The bdnf and il-10 levels in periodontal tissues were negatively correlated (r = 0.691, p = 0.002), whereas no correlation between bdnf and il-17a, tnf-, cxcl10, or clinical parameters was observed (pd, cal, and bop). Following the clinical investigation, the frequencies of polymorphisms (bdnf) were assessed in blood samples of hc and cp subjects (table 2). The frequency of these genotypes agreed with the hardy - weinberg equilibrium (p> 0.05). The distribution of the bdnf polymorphisms was similar between the groups (table 2). We also investigated whether some of these polymorphisms were associated with worse clinical periodontal parameters . As shown in table 3, no differences in clinical parameters were found when comparing the genotypes . The levels of bdnf and the inflammatory mediators cxcl10 and tnf- were increased in gg genotype of bdnf rs6265 polymorphism (figures 3(a), 3(b), and 3(c)), but mpo levels did not alter significantly (figure 3(d)). In bdnf rs4923463 polymorphism the levels of bdnf and mpo did not differ, but the levels of cxcl10 and tnf- were higher in patients with aa genotype (figures 3(e)3(h)). A wide range of nonneural cells in peripheral tissues or in the immune system expresses neurotrophins and their receptors . Thus, the mitogenic and immune regulatory functions of neurotrophins have been discussed [6, 1012]. The neurotrophin bdnf is reported to be involved in inflammatory reactions, and its production is increased in response to proinflammatory cytokines . The present study is the first to demonstrate that bdnf levels were increased in periodontal tissues from chronic periodontitis compared to healthy subjects . In agreement with our findings, bdnf was found in high levels in the plasma of patients with osteoarthritis and in patients with rheumatoid arthritis . While some authors reported that bdnf levels were significantly correlated with self - reported pain, others did not find association between bdnf and clinical parameters of arthritis . Several studies analyzed bdnf rs6265 polymorphisms in psychiatric disorders [1618]. To date, few studies examined the rs4923463 polymorphism [16, 1820]. While one study found correlation between snp rs4923463 and attention - deficit / hyperactivity disorder, schizophrenia, and risk of suicide in bipolar disorder, another study did not find any correlation between schizophrenia and rs4923463 polymorphism . Previously, two studies have evaluated the effects of bdnf polymorphisms in bone [21, 22], but there are no available studies in periodontal disease . In the present study we did not find differences in bdnf genotype distribution between patients with cp and controls . Nevertheless, we found that subjects with gg (rs6265) genotype expressed higher levels of bdnf in periodontal tissues, in agreement with a previous report showing that bdnf - m66 variant alters intracellular trafficking and impairs bdnf secretion . On the other hand, rs6265 polymorphism interestingly, the snp rs6265 was reported as a phossnp, which means this snp regulates protein phosphorylation . The snp rs6265 affects substrate - kinase interaction between bdnf protein and chek2 kinase and regulates bdnf phosphorylation at site t62 . Subjects aa genotype carriers exhibited lower bone mineral density compared to g carriers . Specifically, bdnf - v66 (major allele g at rs6265) transfection significantly increases expression of osteoblast specific markers (opn, bmp2, and alp) and promotes osteoblast differentiation and maturation in cell culture . An association of rs6265 with bone metabolism was also suggested in the largest meta - analysis involving 32,961 individuals of european and east asian ancestry . They found that homozygous minor allele a carriers (aa) have significantly decreased bmd compared to major allele g carriers (ga and gg). It has been reported that bdnf is able to induce an increase in il-10 expression . However, a negative correlation between the production of bdnf and il-10 was observed in samples from patients with periodontitis . Il-10 can inhibit the release of proinflammatory cytokines from monocytes / macrophages and can therefore inhibit the lipopolysaccharide- and ifn--induced secretion of inflammatory cytokines (e.g., tnf-, il-1, il-6, cxcl8, and others). In periodontal disease, il-10 is thought to be associated with lower disease severity . Previous studies demonstrated that bdnf induces periodontal tissue regeneration by activation of cementoblasts differentiation, vascular endothelial cell migration, and also has a positive effect on bone remodeling [5, 28]. This data together suggested that bdnf has a role in bone remodeling and any change in this neurotrophin levels could have an impact in bone repair . Finally, we observe that cp subjects with gg (rs6265) and aa (rs4923463) genotypes demonstrated increased levels of tnf- and cxcl10 . Cxcl10 has several roles, such as chemoattraction of macrophages, t cells, nk cells, and dendritic cells . In addition, previous studies showed that exposure to bdnf substantially and synergistically enhanced tnf- levels in vitro, and tnf- preconditioning increased proliferation, mobilization, and osteogenic differentiation in vitro . We can hypothesize that the concomitant increase of bdnf, tnf-, and cxcl10 in patients with the gg genotype may be an attempt of the host to induce periodontal healing . So, maybe if these patients were examined after periodontal treatment, they could display higher and better levels of tissue regeneration compared to patient who do not exhibit the gg genotype . In conclusion, bdnf seems to be related to periodontal pathogenesis and also involved in tissue repair . The results obtained here provide a benchmark for future studies with a large cohort of patients to help strengthen and understand the influence of neurotrophins in periodontal disease.
We analyzed information about 634 immigrants from latin america seen at the tropical medicine unit of the ramn y cajal hospital in madrid, spain, during april 1989june 2008 . We used 5 strict criteria for diagnosing vlm: 1) positive serologic test for toxocara sp . Roundworm infection, performed by using a commercial elisa toxocara immunoglobulin (ig) g ridascreen (r - biopharm gmbh, darmstadt, germany), following the manufacturer s recommendations; 2) absolute peripheral blood eosinophil count> 500 cells / mm; 3) exclusion of other parasites causing eosinophilia, such as intestinal nematodes, particularly strongyloides stercoralis (excluded by larval culture and serology by elisa igg), schistosoma sp ., fasciola hepatica, trichinella spiralis, taenia solium, echinococcus granulosus, and cutaneous and blood microfilariae; 4) symptoms associated with vlm (respiratory signs, such as asthma, dyspnea, and eosinophilic pneumonia; dermatologic symptoms, including pruritus and recurrent urticaria; and abdominal symptoms, including abdominal pain and hepatomegaly); and 5) response to treatment with albendazole (1015 mg / kg / d in 2 doses orally for 5 days) assessed 6 months after treatment, decreased titers to toxocara sp . The most frequent countries of origin for patients were ecuador 221/634 (34.9%), bolivia 176/634 (27.8%), peru 71/634 (11.2%), and colombia 56/634 (8.8%). Median age was 32 years (range 440 years); 421 (66.4%) patients were male . The median number of months from arrival in spain to first consultation at the tropical medicine unit was 19 months . Concomitant serologic results positive for toxocara sp . Roundworm infection and eosinophilia were found in 28 (4.4%) patients; 606 patients were excluded . Of these 28 patients, 11 were excluded because of other concomitant parasitic infections that also can cause eosinophilia: 8 patients had positive elisa results for s. stercoralis nematodes (not detected in fecal samples or larval culture); 1 had ascaris lumbricoides eggs in feces; 1 had a positive indirect hemagglutination result but negative elisa result for e. granulosus tapeworm; and 1 had a positive elisa serologic result for t. spiralis nematodes . Only 4 of the 5 remaining cases fulfilled the strict inclusion criteria (table); 1 patient was asymptomatic . After 6 months of treatment with albendazole, titers for toxocara sp . Roundworm infection and eosinophil count decreased, and symptoms improved or resolved for the 4 patients . All patients were treated with albendazole (1015 mg / kg / d in 2 doses orally for 5 days). Clinical toxocariasis is rarely diagnosed in western countries as previously described despite evidence of environmental exposure (1). Results of seroprevalence surveys performed in healthy adults in france were positive for 2%5% of persons in urban areas, compared with 14%37% in rural areas (2). In latin america, rates vary from 1.8% to 51.6% (3,4). However, literature references to vlm imported by immigrants are scarce (5), and the disease may be underdiagnosed in the immigrant population, partly because of nonspecific symptoms and the limitations of serologic diagnosis . In our study, toxocariasis is a common cause of eosinophilia in peripheral blood, although its absence does not exclude infection by toxocara sp . Roundworm infection without eosinophilia (6); similarly, 27% of patients with high antibody titers had eosinophil counts within the reference range (7). By including only patients with eosinophilia, our study applied more stringent criteria . Thus, 28 (90%) of 31 patients who had positive serologic results showed an elevated eosinophil count, in accordance with previously described high toxocara sp . Roundworm seroprevalence (<68%) in patients with eosinophilia of unknown cause (8). Eleven of the 28 patients with positive serologic results for toxocara sp . Roundworm and eosinophilia also had positive serologic results for other parasites that cause eosinophilia . One patient who was infected with a. lumbricoides roundworm had asthma, hepatomegaly, and pruritus . The latter is not usually associated with this parasite, which suggests possible co - infection . Serologic tests for toxocara sp . Roundworm infection should be interpreted with caution because commercial elisa kits that use excretory and secretory antigens derived from second - stage larvae of toxocara sp . Roundworms exhibit a sensitivity of 91% and a specificity of 86%; cross - reactivity has also been described with other nematode infections . The positive serologic results for t. spiralis nematodes and e. granulosus tapeworms may have been caused by cross - reactivity (9). These patients had asthenia and asthma, respectively, and symptoms resolved after treatment with albendazole . Eight patients with strongyloides antibodies were also excluded; however, this finding does not exclude co - infection by both parasites . Finally, a limitation of the study was that we could not definitively exclude cryptic strongyloidiasis for 12 patients because of the difficulty in finding s. stercoralis threadworms in feces and because detection of strongyloides antibodies was not possible . This study illustrates the difficulties in diagnosing vlm in immigrants from tropical and subtropical areas of latin america because only a very small proportion of patients in the series (n = 4) had vlm . The most common symptoms were respiratory (3/4); 2 patients had asthma - like syndrome and 1 had chest pain followed by abdominal pain (2/4). Typical manifestations of vlm are abdominal symptoms (pain, hepatomegaly) and respiratory symptoms (severe asthma, eosinophilic infiltrates). In addition to this, evidence points to toxocara sp . Roundworm infection as a risk factor for asthma in some populations (11,12). Albendazole is the treatment of choice for vlm; for practical purposes, it could be recommended for presumptive treatment in immigrants from latin america with eosinophilia in whom strongyloidiasis is suspected (13). However, the superiority of ivermectin over albendazole has been documented in the treatment of chronic strongyloidiasis (14). Vlm may be difficult to diagnose, especially in immigrants from regions in latin america where polyparasitism is endemic . Positive serologic test results, marked eosinophilia, absence of other helminthic infections, compatible clinical signs, and disappearance of symptoms after specific treatment can help establish a vlm diagnosis, especially in areas of low parasitism . Vlm should be included in the differential diagnosis of eosinophilia in immigrants (children and adults) from tropical areas if respiratory or abdominal symptoms are evident . Albendazole is an effective and relatively safe drug that could be used to treat suspected vlm and other concomitant nematode infections, including cryptic s. stercoralis threadworm infections . Empirically described treatment may lead to resolution of clinical symptoms, even though ivermectin is a better treatment for chronic strongyloidiasis.
Although lbp has generally been believed to be uncommon before the age of 20, the prevalence of lbp among school children and adolescents has been reported to be high in different parts of the world, mostly in western countries, where it varies from 1040% . The nhanes ii (national health and nutrition examination survey series) reported the onset of lbp before the age of 20 in 11% of the general population . Lbp prevalence rates were found to be 2831% in 2 studies carried out on school children in kuwait and tunisia . These studies show that the prevalence of lbp in children is high, equaling that of adults by the end of the growth period . Lbp has a significant economic impact on individuals, society, and quality of life . Many studies have analyzed the risk factors associated with lbp in children and adolescents to describe risk factors profiles [3,58]. To our knowledge there have been no reports on nlbp and related factors among school children in turkey . The aim of this study was to investigate possible factors associated with pain intensity among turkish school children with nlbp, aged 1018 years, and to determine the relationship between related factors and pain intensity using the regression tree method . This study was conducted on school children aged 1018 years in the city of denizli, located in the western part of turkey . Out of 88 primary and high schools in the city, fifth and eleventh grade classes were selected from each of 10 schools (8 governmental and 2 private), using a simple random sampling method . The city department of education gave written permission, and all parents of students gave informed consent . The exclusion criteria were having any kind of musculoskeletal, rheumatic, orthopedic, somatic or psychiatric disorders . All of the exclusion criteria were considered to define nlbp more clearly because we studied nonspecific low back pain . In the sampled schools, 624 children were interviewed in total and 292 (46.8%) were reported as having nlbp . After excluding the students who displayed the aforementioned disorders, 222 students (116 girls and 106 boys) the visual analog scale (vas) was used for measuring the intensity of pain . The vas is designed to present to the respondent a rating scale with minimum constraints . This scale, shown below, was reported as the number of cm . From left of line, with range 010: the interviewers used in the study were selected from the final year students in the physical therapy school of pamukkale university . The dependent variable was pain intensity measured in cm, and the independent variables were bmi, sex, regular exercise habit, studying posture, transportation to / from school, duration of studying, bag handling and type of bed the participants were asked to select 1 of the following studying postures: (1) sitting on chair and studying on a table, (2) sitting on ground without chair and table, and (3) lying in prone or supine position on a bed . Two methods of enquiry about nlbp were used, namely a direct question and a pre - shaded manikin question: have you ever had low back pain? (look at the drawing) have you experienced pain in the shaded area which lasted for 1 week, a month, or longer? Students who answered both of the questions as the regression tree method (rtm) was used to determine risk factors that may affect pain intensity . Rtm is a tree - based model, and is more useful than traditional statistical methods when a data set is large and when there are many variables . Moreover, the rtm takes into consideration interactions among variables, and is not affected by high correlations between risk factors . There is no assumption about distribution shapes of risk factors, but outcome variable should be numeric . In rtm the association between risk factors (x) and outcome variable (y) (pain intensity, in this study) are examined by a schematic representation . Homogeneous groups are constituted according to the adequate cut - off values of the risk factors . At the beginning, all individuals are collected in 1 group called a root node . Homogeneous groups that come into being based on recursive binary splitting are termed terminal node . The homogeneous group means that this group is sufficiently homogeneous and cannot be split any more . Splitting continues until the tree reaches maximum size, and then passing the selection of adequate tree structure stage, called pruning . The maximum tree is not used for every data set because of its overfit structure . After pruning, the values that take place in the terminal nodes give the mean and variance of that group . Two methods of enquiry about nlbp were used, namely a direct question and a pre - shaded manikin question: have you ever had low back pain? (look at the drawing) have you experienced pain in the shaded area which lasted for 1 week, a month, or longer? Students who answered both of the questions as the regression tree method (rtm) was used to determine risk factors that may affect pain intensity . Rtm is a tree - based model, and is more useful than traditional statistical methods when a data set is large and when there are many variables . Moreover, the rtm takes into consideration interactions among variables, and is not affected by high correlations between risk factors . There is no assumption about distribution shapes of risk factors, but outcome variable should be numeric . In rtm the association between risk factors (x) and outcome variable (y) (pain intensity, in this study) are examined by a schematic representation . Homogeneous groups are constituted according to the adequate cut - off values of the risk factors . At the beginning, all individuals are collected in 1 group called a root node . Homogeneous groups that come into being based on recursive binary splitting are termed terminal node . The homogeneous group means that this group is sufficiently homogeneous and cannot be split any more . Splitting continues until the tree reaches maximum size, and then passing the selection of adequate tree structure stage, called pruning . The maximum tree is not used for every data set because of its overfit structure . After pruning, the values that take place in the terminal nodes give the mean and variance of that group . The descriptive statistics are shown (table 1) as mean sd and as frequencies and percentages . The overall mean and standard deviation of pain intensity was 2.580.86 (minimum=1, maximum=5). Among the risk factors used in this study, duration of studying, type of bed, transportation to / from school, and bmi score were found to have a significant effect on pain intensity, while sex, studying posture, regular exercise habit, and bag handling were not significant (figure 1). As will be seen from figure 1, 6 homogeneous groups are defined by the rtm according to pain intensity, with an increasing order . These are as follows: group (id=124): studies less than 4 hours, uses school bus for transportation and has 17.13 <bmi <20.9 (mean pain score=1.74). This group is the lowest risk group among all 6 groups, giving the lowest mean . Group (id=4): studies more than 4 hours and sleeps on orthopedic bed (mean pain score=2.48). Group (id=44): studies less than 4 hours and uses public transportation or walks to / from school (mean pain score=2.60). Group (id=125): studies less than 4 hours, uses school buses for transportation and has bmi> 20.9 (mean pain score=2.70) group (id=120): studies less than 4 hours, uses school buses for transportation and has bmi <17.13 (mean pain score=2.86). Group (id=5): studies more than 4 hours and sleeps on wool or cotton beds (mean pain score=3.11). This group is the highest risk group among the 6 groups, giving the highest mean . It has become clear that a high prevalence of lbp occurs not only in adults, but also in children / adolescents . More recently, cross - sectional and longitudinal studies have focused on nlbp in children . The prevalence has been reported to vary from 10% to 40% in the literature, but the authors found it to be 46.7% in their previous cross - sectional study of 624 school children / adolescents 1018 years old . Among the 8 risk factors included in the study, these factors are as follows: transportation to / from school: prista et al found that school children walking> 30 min per day to and from school are associated with an increased risk factor of lbp . They also showed that long distance walking to / from the school might lead to muscle fatigue resulting in back pain . In our study, the transportation to / from school had an important effect on lbp . Bmi: the bmi score was also a significant risk factor affecting pain intensity in our study . As is well known, increased bmi score increases pain intensity in subjects with low back pain . Type of bed: jacobson et al . In 2002 reported that an experimental bedding system (ameri - spring) reduced back pain and improved the quality of sleep . In our study we found also a significant relationship between the type of bed and low back pain intensity . This shows that quality of sleep is a very important factor affecting pain intensity for subjects who suffer from low back pain . Duration of studying: we also found a significant relationship between duration of studying and nlbp intensity . Korovessis et al . In 2004 reported that dorsal pain increased with increasing backpack weight among children . Lee and chiou found that poor sitting habit were statistically associated with lbp . In our study, studying posture was not found to be an important factor . We also found that the sex of the children was not an important factor in nlbp intensity . Results from the literature, as well as our study, show that taking parents and teachers concerns seriously is of vital importance . Therefore, health care providers should evaluate school children carefully and make accurate observations in terms of risk factors, including duration of studying, type of bed, transportation to / from school, and obesity, to predict any severe musculoskeletal problems, especially nlbp . Finally, physical factors and musculoskeletal risk factors are especially important in terms of nlbp in school children . Further studies are needed to investigate psychosocial risk factors and their relationships with nlbp in school children.
This study was developed and approved by the steering committee of the translating research into action for diabetes (triad) study and conducted in one of triad's six translational research centers, kpnc . Kpnc is a group practice, prepaid health plan that provides comprehensive medical services through 17 hospitals and 23 outpatient clinics to> 3 million members located in a 14-county region in northern california (30% of the general population in the geographic areas covered). The kpnc membership closely approximates the population living in the same geographic area demographically except with respect to income: kpnc members underrepresent the very poor and the very wealthy (9,10). Upon comparison with regional birth certificates over a 14-year period, there were no meaningful differences between women who delivered at a kpnc hospital and women who delivered in the underlying region regarding age at delivery or race, except that women who delivered at a kpnc hospital were slightly less likely to be hispanic (25.8 vs. 32.0%). We used the kpnc gdm registry (11) to identify women with gdm who delivered between 1 january 1995 and 31 december 2006 . During this 12-year period, the proportion of women who had been screened for gdm with a 50-g, 1-h oral challenge test during the second trimester increased by 3% over time (age- and race / ethnicity - adjusted proportions 92.5 [95% ci 92.392.7] in 19951997 vs. 95.7 [95.695.9] in 20042006). If results were abnormal (1-h plasma glucose levels 7.8 mmol / l [140 mg / dl]), this test was followed by a standard diagnostic 100-g, 3-h ogtt . We identified 14,448 pregnancies that had a diagnosis of gdm from a health provider and with plasma glucose results during the index pregnancy that met the national diabetes data group (nddg) criteria on the 3-h 100-g ogtt for gdm, i.e., 2 glucose values at or exceeding the following thresholds: fasting, 105 mg / dl; 1 h, 190 mg / dl; 2 h, 165 mg / dl; and 3 h, 145 mg / dl (12,13) without recognized preexisting diabetes (14). We included only women who met the nddg criteria of gdm because, in this clinical setting, the nddg criteria were used for diagnosis of gdm until january 2007 . Approximately 550 women had no diagnosis of gdm but met the nddg criteria (equivalent to 4% of the size of our gdm cohort with a diagnosis); women without a diagnosis of gdm were excluded from the analysis . For the first and second aims, the primary outcome was performance of postpartum screening for diabetes by either an fpg test alone or a 75-g, 2-h ogtt . Because several weeks may elapse before glucose metabolism returns to normal in most women with gdm (15), the american diabetes association (16) and the american college of obstetricians and gynecologists (17) both recommend that postpartum glucose screening be performed at 6 weeks postpartum or later . We considered the postpartum screening performed only if it was done during the first year after delivery starting from 6 weeks and if the woman was not pregnant again . For the third aim, the outcome was the proportion of women identified with either ifg (defined as an fpg 100 mg / dl but <126 mg / dl) or diabetes diagnosed by fpg 126 mg / dl (18). Among women who had postpartum screening, therefore, when we report on trends in postpartum conversion to diabetes, we report only on the fasting values detected by either an fpg or an ogtt . For the fourth aim, we examined the proportion of women who were identified as having pre - diabetes on the postpartum screen (that included ifg as defined above and impaired glucose tolerance [igt] defined as a 2-h plasma glucose value 140 mg / dl) or diabetes (defined as an fpg> 126 mg / dl or a 2-h plasma glucose value 200 mg / dl) (18). Age, gestational age at delivery, race / ethnicity, maternal body weight during the beginning of the second trimester, and gestational age at gdm diagnosis were ascertained from the computerized medical records at birth . Use of insulin or glyburide during pregnancy was obtained from the pharmacy database . Because we had data on body weight but not on height, a woman was considered obese if her weight was 90th percentile of the weight distribution of women of her race / ethnicity in this study population . Macrosomia was defined as birth weight> 4,000 g. data on annual household income were based on census block data . Education and parity were obtained by linkage with the state of california birth certificate database . Because the lag time before state birth certificates became available is 3 years, we have these variables for women who delivered between 1995 and 2004 . The yearly age- and race / ethnicity - adjusted proportion of women with gdm who had postpartum glucose screening and 95% cis were calculated by the direct method, in which the age and race / ethnicity distribution of the entire study population was used as the standard . Among women who had postpartum screening, the direct method was used to calculate the yearly age- and race / ethnicity - adjusted rates of ifg and diabetes diagnosed by fpg . Predictors of postpartum screening were examined in a multivariable logistic regression model adjusted for age, race / ethnicity, education, income, obesity, parity, gestational age at gdm diagnosis, glyburide and insulin use during pregnancy, a macrosomic infant at the index pregnancy, visits to an internal medicine or obstetrics / gynecology provider during the postpartum period, year of delivery, and medical facilities . Sas (version 9.1; sas institute, cary, nc) was used for all analyses . This study was approved by the human subjects committee of the kaiser foundation research institute . The yearly age- and race / ethnicity - adjusted proportion of women with gdm who had postpartum glucose screening and 95% cis were calculated by the direct method, in which the age and race / ethnicity distribution of the entire study population was used as the standard . Among women who had postpartum screening, the direct method was used to calculate the yearly age- and race / ethnicity - adjusted rates of ifg and diabetes diagnosed by fpg . Predictors of postpartum screening were examined in a multivariable logistic regression model adjusted for age, race / ethnicity, education, income, obesity, parity, gestational age at gdm diagnosis, glyburide and insulin use during pregnancy, a macrosomic infant at the index pregnancy, visits to an internal medicine or obstetrics / gynecology provider during the postpartum period, year of delivery, and medical facilities . Sas (version 9.1; sas institute, cary, nc) was used for all analyses . This study was approved by the human subjects committee of the kaiser foundation research institute . We identified 14,448 pregnancies complicated by gdm occurring between 1995 and 2006 among kpnc members who were aged 1544 years and delivered live infants or had still births . These pregnancies occurred among 13,547 women, because 901 had more than one pregnancy during the 12-year study period . The percentage of women with gdm pregnancies who received a postpartum glucose screening test increased from 1995 (20.3%) to 2006 (55.9%). Between 1995 and 2006, the age of women with gdm increased slightly (28.2 5.7 to 28.8 6.0 years) and the proportion of women with gdm who were hispanic increased markedly . The race / ethnicity distributions in 1995 versus 2006 were as follows: 60.7 and 43.4% white, 14.5 and 15.4% asian, 14.1 and 24.9% hispanic, 6.7 and 6.7% african american, 2.0 and 4.4% other, and 2.0 and 5.2% unknown . Changes in the demographics represent changes in the entire population with gdm, regardless of performance of diagnostic screening . After adjustment for age and race / ethnicity, the proportion of women with gdm who received a postpartum glucose screening increased steadily over time from 20.7% (95% ci 17.823.5) in women who delivered in 1995 to 53.8 (51.356.3) in women who delivered in 2006 (fig . Unadjusted characteristics of women by postpartum screening status are illustrated in table 1 . In a multiple - adjusted logistic regression model (table 2), older age, asian or hispanic race / ethnicity, higher education, at least two prior births, earlier gestational age at gdm diagnosis, use of insulin or glyburide during pregnancy, and visits to an internal medicine or obstetrics / gynecology provider during the postpartum period were independent and significant predictors of postpartum screening . Obesity and higher parity were significantly associated with less frequent screening . On the basis of fpg (either performed alone or as part of the ogtt), 3.5% (n = 191) had diabetes and 22.0% (n = 1,228) had ifg . The proportion of women with ifg postpartum remained similar over time, but the proportion of women with diabetes decreased from 19951997 to 19982000 and then leveled off (fig . 564 were screened at postpartum, and 131 had ifg and 32 had diabetes by fpg (age- and race / ethnicity - adjusted rates 24.2 [95% ci 20.27.8] and 6.1 [4.28.1], respectively). Among women who delivered in 20042006, 2,381 women were screened, and 583 had ifg and 80 had diabetes by fpg (age- and race / ethnicity - adjusted rates 24.3 [22.626.0] and 3.3 [2.64.0], respectively). The proportion of women receiving an ogtt increased from 1995 (5.0%) to 2005 (16.6%) and markedly increased in 2006 (71.5%). In 2006, kpnc instituted a nurse managed care program that included greater attention to postpartum screening guidelines . Among the 600 women who underwent a 75-g ogtt in 2006, 16 had diabetes at postpartum . Diabetes was diagnosed in 4 (25%) by fpg alone . Of the remaining 12 women, of the 188 women who were found to have pre - diabetes (i.e., either ifg or igt according to the fasting or 2-h glucose values measured during the 75-g ogtt), only 114 (60%) were diagnosed with ifg; 74 (40%) would have been classified as having normal glucose tolerance on the basis of fpg alone . Therefore, 78 (38%) of the 204 women with either diabetes or pre - diabetes were identified only by the 2-h glucose measurements . We identified 14,448 pregnancies complicated by gdm occurring between 1995 and 2006 among kpnc members who were aged 1544 years and delivered live infants or had still births . These pregnancies occurred among 13,547 women, because 901 had more than one pregnancy during the 12-year study period . The percentage of women with gdm pregnancies who received a postpartum glucose screening test increased from 1995 (20.3%) to 2006 (55.9%). Between 1995 and 2006, the age of women with gdm increased slightly (28.2 5.7 to 28.8 6.0 years) and the proportion of women with gdm who were hispanic increased markedly . The race / ethnicity distributions in 1995 versus 2006 were as follows: 60.7 and 43.4% white, 14.5 and 15.4% asian, 14.1 and 24.9% hispanic, 6.7 and 6.7% african american, 2.0 and 4.4% other, and 2.0 and 5.2% unknown . Changes in the demographics represent changes in the entire population with gdm, regardless of performance of diagnostic screening . After adjustment for age and race / ethnicity, the proportion of women with gdm who received a postpartum glucose screening increased steadily over time from 20.7% (95% ci 17.823.5) in women who delivered in 1995 to 53.8 (51.356.3) in women who delivered in 2006 (fig . Unadjusted characteristics of women by postpartum screening status are illustrated in table 1 . In a multiple - adjusted logistic regression model (table 2), older age, asian or hispanic race / ethnicity, higher education, at least two prior births, earlier gestational age at gdm diagnosis, use of insulin or glyburide during pregnancy, and visits to an internal medicine or obstetrics / gynecology provider during the postpartum period were independent and significant predictors of postpartum screening . On the basis of fpg (either performed alone or as part of the ogtt), 3.5% (n = 191) had diabetes and 22.0% (n = 1,228) had ifg . The proportion of women with ifg postpartum remained similar over time, but the proportion of women with diabetes decreased from 19951997 to 19982000 and then leveled off (fig . 564 were screened at postpartum, and 131 had ifg and 32 had diabetes by fpg (age- and race / ethnicity - adjusted rates 24.2 [95% ci 20.27.8] and 6.1 [4.28.1], respectively). Among women who delivered in 20042006, 2,381 women were screened, and 583 had ifg and 80 had diabetes by fpg (age- and race / ethnicity - adjusted rates 24.3 [22.626.0] and 3.3 [2.64.0], respectively). The proportion of women receiving an ogtt increased from 1995 (5.0%) to 2005 (16.6%) and markedly increased in 2006 (71.5%). In 2006, kpnc instituted a nurse managed care program that included greater attention to postpartum screening guidelines . Among the 600 women who underwent a 75-g ogtt in 2006, 16 had diabetes at postpartum . Diabetes was diagnosed in 4 (25%) by fpg alone . Of the remaining 12 women, 8 (50%) had ifg and 4 (25%) had normal fpg . Of the 188 women who were found to have pre - diabetes (i.e., either ifg or igt according to the fasting or 2-h glucose values measured during the 75-g ogtt), only 114 (60%) were diagnosed with ifg; 74 (40%) would have been classified as having normal glucose tolerance on the basis of fpg alone . Therefore, 78 (38%) of the 204 women with either diabetes or pre - diabetes were identified only by the 2-h glucose measurements . In a managed care plan with a large number of women with gdm pregnancies, we found that between 1995 and 2006, screening for postpartum diabetes increased from 20.7 to 53.8% . The increase in screening performance is not likely to be due to advancing maternal age or changes in the racial / ethnic composition of women with gdm, as this trend in screening performance was similar after adjustment for age and race / ethnicity, and almost the entire population of pregnant women was screened for gdm between 1995 and 2006 . Although the proportion of women with ifg on their postpartum screen did not significantly change over time, the proportion of women with diabetes (diagnosed by fpg levels) at postpartum decreased by 50% . This observed decrease in diabetes among women with postpartum screening is not likely to be a consequence of the small increase (3%) in gdm screening over time . The decrease is more likely because of better identification of diabetes before pregnancy, as suggested by the reported increase in postpartum screening among women with gdm and because of an increase in glucose screening in postpartum women without gdm (1.9% in 1995 vs. 8.2% in 2006). As in other reports (58), the majority of women in our cohort did not undergo postpartum diabetes screening in the early years of the study . It is possible that health care providers might have recommended more postpartum screening among these racial / ethnic groups, given their higher prevalence of diabetes (19). It is also possible that asian women were more likely to have had a recent physical examination, giving the health care provider the opportunity to recommend screening, as suggested by racial / ethnic differences in access to care among gdm women (20). Similar to other reports, we found that greater contact with medical care, either through a postpartum visit or other contacts, was associated with greater postpartum screening and may have provided additional opportunities to perform screening (6,7). Women who were more likely to be screened also were older and had higher educational attainment . Although reasons are speculative, these women may have had greater awareness of their diabetes risk and the recommendation for screening . In women who were screened postpartum, gdm was diagnosed earlier in their index pregnancy, and they were more likely to have been treated with medications, which may have increased their and their provider's awareness of their diabetes risk . Glucose levels on the diagnostic 3-h ogtt during pregnancy were similar in women who were and were not screened, suggesting that these were not used to guide testing . As shown by others (21), there was a suggestion that some of the women with a history of gdm who might have had a higher risk of developing diabetes during the postpartum period, such as those who were obese or with higher parity, were less likely to perform postpartum screening . The american diabetes association (16), the american college of obstetricians and gynecologists (17), and the fifth international congress workshop for gestational diabetes (15) endorse the postpartum ogtt and fpg to different extents . If only fpg were used in postpartum screening, 74 (40%) cases of igt and 16 (75%) cases of diabetes would have been missed . Kitzmiller et al . (19) reported that among 527 women with gdm, at postpartum 16.5% had isolated igt, only 16% of women in whom diabetes was diagnosed met the criteria for both elevated fpg and 2-h values, and 21 of 25 women met the criteria for diabetes according to their 2-h values alone . Hunt and conway (21) also reported that one - third of their postpartum gdm cohort undergoing the ogtt and who had diabetes or pre - diabetes had isolated 2-h elevations . Their results are very similar to those found in this study: 78 of 204 women compared with 41 of 117 (or 38% vs. 35%). Therefore, the greater convenience of the fpg needs to be weighed carefully against its decreased sensitivity, particularly among women with a history of gdm . We were not able to distinguish whether the lack of screening occurred because of a lack of provider order or other reasons . However, provider orders for screening might occur only after negotiation with the patient, and a lack of provider order may, at least in part, reflect women's objections to the test . We defined obesity by using race / ethnicity - specific percentiles, rather than height - to - weight ratios, thus introducing the possibility for misclassification and artificially decreasing the association between obesity and screening to the null . Information on other confounders, such as family history of diabetes, was not available from electronic records . Because the population of women with gdm is of reproductive age, postpartum screening and subsequent diagnoses of diabetes affect not only the mothers but also future pregnancies . The risk of complications, particularly stillbirths and congenital abnormalities, may be reduced with optimal glycemic control before the subsequent pregnancy (1). Prepregnancy glycemic control might also reduce the risk of the infant to the in utero exposure to hyperglycemia that might lead to childhood obesity and diabetes (22). A diagnosis of pre - diabetes would identify women at high risk of future maternal diabetes, but this risk could be reduced through the application of interventions such as thiazolidinediones, metformin, or intensive lifestyle modification (3,4). We conclude that, among women with a gdm history, postpartum diabetes screening has increased, but screening is still suboptimal . Performance of an fpg alone, as opposed to the ogtt, will miss a subpopulation of women at risk.
Protein phosphorylation and dephosphorylation are central events in cell recognition of external and internal signals, leading to specific responses . While protein kinases transfer a phosphate group from atp to a protein (i.e., phosphorylate), protein phosphatases catalyze the removal of phosphate groups from specific residues of proteins (i.e., dephosphorylate) [1, 2]. The balance between the antagonistic activities of protein kinases and phosphatases are responsible for many cellular functions, including metabolic pathways, cell - cell communication, proliferation, and gene transcription . The complete genome sequencing of various microorganisms made it possible to assemble the kinome and phosphatome of a few trypanosomatids [4, 5]. These strategies have brought new perspectives of researches in the areas of biochemistry, physiology, and genetics, providing knowledge about the microorganisms' life cycles, as well as predicting diagnostic biomarkers, novel drug targets and vaccine candidates against parasitic infections . Parasites engage a plethora of surface and secreted molecules in order to attach and enter mammalian cells . Some of these molecules are involved in triggering specific signaling pathways both in the parasite and the host cell, which are critical for parasite entry and survival . Plasma membranes of cells contain enzymes that are oriented with their active sites facing the external medium rather than the cytoplasm, which are important for host - parasite interactions [7, 8]. In the case of an ectoenzyme other criteria can be included as: (1) the enzyme has to act on extracellular substrate, (2) cellular integrity is maintained during enzyme activity, (3) the products are released extracellularly, (4) the enzyme is not released to the extracellular environment; and (5) the enzyme activity can be modified by nonpenetrating reagents [7, 8]. Supporting this idea, the presence of surface - located phosphatases, called ecto or extracytoplasmic phosphatases have been characterized in several microorganisms . However, the physiological roles of these enzymes in these cells are not well established yet . In eukaryotes, thus, catalytic signature motifs and substrate preferences classified these proteins into four major groups: phosphoprotein phosphatases (ppps), metallo - dependent protein phosphatases (ppms), aspartate - based phosphatases with a dxdxt / v motif (the members of these three groups are ser / thr specific phosphatases) and the distinct group of protein tyrosine phosphatases (ptps). Protein tyrosine phosphatases belong to three evolutionarily unrelated classes: protein tyrosine phosphatases (ptps), cdc25 and low molecular weight phosphatases (lmw - ptps), which have a common motif (cx5r) in their catalytic sites . The classical ptps are classified, depending on the presence or absence of transmembrane domains, into receptor or nonreceptor type phosphatase groups . The use of inhibitors, divalent cations, metal chelators and different ph range has also been an important tool for classification of these enzymes . Likewise, phosphatases may be acid or alkaline according to their ph range for activity . The optimum ph for acid ectophosphatases lies on the acid range (ph values between 4.5 and 5.5), while the optimum ph for alkaline ectophosphatases lies on the alkaline range (ph values between 8.0 and 9.0) [9, 10]. The inhibitors classically used include: phosphotyrosine phosphatase inhibitors ammonium molybdate and sodium orthovanadate; acid phosphatase inhibitor sodium fluoride (naf); secreted phosphatase inhibitor sodium tartrate; alkaline phosphatase inhibitor levamisole and phosphoserine / threonine phosphatases inhibitors okadaic acid and microcystin - lr [1113]. These enzymes may provide microorganisms with a source of inorganic phosphate by hydrolyzing phosphomonoester metabolites [1315] protect them upon entering the macrophage by suppressing the respiratory burst, as well as play a role in cell differentiation, infection of host cells [1820] and protecting the cells from acidic conditions by buffering the periplasmic space with phosphate released from polyphosphates . Some protein phosphatases have been described as being active towards low molecular weight nonproteic phosphoesters, such as alkyl and aryl phosphates, including the phosphotyrosine analog, p - nitrophenylphosphate . From a general standpoint, the surface accessibility of ectophosphatases, along with protein phosphorylated on serine / threonine / tyrosine residues at the cell surface make this set of enzymes a key tool for the survival of pathogens in hostile environments and escaping the host immune responses [19, 2224]. In this review, we describe the role of ectophosphatase activities in host - parasite interactions, particularly ectophosphatases in parasitic protozoa and fungi . Little is still known about the physiological role of protein phosphatase activity in trypanosomatids, even though the first demonstration of this activity in trypanosoma brucei and t. cruzi took place in 1972 . The kinetoplastid parasites have complex life cycles and some of their life forms are difficult to grow in culture, which may represent a problem for studying ectophosphatases . Pathogenic trypanosomatids have at least two different host environments in their life cycles, an insect vector and a mammal . Also, each trypanosomatid genus has different abilities to survive and reproduce in such hosts . T. cruzi invades and replicates in many cell - types, including macrophages, fibroblasts and myocytes . T. brucei is an exclusively extracellular parasite that resides in the bloodstream of the mammalian host . As the life cycles of these parasites take place through widely different environments, frequent and substantial adaptive changes are required in many cell processes, resulting in changes in gene expression, protein levels and protein modifications [26, 27]. Along with those, cell surface components play a key role in the survival of protozoan parasites in hostile environments and in confrontation with host immune responses . Since, these flagellates have an unusual composition of phosphatases with the ptp family being greatly reduced while the stp family is expanded by comparison with human phosphatases . The low similarity to their vertebrate counterparts indicates that these enzymes may be potentially suitable targets for development of potent inhibitors with minimal effects on the physiology of mammalian hosts . Under these conditions, ectophosphatases play an important role in the interaction of cells with their surroundings, especially because their catalytic sites face the extracellular milieu . Ecto - phosphatases has been reported in some protozoa parasites, such as t. rhodesiense, t. congolense, t. brucei [30, 31], t. cruzi, t. rangeli [13, 33], some leishmania species [11, 34], herptomonas muscarum muscarum, phytomonas spp . [36, 37], entamoeba histolytica, giardia lamblia and trichomonas vaginalis . In general, these ectoenzymes are usually reported to have optimum activities in the acidic ph range, and they are therefore also known as membrane - bound acid phosphatases [28, 29]. In trypanosomatids, the low optimum ph and the surface location of these enzymes suggest its role in an acidic microenvironment and/or a close relationship with lysosomal digestion, possibly reflecting an adaptation of the parasite to the intracellular or phagosomal environment [41, 42]. Cloning and purification of an acidic phosphatase in t. brucei suggest that these enzymes may represent a new ectophosphatase class lacking homology to other known phosphatases . It seems that these proteins are related to the regulation of t. brucei development, since these acidic phosphatases are expressed in bloodstream forms, but not in the insect procyclic form . Likewise, an ectophosphatase activity on the surface of intact procyclic and bloodstream forms of t. brucei was demonstrated by fernandes et al . [43, 44]. Similarly, an ectophosphatase was also cloned and purified in l. mexicana, where it was located in the endosomal / lysosomal compartment between the flagellar pocket and the nucleus in wild - type promastigotes, and the overexpression of this protein leads to its abundant exposure on the cell surface [45, 46]. The same was seen with membrane - bound acid phosphatase from the bloodstream form of t. brucei, where the enzyme is supposed to participate in the maintenance of endocytosis / exocytosis and in differentiation to the insect stage . The wide distribution of acid phosphatases on the cell may reflect some physiological adaptation for parasite survival within the host . In this scenario, ectophosphatase activities were identified at the cell surface of all t. cruzi development stages: epimastigote, trypomastigote and amastigote forms [18, 32]. It seems that in amastigote forms these enzymes are magnesium - dependent and can hydrolyse phosphoaminoacids and phosphoproteins under physiological conditions [18, 32]. This behavior could facilitate the interaction between parasite and host cells, once t. cruzi phosphatases leads to dephosphorylation of proteins important in the signal transduction pathway or cycle regulation of this protozoan parasite . Supporting this idea, y strain presents mg - dependent ectophosphatase activity, while colombiana strain expresses mg - independent activity . Among other characteristics, members of these two groups parasites from the colombiana strain appeared to be more infective to myoblasts than those from the y strain, while the latter is more infective towards macrophages than the parasites of the colombiana strain . Intriguingly, platelet - activating factor (paf), a phospholipid mediator involved in differentiation cellular in t. cruzi, induces the secretion of an ectophosphatase in these parasites, associating this event with the infectivity of the parasite . Addition of sodium orthovanadate (a protein tyrosine phosphatase inhibitor) in the interaction medium from l. amazonensis and macrophages significantly increased parasite binding and internalization, suggesting that leishmania induces tyrosine phosphorylation [24, 51]. Under these conditions, protein tyrosine kinase - linked pathways regulate the leishmania promastigote invasion, which ectophosphatase activity upregulate l. amazonensis binding ligands for macrophage receptors and intracellular survival within these cells [24, 51, 52]. It seems that during macrophage infection by leishmania the parasite attenuates map kinase signaling, as well as c - fos and inos expression in macrophages, stimulating the phosphotyrosine phosphatase activity in these cells [5355]. Possibly by other intracellular pathogens as a strategy of the parasites to interact and survive within their hosts . In l. donovani tyrosine phosphatase activity was also detected, suggesting that tyrosine phosphorylation occurs, though not via receptor tyrosine kinase or tyrosine kinase - like activities but very likely due to the activity of atypical and/or dual specific kinases . Futhermore, a membrane - bound ptp has been describe in l. major metacyclic promastigote forms, which is translocated to the cytoplasm in promastigotes . In spite of the increased level of the molecule in metacyclic promastigotes compared to the procyclic forms, the specific activity of the enzyme was lower in metacyclic than in procyclic promastigotes . Interestingly, a protein tyrosine phosphatase, has been identified in l. major (lmptp1) that allows amastigotes forms to survive in mice . Although its biological function is unclear, this may be an important factor in virulence, enabling the invading pathogen to survive in a host . Ecto - phosphatase isolated from l. donovani promastigotes inhibits the production of superoxide anions in intact human neutrophils . This activity could contribute to the survival of the parasite within the host, we can hypothesize that parasites with greater ectophosphatase activity would be more resistant to oxidative bursts from the host's immune system . The role of ectophosphatases in invasive amoebiasis is still unknown, even though two acid phosphatases have been characterized in these parasites: a membrane - bound acid phosphatase (map) [58, 59] and a phosphatase that is secreted to the culture medium (sap), as well as to the cell interface in amoebic liver abscess [60, 61]. These enzymes may be associated with cellular adhesion processes, since the invasive e. histolytica showed much higher ectophosphatase activity when compared to the noninvasive counterpart and the free - living e. moshkovskii . The fungal cell wall is a compact albeit dynamic structure that plays important roles in several biological processes determining cell shape, morphogenesis, reproduction, cell - cell and cell - matrix interactions, osmotic and physical protection . Several different cell wall components have been characterized such as specific enzymatic activities, heat - shock proteins, glycosphingolipids (gsl), melanin, histone and integrin - like proteins . Even though the roles of ectophosphatases in fungi are still largely unknown, the cellular distribution of ectophosphatases, together with their ability to interfere with physiologic processes through the removal of phosphate groups of regulatory proteins, suggest a task for these molecules during the infection of host cells . The presence of surface - located acid phosphatases, called ecto or extracytoplasmic phosphatases has been demonstrated in nonpathogenic yeast saccharomyces cerevisiae and in pathogenic species such as candida albicans, candida parapsilosis [19, 65], sporothrix schenckii, aspergillus fumigatus, fonsecaea pedrosoi [22, 68], cryptococcus neoformans and pseudallescheria boydii . Futhermore, most of the phosphatases synthesized under pi - limiting conditions are either located on the extracellular medium or are associated with the plasma membrane or cell wall [15, 22]. Corroborating with this hypothesis, kneipp et al . Demonstrated that conidial forms of f. pedrosoi has an ectophosphatase activity modulated by exogenous phosphate . It seems that in f. pedrosoi, conidial cells that were cultivated in a pi - depleted medium had an ectophosphatase activity significantly higher than that of fungal cells grown in the complete medium . These cells expressing high phosphatase activity were significantly more capable of adhering to epithelial cells and fibroblasts than fungi expressing basal levels of enzyme activity . It was then proposed that the removal of phosphate groups from surface proteins in host cells could result in conformational transitions and in an attenuated electrostatic repulsion between fungal and epithelial cells . Probably, the removal of inorganic phosphate could therefore expose at the host surface additional sites for interaction with infectious agents . It seems that ectophosphatases may contain adhesive domains that could directly promote the attachment of fungal cells to their hosts, therefore functioning similarly to the well - characterized microbial adhesins . Probably, they could regulate the functional activation of surface adhesins, which would be the key structures mediating fungal attachment . Intriguingly, known activators of signaling pathways and cell differentiation, paf and propanolol, promoted an enhancement of f. pedrosoi ectophosphatase activity suggesting that f. pedrosoi ectophosphatase may be considered a surface marker for morphological transition and infection . In the fungus c. neoformans a thick capsule composed of neutral and charged polysaccharides, can be modulated by different environmental conditions, including the sites of fungal infection inside the host . It seems that the molecules coating the outer layer of the cell wall could be relevant during the interaction of poorly encapsulated cells with host tissues . Ectoenzymes possibly have their accessibility to external receptors masked by the capsule polysaccharides of c. neoformans, diminishing the potential of these structures to be surface molecules influencing the interaction between fungal and host cells . . However, the levels of enzyme activity, varied considerably among the isolates and no correlation between enzyme activity and capsular size or serotype was observed . Evidences show that isolates with capsular polysaccharides of the same serotype varied greatly in ectophosphatase activity . In addition, the strain, which is poorly encapsulated, removed phosphate groups much more efficiently than strain, which expresses a large capsule, indicating that the presence of the capsule impairs enzyme activity in this process . On the other hand, some encapsulated strains presented levels of ectophosphatase activity higher than that observed in the acapsular mutant . Moreover, some strains that had very similar levels of enzyme activity, but differ greatly in capsule size were also found . Taken together, these data indicate that differences observed in enzyme activity should be derived from natural variation of ectophosphatase expression in different c. noeformans strains . Corroborating with the previous findings, kiffer - moreira et al . Investigated three different isolates of c. parapsilosis, including a laboratory - adapted strain (cct 3834) and two recently isolated strains (rfo and h297). They observed that the rfo strain exhibits the highest levels of enzyme activity and adhesion to cho cells, followed by the h297 and the cct 3834 isolates . Pretreatment of yeasts with the irreversible inhibitor sodium orthovanadate caused a significant reduction in the ability of these fungi to attach to epithelial cells . Although sodium orthovanadate can affect different biological processes and inhibit atpases involved in cation transport [72, 73], its major biological activity in living cells occur on the cell surface, as the oxidation reduction reactions that take place in the cytoplasm diminish its inhibitory effect . Similarly, c. albicans isolate from oral cavities of hiv - infected children (hiv) present an ectophosphatase activity significantly higher than the hiv - negative children (hiv). The c. albicans yeasts from hiv patients showed higher indices of adhesion to epithelial cells, which suggests that the activity of fungal acidic surface phosphatases may contribute to the early mechanisms required for disease establishment . It is reasonable the hypothesis that ectophosphatases represent a virulence marker, since these enzymes represent part of the outer layer and are linked to cell differentiation and host cell - pathogen interactions . The balance of phosphorylation - dephosphorylation of serine, threonine and tyrosine residues modulates signaling pathways critical for determining the outcome of multiple cellular functions . Further studies are warranted to resolve the roles of ectophosphatases in host - pathogen interactions, as well as the possible correlations between the expression of these enzymes and the clinical manifestation of the diseases.
During the recent decade, a number of researchers have described the growing psychosocial risk factors for coronary heart disease (chd), which include depression, anxiety, stress, and alexithymia that decrease the health - related quality of life and protective factors such as social support . Studies carried out internationally highlight that besides negative psychological factors like depression, anxiety, and stress, positive factors such as social support play a protective role in the onset and progress of chd . In 1973, sifnoes proposed the term alexithymia to imply a cognitive affective disturbance, characteristic of persons who cannot describe their feelings or elaborate their fantasies . It has been characterized more generally as an emptiness of feeling, a poverty of imagination or a life of fantasy, difficulty in communicating with others, and a lack of positive emotions with a high prevalence of negative emotions . Alexithymia is associated with states of negative affect . Which increase the possibility of somatic complaints . The assumption is that these characteristics would lead to a deficiency in cognitive processing and regulation and adjustment of emotions, and are associated with starting and/or continuing some of the psychiatric and medical disorders . Some researchers believe that dysfunction in the limbic system, brain abnormal lateralization, and/or difficulty in the efficiency of hemispheric communication are the causes of its emergence . The prevalence of alexithymia in working age populations has been shown to be about 9 - 17% for men and 5 - 10% for women . At the population level, alexithymia is associated with older age, lower socioeconomic status, fewer years of education, single marital status, and poorer perceived health . Alexithymia has been shown to be associated with several medical conditions and mental health problems . An association between alexithymia and dissatisfaction with life has been found in some studies of coronary heart disease patients . One study investigated factors associated with alexithymia in patients (n = 153) with coronary heart disease verified by coronary angiography . In chd patients, alexithymia was unrelated to cardiovascular diseases, but was related to depression and decreased life satisfaction . In the kuopio ischemic heart disease risk - factor study, 2682 middle - aged men were studied using the 26-item toronto alexithymia scale (tas). Alexithymia was associated with greater self - reporting of symptoms during exercise - tolerance testing and it was found that the extent of carotid atherosclerosis was inversely related to the degree of alexithymia, suggesting a biological connection to atherosclerosis . In this study, men in the highest alexithymia quintile were at a two - fold greater risk of death from any cause, while no association was found between behavioral or physiological risk factors and mortality . Lumley et al ., observed that the degree of alexithymia (tas-20) was associated with the presence or absence of chest pain during exercise testing, but not with chd (n = 180). They concluded that alexithymia was associated with an increased risk of illness - related behavior, but not necessarily with the presence or severity of chd . One study to testing the validity of existing conceptualizations of the alexithymia concept in the general adult population described that older adults engaged in less introspective thoughts, traditionally thought to denote increased alexithymia . Difficulty in identifying and describing emotions did not differentiate older and younger adults, but they were both associated with heightened depression, anxiety, and poor perceived quality of life . Alexithymia in somatoform and depressive disorders (2003) was investigated in one study (two groups of 30 subjects each, bearing diagnoses of somatoform disorder and depressive disorder, respectively, and one group of 30 normal controls) and the results showed that the mean of alexithymia scores in the somatoform (60.4) and depressive disorder groups (62.5) were higher than in normal subjects (54.2). Even as the total alexithymia scores did not differentiate somatoform from depressive disorders, the depressed subjects had greater difficulty in expressing their feelings . Kauhanen et al ., noted more physical symptom - reporting with high alexithymia, and myers observed alexithymia - elevated anxiety scores . Linden et al ., noted that high alexithymia showed smaller heart rate responses to the stress task and more anger in behavior . 2009) investigated the relationship between alexithymia and the health - related quality of life (hrqol) in a nationally representative population sample of 5,418 subjects, of age 30 to 97 years, and concluded that alexithymia could be a predisposing factor to a poorer hrqol . A study investigated the relationship between alexithymia and the health - related quality of life (hrqol) and concluded that the alexithymic group had significantly (p <0.001) lower mean scores on every dimension of hrqol, even after controlling for confounding demographic variables, somatic diagnoses, and depressive and anxiety disorders . This research was a cross - sectional study that examined the causal relationship between alexithymia and dass (depression, anxiety, stress), quality of life, and social support . The sample consisted of 398 chd patients (166 females and 232 males and age: 40 - 70 years), who were randomly selected from the isfahan chamran heart center and isfahan cardiovascular research center (icrc) (2013) and the other heart clinical center . The patients filled in five questionnaires assessing depression, anxiety, stress (dass-21), alexithymia (tas-20), health - related to quality of life (whoqol-26), multiple scale of perceived social support (mspss-12), and another questionnaire, which obtained demographic information . Before attending the study, each patient had been examined by a cardiologist, who had confirmed the diagnosis as coronary heart disease . Alexithymia - tas-20 scale: the tas is a 20-item instrument that is one of the most commonly used measures for alexithymia . The tas-20 has three subscales: 1.1 . - difficulty describing feelings subscale (ddf) is used to measure the difficulty in describing emotions ., which has five items: 2, 4, 7, 12, 17 . 1.2 . - difficulty identifying feelings subscale (dif) is used to measure the difficulty in identifying emotions ., which has seven items: 1, 3, 6, 11, 9, 13, 14 - externally - oriented thinking subscale (eot) is used to measure the tendency of individuals to focus their attention externally, which has eight items: 5, 8, 10, 15, 16, 18, 19, 20 . The items are rated using a five - point likert scale whereby 1 = strongly disagree and 5 = strongly agree . The total alexithymia score is the sum of responses to all the 20 items, while the score for each subscale factor is the sum of the responses to that subscale . The tas-20 uses cutoff scoring: equal to or less than 51 = non - alexithymia, equal to or greater than 61 = alexithymia . It demonstrates good internal consistency in the normal iranian sample (cronbach's alpha = 0.81) and test retest reliability (0.77, p <0.01). The internal consistency reported in a clinical sample from iran, using cronbach's alpha for the total tas-20 scale and subscales of dif, ddf, and eot, were . 66, respectively, and the total validity using test retesting for tas-20 and its subscales dif, ddf, and eot were . 2-dass scale-21: the dass scale is a set of three self - report scales designed to measure the negative emotional states of depression, anxiety, and stress . Each of the three dass scales contains seven items, divided into subscales of two to five items with a similar content . The depression scale assesses dysphoria, hopelessness, devaluation of life, self - deprecation, lack of interest / involvement, anhedonia, and inertia . The anxiety scale assesses autonomic arousal, skeletal muscle effects, situational anxiety, and subjective experience of anxious affect . 2.3 . It assesses difficulty in relaxing, nervous arousal, and being easily upset / agitated, irritable / over - reactive and impatient . Subjects are asked to use the four - point severity / frequency scales to rate the extent to which they have experienced each state over the past week . Scores for depression, anxiety, and stress are calculated by summing the scores for the relevant items . The assumption on which the dass development has been based (and which has been confirmed by the research data) is that the differences between depression, anxiety, and stress experienced by normal subjects and the clinically disturbed, are essentially differences of degrees . The validity and reliability of this scale was reviewed by samani and joker (2007), who reported that reliabilities for depression, anxiety, and stress scale were . 3 . Multiple scale perceived social support: the 12-question multidimensional scale of perceived social support (mspss) was used to measure the degree of social support each participant felt he or she had . High levels of perceived social support are generally associated with low levels of depression and anxiety . Family; 2 . Friends; 3 . Significant others) based on the seven - point likert measures, which measures the respondents perceptions of the availability of various types of social support such as family, friends, and significant others the remaining 24 items comprise four dimensions of health including, physical, psychological, social, and environmental . All scores are transformed to reflect 4 - 20 for each domain, with the higher scores corresponding to a better qol . The individual's perception of quality of life is measured by summing the total scores for each particular domain . All domain scores are scaled in a positive direction (higher score indicates a higher qol). This questionnaire was standardized on a sample of 2,956 healthy and 2,936 unhealthy rural and urban inhabitants (age 30 years and above) from isfahan, najaf - abad, and arak, who participated in the isfahan healthy heart program (ihhp) from two dissimilar iranian provinces, during 2006 . Demographic questionnaire: this questionnaire consists of questions about age, sex, marital status, education, and so on . Table 1 shows the patients sociodemographic characteristics based on age, sex, marital status, and education . Patients sociodemographic characteristics (n = 398) table 2 shows patients descriptive characteristics such as mean and standard deviation . Patients descriptive characteristics cronbach's alpha was calculated as 0.74 for the toronto alexithymia scale (tas), 0.89 for the depression, anxiety, and stress scale (dass), 0.87 for the multidimensional scale of perceived social support (mspss), and 0.90 for the world health organization quality of life questionnaire (whoqol). Based on the conceptual model [figure 1] of the study, the structural model of variables (i.e., tas, dass, mspss, and whoqol scores) was presented using the outputs of amos . Although in traditional statistical methods, the researcher uses a single measure to verify the null hypothesis, the complexity of the structural equation modeling eliminates this single measure . Therefore, three main groups of indices (absolute fit, comparative fit, and parsimonious fit indices) are used to fit a model . The conceptual model of alexithymia according to table 3 [relative chi - square (cmin / df) = 2.580 and df = 58 for default model], the model fitted well . Even as most experts consider relative chi - square values of 2 - 3 to show a good fitting model, schumacher and lomax expanded the range to 1 - 5 . Chi - square (cmin) values and number of parameters table 4 shows the absolute fit indices . Goodness of fit index (gfi = 0.94) and adjusted goodness of fit index (agfi = 0.91) suggested good fitting of the default model (values between 0 and 1 for these two indices indicate good fitting). Table 5 shows the comparative fit index (cfi), parsimony comparative fit index (pcfi), and root mean square error of approximation (rmsea). As is seen, the calculated values (cfi = 0.96 and pcfi = 0.71) suggest a good fit for the default model (cfi> 0.90 and pcfi> 0.50 are favorable). Rmsea can be calculated for confidence intervals, to determine the significance of the difference between the obtained value for the model and zero . As rmsea <0.08 is acceptable, our default model fitted well (rmsea = 0.06). In addition, the holter index of 204 was again indicative of a well - fitting model . Comparative and parsimonious fit indices according to all the mentioned indicators, the default model presented a good fit for the relationship between alexithymia and depression, anxiety, stress, quality of life, and social support . Based on the conceptual model [figure 1] of the study, the structural model of variables (i.e., tas, dass, mspss, and whoqol scores) was presented using the outputs of amos . Although in traditional statistical methods, the researcher uses a single measure to verify the null hypothesis, the complexity of the structural equation modeling eliminates this single measure . Therefore, three main groups of indices (absolute fit, comparative fit, and parsimonious fit indices) are used to fit a model . The conceptual model of alexithymia according to table 3 [relative chi - square (cmin / df) = 2.580 and df = 58 for default model], the model fitted well . Even as most experts consider relative chi - square values of 2 - 3 to show a good fitting model, schumacher and lomax expanded the range to 1 - 5 . Chi - square (cmin) values and number of parameters table 4 shows the absolute fit indices . Goodness of fit index (gfi = 0.94) and adjusted goodness of fit index (agfi = 0.91) suggested good fitting of the default model (values between 0 and 1 for these two indices indicate good fitting). Table 5 shows the comparative fit index (cfi), parsimony comparative fit index (pcfi), and root mean square error of approximation (rmsea). As is seen, the calculated values (cfi = 0.96 and pcfi = 0.71) suggest a good fit for the default model (cfi> 0.90 and pcfi> 0.50 are favorable). Rmsea can be calculated for confidence intervals, to determine the significance of the difference between the obtained value for the model and zero . As rmsea <0.08 is acceptable, our default model fitted well (rmsea = 0.06). In addition, the holter index of 204 was again indicative of a well - fitting model . Comparative and parsimonious fit indices according to all the mentioned indicators, the default model presented a good fit for the relationship between alexithymia and depression, anxiety, stress, quality of life, and social support . Alexithymia, a condition of inability to use words to understand the emotional states of oneself or others, prevents individuals from adjusting to their emotions . The present study has examined the predictive value of alexithymia in changes in depression, anxiety, stress, social support, and quality of life among patients with coronary heart disease . After fitting all the data, alexithymia shows significant positive relationships with depression, anxiety, and stress, as previous research has described . In other words, increasing levels of alexithymia and its two dimensions would worsen depression and anxiety in patients . Sarrijarri has shown that exacerbation of depressive symptoms will cause difficulty in identifying, describing, and sharing their feelings with others . Similarly, research has shown that alexithymia is more common in people with anxiety disorders and that anxiety is related to the two mentioned aspects of alexithymia . Alexithymia is a cognitive - emotional disorder that impairs the adjustment of emotions, concentration, processing, and evaluation of cognitive - emotional information . Therefore, individuals with alexithymia fail to manage their cognitive - emotional system and properly deal with stressful situations, particularly diseases . This strengthens the feelings of inadequacy and insufficiency in this group and intensifies negative emotions such as anxiety and depression . Moreover, since emotional arousal is associated with physiological arousal, negative emotions (e.g. Anxiety and depression) will bring about physical symptoms including pain, fatigue, and dizziness . Finally, disruption of psychological defenses in subjects with alexithymia leads them to deny their emotions and makes them incapable of describing them . We also found difficulty in identifying feelings and difficulty in describing feelings to be related to depression and anxiety . Matn and jenakar suggested difficulty in identifying feelings as the only aspect of alexithymia that predicts depressive symptoms . Furthermore, they reported difficulty in both identifying and describing feelings as predictors of anxiety symptoms . Previous studies have indicated consistent results in terms of the relationship between alexithymia and stress and inadequacy of coping styles with stress . This finding can be justified by considering the poor emotional intelligence of people with alexithymia, which combines with difficulties in identifying and describing feelings and reduces the ability of dealing with stressful situations . As the inability to cope with difficult situations imposes a lot of stress on the individual, a positive relationship exists between alexithymia and stress . Another finding of this study was the negative relationship between alexithymia and social support and its aspects, that is, increasing alexithymia decreases social support . In fact, patients with alexithymia cannot receive adequate social support from their family, friends, and/or significant others . Seemingly, impaired emotional intelligence in these individuals results in their poor interpersonal skills and inability to receive social support . The results indicated that there is a negative relationship between alexithymia and quality of life . The strongest relationships were identified between difficulty in identifying feelings and psychological aspect of the quality of life (r = - 0.48; p <0.01), physical aspect of quality of life (r = - 0.43; p <0.01), social aspect of quality of life (r = - 0.34; p <0.01), and environmental aspect of quality of life (r = -0.24; p <0.01). Available evidence about the association between the difficulty in identifying feelings and somatoform disorder can explain the negative effects of alexithymia on the quality of life . It also seems that difficulty in identifying feelings results in a false interpretation of physical feelings and decreases the health - related quality of life . In other words, alexithymia is not only an inner state, but is also associated with health outcomes . Research has shown that people with alexithymia do not use normal coping styles, and may hence, have a higher tendency toward unhealthy behaviors such as suppression of emotions and drinking . Patients might experience problems in different aspects of life, that is, negative emotions may cause psychological problems, wrong interpretation of physical health may lead to physical problems, social relations may be affected by difficulty in establishing good social relations, and environmental quality of life can be decreased due to inappropriate interactions with the environment . According to the results of the present study it is important to pay attention to alexithymia as an important variable associated with (depression, anxiety, stress), social support, and quality of life of patients with coronary heart disease . It can be concluded that alexithymia is connected with the pathological indicators of cardiac disease (depression, anxiety, and stress) and its protective parameters (social support and quality of life). Most patients with heart disease are elderly and alexithymia is also more common among older people . Therefore, conducting a longitudinal study to determine the precise role of alexithymia in the psychiatric pathology of cardiac diseases is essential . This research showed the relation between alexithymia and (depression, anxiety, stress), social support, and quality of life, as a psychological structural model in chd . Most of the relationships were associated with alexithymia, depression, anxiety, and stress (dass: 49%) and the lowest relationship was between alexithymia and social support (mspss: 09%). Of particular interest in the findings if alexithymia is considered to be a personality trait or state - dependent trait, alexithymic individuals are at risk for medical or psychiatric disorders and negative emotions . Increasing our knowledge regarding the role of a negative affect may have a major implication on how future interventions are targeted, for example, if evidence supports the lack of emotional awareness (e.g. Depression, anxiety, and stress) as an underlying factor for a subset of patients seeking interdisciplinary treatment, then intervention can be modified to focus on processes related to increasing emotional awareness and identification, with a hope to improve the quality of life in patients with coronary heart diseases . The limitations of the study were: inability to control the behaviors that were associated with alexithymia and could thus affect the relationship between alexithymia and depression, anxiety, stress, social support, and quality of life . As the study population was limited, generalization of the results to other patients had to be made with caution . The present study showed the structural relations between the psychological factors associated with alexithymia in coronary heart disease . The results highlight the importance of alexithymia as a risk factor associated with neuroticism (anxiety, depression, and stress) and social support which reduces the quality of life among coronary heart disease patients . In this regard, psychological interventions are recommended to reduce the consequences of this disorder, especially among coronary heart disease patients . Conducting longitudinal studies in this area may lead to more interesting and favorable results, which is recommended to the researchers interested in this field.
Tumor - induced osteomalacia (tio) is a rare paraneoplastic disease in which causative tumor (generally of mesenchymal origin) gives rise to an oncogenic osteomalacial syndrome, whose underlying pathogenetic mechanism is related to the tumor s production of a phosphaturic factor that reduces the renal reabsorption of phosphates and causes renal phosphate leak . The tumors are generally small, slowly growing and non - invasive . Most are localized at long bone level, but they are frequently impossible to locate and the correct diagnosis may take 5 - 7 years . The clinical picture is characterized by non - specific symptoms, such as hyposthenia, worsening myalgia and bone pain (mainly affecting load - bearing areas), and gait alterations (anserine gait). Until a correct diagnosis has been made patients usually undergo symptomatic treatments without success . Those patients history is characterized by numerous pathological fractures, periprothesical fractures and prothesis mobilizations . The elective surgical treatment of tio is tumor resection, which normally leads to a complete regression of the osteomalacial syndrome . The consequent rehabilitation treatment must consider the complexity of the clinical - functional picture of the patient, especially if a substitution megaprothesis surgery is needed . The clinical picture is characterized by non - specific symptoms, such as hyposthenia, worsening myalgia and bone pain (mainly affecting load - bearing areas), and gait alterations (anserine gait). Until a correct diagnosis has been made patients usually undergo symptomatic treatments without success . Those patients history is characterized by numerous pathological fractures, periprothesical fractures and prothesis mobilizations . The elective surgical treatment of tio is tumor resection, which normally leads to a complete regression of the osteomalacial syndrome . The consequent rehabilitation treatment must consider the complexity of the clinical - functional picture of the patient, especially if a substitution megaprothesis surgery is needed . The clinical history of pm (born in 1945) began in 2000, when she consulted her general practitioner because of widespread costal and vertebral pain of no known traumatic cause, mainly localized in the dorso - lumbar region . The x - ray findings were negative, and she was prescribed non - steroidal anti - inflammatory drugs treatment without receiving any benefit . The picture remained unchanged until 2003, when bone scintigraphy revealed areas of the pathological accumulation of the radiolabeled tracer in the proximal third of the right femoral diaphysis, some costal arches and an area of bone rarefaction in the right humeral diaphysis . Further laboratory tests revealed hypocalcemia, and so the patient started pharmacological treatment with bisphosphonates, calcium and vitamin d3 for suspected advanced osteoporosis . Because of the persistence of the painful symptoms, a bone biopsy of the humeral lesion was performed; that led to the diagnosis of a spindle cell mesenchymal tumor, morphologically arising from a glomus tumor . Given the suspicion of tio, the patient underwent octreotide scintigraphy, which revealed increased tracer uptake at the level of the medio - proximal third of the right humerus . In january 2005, she underwent a first surgical intervention that involved curettage of the lesion, an autograft from the ipsilateral tibia, and a bone biopsy, thus allowing the final diagnosis of a phosphaturic mesenchymal tumor . As a result of an accidental fall in july 2005, the patient suffered a subcapital fracture of the right femur and a bifocal fracture of the previously treated right humeral diaphysis . Laboratory tests once again revealed hyperphosphaturic hypophosphatemia, and so she therefore underwent dual surgery involving the implantation of a bi - articular endoprosthesis cemented to the femur, an osteosynthesis of the humerus with a screwed plate and then sent to the rehabilitation department . In 2007, worsening humeral pain associated with disease recurrence (confirmed by means of instrumental investigations) led to the patient being readmitted to hospital to undergo to a segmental resection of the diseased humerus and its reconstruction by means of the insertion of a cemented 8-holed plate which, soon afterwards, were replaced with a new ipsilateral tibial graft in order to provide medial reinforcement after a distal screws displacement (figure 1a). The patient s clinical condition remained stable until april 2009 when, during an outpatient follow - up examination, she complained pain in her right hip radiating to the knee and accentuated by weight bearing . X - rays revealed loosening of the femoral stem with bone reabsorption around the cemented area . The prosthetic stem was revised and a new femoral stem was implanted (figure 1b). In march 2010 the pain in the right hip and femur reappeared and subsequent x - rays showed partial progressive subsidence of the prosthetic stem to a depth of about 2.5 cm (figure 1c). Furthermore, examinations of the humerus revealed the partial mobilization of the proximal screws (figure 1d). In september 2010 the patient underwent to a new revision surgery, and due to a poor cortical bone resistance a 22 cm resection from the apex of the great trochanter were executed and a large - resection modular prosthesis was implanted . The stem was cemented and trevira mesh was used proximally to attach the extra - rotary thigh musculature in order to reconstruct the articular capsule (figure 1e - f). The postoperative course was normal and the patient started rehabilitation training as per ad hoc protocol (table 1). The functional outcomes and efficacy of the rehabilitation program were evaluated using the following scores (table 2): range of motion, visual analogue scale, barthel s index, toronto extremity salvage score, and musculoskeletal tumor society scale . Since the 1 week of permanence in the g. pini oncological orthopedic surgery the patient started distal mobilization exercises of the treated limb to prevent venous and lymphatic pooling, selective isometric exercises for maintaining the tone and trophism of the main anti - gravitational muscles (gluteal muscles from day 3 - 4), and self - mobilization and strengthening exercises of the body districts not involved in the surgery . From the 2 week, the patient has substituted a lower limb splint with a newport hip orthosis (figure 2) locked in position 0. the patient and her relatives were trained in correct wearing of the support by technical staff and nurses . From day 12, the by now clinically stable patient started the training for the recovery of orthostasis and short - distance transfers (from bed to wheelchair) with no weight bearing . The back of the wheelchair used was fully reclined in order to avoid the patient to flex the hip . It was necessary to make these movements for keeping the hip in position 0, the on - bed transfer has been made on the operated limb side, with maximum assistance of the therapist in order to avoid any rotation of the lower limb . Together with the re - education of postural transfers, the patient was trained in how to reach and maintain a standing position with the support of a 4-legged walking frame (to guarantee the greatest stability) with the treated limb just touching the floor . From the 3 week were added exercises using a 3-stage technique . The forward movement of the limb was compensated by a sagittal oscillation of the trunk to avoid hip flexion - extension . In order to allow the patient to adequately oscillate the operated limb without flexing the hip, she was initially asked to provide contralateral support in an equine manner (to avoid a swinging forward movement) balanced by the counter - oscillation of the trunk on the coronal plane . The therapeutic exercises proposed to the patient in a prone lying position had the aim of consolidating the objectives set from the 1 week and mobilizing the knee while keeping the hip extended . In addition to guaranteeing the early recovery of walking abilities, the knee mobilization exercises had the aim of improving the tone and trophism of the femoral quadriceps and the stiffness of the ilotibial band, which, as this was the site of surgical access, was at risk of adherences that could compromise the knee movement . On the 21 day after surgery, the patient was transferred to gaetano pini rehabilitation centre where the orthosis was gradually released (starting on post - surgery day 30) until it reached 90 of flexion by means of passive and assisted active sagittal mobilization under the pain threshold . She also performed progressive exercises of assisted active mobilization with the hip in abduction, and strengthening exercises for her right arm overloaded by the heavy use of walking aids . The walking re - education exercises continued with the gradual replacement of the walking frame by elbow crutches: her gait showed slight abduction of the hip (which gradually regressed) during both the advancement and fleetingly light weight - bearing phases . Once capable of walking autonomously for albeit short distances, the patient was given functional exercises in preparation for her return home, including stairs training, re - education in the activities of daily living, and the postural movements involved in entering and leaving a car . A few days before being discharged, she began the weaning from the orthosis and started step training exercises in water (with the water up to her chest - 25% of her weight borne by the limb). This hydrokinetic therapy gave the treated limb an important proprioceptive stimulus in terms of weight bearing without aids, but in absolute safety conditions . Throughout her stay in the rehabilitation center, when she was discharged 60 days after surgery, she was able of walking autonomously for long distances with the aid of two elbow crutches . She was instructed to continue the exercises at home in order to maintain and consolidate her acquired abilities . At the orthopedic follow - up examination 2 weeks after discharge the 6-months physiatric follow - up examination showed a clear improvement in the functional outcome measures (table 2). The tumor - induced osteomalacia is a paraneoplastic syndrome, which causes osteomalacy, causing small dimension lesions, characterized by a slow growth without any specific anatomic localization . Those lesions can lead to pathologic fractures, which, if treated without removing the neoformation or with a partial excision, may recur subsequently, along with loosening or displacement of prothesis components . The lesions can be studied with x - rays, computed tomography, magnetic resonance imaging, concreotide tri - phasic whole - body scintigraphy and f-18 fluorodeoxyglucose positron - emission tomography . Having confirmed the nature of the lesion, treatment of a phosphaturic - secreting mesenchymal tumor is its surgical removal with broad margins by means of unconventional techniques such as a large resection and prosthetic implantation . In absence of a correct diagnosis, the rehabilitation treatment of patients with tio was initially based on reducing / containing the painful symptoms and asthenia, and is naturally not beneficial . For this reason, the worsening pain and progressive loss of functional capacities can reduce the compliance with treatment and affect patient mood . The loss of bone mass due to this type of tumor is not associated with the muscle tissue loss, but the overall balance is usually characterized by asthenia of the main anti - gravitational, stabilizing and mobilizing muscles as a result of long pre- and post - surgery periods of immobility . The adherence of scars due to repeated revision surgeries can also give rise to articular and functional limitations (figure 3) unless they are appropriately treated . The time necessary to resume everyday living activities is longer than after conventional surgery, and requires specific techniques strictly related to the highly invasive surgery . The requirements are: i) special care during the first four weeks of rehabilitation and, in any case, a longer period of hospitalization that that required after a normal hip replacement procedure (the protocol lasts of 60 days); ii) the use of specific aids (hip orthosis and reclinable wheelchair) and training the patient and caregivers by nurses and technical staff in how to use them; iii) learning specific movement techniques to get an early recovery of a standing position and making positional changes aimed at protecting the implant stability while developing the patients residual functional capacities also by means of compensatory strategies; iv) particular care in treating the surgical wounds, which are more extensive than those associated with traditional prosthetic implants . The functional pictures which result are often complex for the long diagnostic - therapeutic pathway and for the highly invasive surgery needed . The scientific research has not validated any standard rehabilitation protocol or any specific guidelines yet . The protocol developed at the gaetano pini orthopedic institute with the collaboration of the rehabilitation and orthopedic team has demonstrated to be effective in the case above described.
Human neurocysticercosis (ncc) is caused by larval stage of zoonotic tapeworm taenia solium (pork tapeworm) which remains a major public health problem in developing and some developed countries . Porcine cysticercosis is the cause of human taeniasis and neurocysticercosis is a consequence of taeniasis . Based on the available information, a very conservative and rough economic estimate indicates that the annual losses due to porcine cysticercosis in 10 west and central african countries amount to about 25 million euros . . Also stated that, in china, the amount of pork discarded in the whole country due to cysticercosis annually has been estimated as 200,000,000 kg with a value of more than us $120,000,000 . Stated that notably data on myanmar are lacking, although there are several reports of porcine cysticercosis based on meat inspection in the abattoirs in neighboring countries, 9.3% in india, 32.5% in nepal, 5.4% in china, 0.022.63% in indonesia, and 0.04 to 0.9% in vietnam . Although most of myanmar culinary habits are based on thorough cooking, new food style such as barbecue and dishes based on raw or undercooked pork or pork product becomes popular among customers . Moreover, small - scale pig husbandry has become one of the major sources of income in myanmar farmers . So it may be high risk of getting food - borne zoonotic diseases according to the new food style and traditional husbandry method . Due to lacking of information on porcine cysticercosis in myanmar up to now, it is important to investigate the prevalence and associated risk factors . Nay pyi taw area, the capital of myanmar, has big population of pigs (about 200,000 pigs) to support the demand of pork consumption in this area . Most of the pig farmers are smallholders and most of pig husbandry systems are free ranging or semi - intensive with lack of proper sanitation . One of the main obstacles to control the t. solium infections is the lack of adequate epidemiological data on cysticercosis / taeniasis . Therefore, the objectives of this community - based study were to investigate the prevalence of porcine cysticercosis and associated risk factors in pigs within study area . Moreover, findings of this study will assist to develop the control strategies of porcine cysticercosis for the public health aspect . The cross - sectional studies were conducted from january to march and june to july 2014, to investigate the prevalence of taenia solium cysticercosis in slaughtered and farmed pigs within pyinmana, lewe, and tatkon townships, nay pyi taw area . It is located between latitude 1945n and longitude 966e and with climate data; the altitude is 115 m above sea level, annual rainfall is 115 mm, and annual temperature is 21.232.5c . An expected prevalence of 30% with a confidence level of 95% was used in this unit . In this study, 300 slaughtered pigs and 364 farmed pigs from the study area were examined although calculated samples were 298 and 323, respectively (table 1). Blood collected from the jugular vein of farmed pig was conducted for the seroprevalence and a structured questionnaire with both closed and open - ended questions was administered to owners to obtain management practices in pig husbandry . Piglets younger than two months, pregnant sows, and nursing sows with litters less than two months old were excluded from this study to overcome the stress which causes adverse effect in animals . Meat inspection was carried out as described by boa et al . In the three slaughterhouses of these townships . There were 300 randomly selected pigs recruited and 9 different muscles (tongue, masseter, brain, shoulder, diaphragmatic, heart, skeletal, fore limb, and hind limb muscle) from each pig in meat inspection . Briefly, long and parallel incision into the masseter muscles on both sides of face in an upward direction was made . A deep longitudinal incision covering about 3/4 the thickness of the tongue and covering the whole length of the tongue was made to examine the cysts . After opening the pericardium, the heart was also visually examined for the presence of cysts . The heart was cut open and a deep (3/4 the thickness of septum) incision into the septum was made to expose any metacestodes . All the other muscles were viewed, palpated, incised by surgical blade, and visually examined . The pig was kept under restraint at standing position and blood samples were obtained from the external jugular vein by using sterile disposable syringes and put into vacutainers with clot activators . Those vacutainers were kept in cold boxes with ice and transported to department of pharmacology and parasitology, university of veterinary science, nay pyi taw, and allowed overnight at 4c to clot . To obtain serum, the clear sera were transferred to 1.5 ml microvial tubes and stored in labeled wails and kept at 20c until analysis . Detection of igg antibody of t. solium cysticerci was carried out by using antibody - elisa kit (novatec immundiagnostica gmbh co., belgium) according to manufacturer's instruction . Briefly, all thawed samples were diluted as 1 + 100 with igg sample diluent (phosphate buffer) before assaying . The 100 l controls and diluted samples were dispensed into their respective wells and the foil was covered . After incubation for 1 hour at 37c and the foil being removed, the contents of the wells were aspirated and washed three times with washing solution . And the 100 l protein a conjugate (horseradish peroxidase) was dispensed into all wells except a1 and covered with foil and incubated for 30 min at room temperature . After washing three times, 100 l tmb (3,3,5,5-tetramethylbenzidine) substrate solution was dispensed into all wells and incubated for exactly 15 min at room temperature in the dark . The reaction was stopped by adding 100 l stop solution (0.2 m h2so4). The absorbance was determined at 450/620 nm using an elisa reader (stat fax). In each elisa kit testing, there are two cut - off controls (c1 and d1). The mean absorbance of these cut - off controls was used as cut - off value . Samples are considered positive if the absorbance value is higher than 10% over the cut - off and samples are considered negative if the absorbance value is lower than 10% below the cut - off . The sensitivity and specificity of these kits to diagnose swine cysticercosis are 93.8% and> 95%, respectively . A questionnaire was developed and used to collect information on hypothesized risk factors and other related pieces of information from sampled pig owners . Households in each township were selected by using the snowballing technique from those farmers willing to participate in the study . It is a technique for developing a research sample where existing study subjects recruit future subjects from their acquaintances . The questionnaire interviewed data were analyzed for the relationship between the prevalence of t. solium cysticercosis and hypothesized risk variables such as age, gender of pigs, husbandry system, feed type, environment of pig farm (accessibility of human feces), personal hygiene of owners, pork consumption, cooking and eating habit of pork, use of anthelmintics in pigs and owners, and knowledge on taeniasis . They were examined for testing its significance by pearson chi - square test at = 0.05 . Seroprevalence of porcine cysticercosis in farmed pigs was 15.93% (58/364) in the study area . Prevalence of households with pigs infected with t. solium cysticerci by ab - elisa examination was 23.15% (47/203 households). The households with porcine cysticercosis in pyinmana, lewe, and tatkon were 0/12 (0%), 13/124 (10.48%), and 34/67 (50.75%), respectively . All the infected pigs presented parasites located in the tongue . Only in one pig, the prevalence in slaughterhouses of pyinmana, lewe, and tatkon townships was 22% (44/200), 23.33% (7/30), and 28.57% (20/70), respectively . Univariate analysis of hypothesized risk factors of gender (or = 3.0; 95% ci = 1.75.4), increased age (or = 2.3; 95% ci = 1.24.2), husbandry system (or = 5.1; 95% ci = 2.411.2), feed type (or = 16.9; 95% ci = 2.3124.3), no hand washing habit before feeding (or = 31.5; 95% ci = 4.3230.9), not using anthelmintic in pigs (or = 11.9; 95% ci = 5.028.5) and owner (or = 2.5; 95% ci = 1.44.4), and pork consumption of owner (or = 37.4; 95% ci = 9.0156.1) was significantly associated with cysticercus cellulosae infection (p <0.05). The distribution and odds ratio of significant risk factors concerning porcine cysticercosis are shown in table 2 . In southeast asia, pigs are an important source of food and economic important for smallholder farmers . Older pigs may be penned or tethered although common raising practice of pigs is freely roaming in the village . In myanmar, most of the pig farmers are smallholders and practice as free - range or backyard farming . In myanmar, most of the pig farmers usually keep the weaned pigs until six to eight months of age and then send to slaughterhouse . In the village, every household keeps at least one pig not only for table waste feeding to pigs but also for extra income . Most farms are having the habit of feeding waste materials such as swill and kitchen leftover, broken rice, rice bran, groundnut meal, sesame meal and local forage, and poor sanitation . The present study is the first report of t. solium cysticercosis in pigs in myanmar . Pigs in the study area positive for cysticercosis have been exposed to t. solium eggs . Among the 17 hypothesized risk factors, the gender of pigs (being female) was significantly associated with porcine cysticercosis in this study . It can be explained that female pigs were for kept long time for breeding purpose than male and so they have more risk to get exposed to t. solium eggs . However, jayashi et al . Reported that gender was not a significant risk factor for porcine cysticercosis . The present study demonstrated that the older the pigs, the greater the chance to get infection . These results are in agreement with those reported by pouedet et al ., jayashi et al ., sarti et al ., garca et al ., and pondja et al . Older pigs might also have greater chance to get exposed to t. solium eggs than younger ones . They might have much time to develop cyst and trigger the production of circulating antibodies . Besides, it could be possible that younger pigs are protected during their first months of life against parasite infection, due to the presence of maternal cysticercus antibodies and they become susceptible later after the slow clearance of those antibodies . The result showed that pigs from households practiced semi - intensive system (the pigs are allowed to roam freely in the environment and only panned or tethered at feeding time and night) were more likely to have porcine cysticercosis than intensive (the pigs are kept in the backyard or corral and not allowed to roam) pigs . Therefore, semi - intensive management system represented as an important risk factor for porcine cysticercosis in the study area as the pigs in this practice could access the infected human faeces . Accessibility of infected human faeces is the main source for porcine cysticercosis [17, 19, 20]. Among the feed types used in pig farms, this might be contaminated with t. solium eggs from infected food preparers of swill collected houses . So the collected swill should be cooked thoroughly before feeding to prevent infection including cysticercosis . Use of anthelmintic in pigs and owners was significantly associated in this study . By interviewing the farmers and township veterinary officers, although ivermectin cannot kill any larvae of cestode, albendazole can kill these larvae . Although all the farmers wash their hands after feeding the pigs, only 21.2% famers (43/203) wash their hands before feeding . All cysticercosis positive samples were from those who do not practice hand - washing habit . Pork consumption of owners is also one of the risk factors in survey of porcine cysticercosis . Nine hypothesized risk factors not included in analysis were breed of pigs, place of purchase, presence of latrine, hand - washing after feeding the pigs, source of water for pigs, cleanliness of water, knowledge on taeniasis and cysticercosis, and occurrence of cyst in pork . In this study, all pigs are indigenously bred . All farmers have latrines using water, but the children do not use latrine and are used for defecation out of latrine . Some farmers washed the hands before feeding the pigs and all farmers washed their hands after feeding . All farmers did not have the knowledge on taeniasis and cysticercosis and they have never seen the cysts in the pork in the study area . The presence of zoonotic agent, cysticercus cellulosae, may depend on intrinsic factors: age, gender, and extrinsic factors: pig husbandry system, hand - washing habit of owner, use of kitchen waste as pig feed, not using anthelmintic in pigs and owners, and pork consumption of owner in the study area . Presence of this infection is of public health importance because it may lead to the occurrence of neurocysticercosis in human . Although the occurrence of human neurocysticercosis has not been reported yet in myanmar, all public should take awareness of potential risk factors due to the prevalence with high percentage observed in this study . Myanmar has no national monitoring program for t. solium cysticercus spp . In these animals yet . Therefore, it is advisable to monitor whether there is high or low prevalence of t. solium cysticercosis in the whole country . It could also be suggested that confinement housing system should be developed in pig industry of myanmar to efficiently prevent porcine cysticercosis . For practicing sanitary and culinary habit, thorough cooking education programs should also be implemented for both swine breeders and consumers so as to prevent taeniasis in human and porcine cysticercosis and also other zoonotic helminth diseases in myanmar . This prevalence with relatively high percentage of porcine cysticercosis (15.93%) in ab - elisa and 23.67% in slaughtered pigs indicates the presence of human taeniasis and it also leads to the associated risk of human cysticercosis and neurocysticercosis.
Organized by nei dan school, european school of taiji quan and by the tao and science studies centre, under the aegis of the provincia di bologna and the partnership of asi (alleanza sportiva italiana) and luni editrice . Goodwill of the conference is to act as a starting point to develop a net of experts, doctors and scientists, who will investigate the dynamic interactions between spiritual insight and scientific analysis to come to the creation of a new paradigm of modern science . Science, philosophy, medicine and body arts of the ancient east are reunited together to create a new ecological awareness of body and mind . Modern science, which paved the way for an outlook of reality considering the universe as a whole, in which all parts and phenomena are connected among them, can be integrated to the ancient eastern wisdom for the control of the mind and to the body arts (taiji quan, qi gong, yoga) to develop a new ecological awareness, an awareness based on nature and on the dynamic relation among all living creatures . The conference was divided in two sections: a gathering of experiences, of paths where science meets metaphysics to have a new language born, made of images and movement, and a panel to understand how taiji quan, the arts of movement and meditation, can prolifically meet cognitive sciences and neurosciences . Speakers during the tao and science conference were: andrea pezzi (presenter and tv author); professor edwin l. cooper professor, laboratory of comparative neuroimmunology, department of neurobiology, david geffen school of medicine, university of california los angeles, editor - in - chief, the journal: evidence based complementary and alternative medicine, oxford university press; professor carlo ventura (professor of molecular biology at the faculty of medicine, university of bologna; director of the laboratory of molecular biology and stem cell bioengineering, national institute of biostructures and biosystems by the institute of cardiology of santorsola malpighi hospital in bologna); professor angelo marzollo (professor of systems theory, faculty of sciences, university of udine; vice general secretary of the international centre for mechanical sciences; unesco ex - person in charge for mathematics and now consultant); professor giovanni sambin (professor of logic mathematics, university of padova); dottor matteo luteriani (publisher, journalist and master of martial arts), dottor massimo mori (doctor, poet and master of taiji quan) and eng . Flavio daniele (writer and master of taiji quan) the following guests took part in the conference through their representatives: professor james k. gimzewski (department of chemistry and biochemistry, university of los angeles ucla; director of the pico lab laboratory at ucla); professor aldo stella (teacher of medical psychology, university of urbino; teacher of psychology of cognitive processes, university for foreigners of perugia); professor carmelo di stefano (teacher of teaching didactics of the adapted movement and sports activity, faculty of motor sciences, university of bologna). Carlo ventura, md, phd: western science has long been entangled with increasing reductionism and the development of field restricted approaches to understand cell biology and the molecular basis of disease . It is now becoming increasingly evident that reductionism is a remarkable bias in pursuing some of the major goals of modern biology and medicine . Complex problems, including cell growth and differentiation under normal or malignant conditions (cancer), and the adaptive mechanisms of humans to multiple changes in cell signaling networks now pose the need for holistic approaches both at the molecular biology and medical levels . Such a requirement is even more urgent in spite of the emerging interest in stem cell biology, since taking a glimpse at the mechanisms underlying cell commitment and fate specification may hold promises for a revolutionary field, the so - called regenerative medicine. While moving from reductionism to holistic approaches, the cell is studied as an integrated system, behaving as a neural network with complex and sophisticated logics . Awareness of these features has progressively led to wide - ranging strategies in the investigation of gene and protein expression . Techniques such as the dna microarray and the serial analysis of gene expression (sage) are now able to follow the expression of thousands of genes and signaling molecules at a time, attempting to uncover the overall plans that underlie molecular patterning and cellular decisions . Omics era (genomics, proteomics) and will hopefully form the scientific underpinning for moving from basic science to a clinical practice in which physicians will learn how to deal with illness rather than disease (or even worst, diseased organs). A major sign of these cultural changes is provided by the ongoing development of nanobiotechnologies . In both the philosophical and visual sense, seeing is believing does not apply to nanotechnology, for there is nothing even remotely visible to create proof of existence . On the atomic and molecular scale, data is recorded by sensing and probing in a very abstract manner, which requires complex and approximate interpretations . More than in any other science, visualization and creation of a narrative becomes necessary to describe what is sensed, not seen . We have growing needs for separating the informational content of life from its material substrate. Information is thought to be the essence of life, as in the dna code (james k. gimzewski, university of california at los angeles, department of chemistry and biochemistry, director of the pico lab at ucla). Edwin cooper highlighted how different alternative medicines if integrated can be useful to reclaim that holistic view of the diseased person, that an excessive specialization has made modern western medicine lose and he illustrated the work done in this direction by the biomedical journal ecam . Disease has always been of enormous concern in human society . From prayers and spells to the birth of medicine as a rational science, man has developed all sorts of medical treatments to combat different illnesses and chronic ailments: according to the chinese proverb: life is worth more than a thousand gold pieces. The first objective in a serious approach to complementary and alternative medicine (cam) should be to obtain a broad understanding, with a minimum of detail, of how cam fits into the pattern of biology of the way in which the nervous, endocrine and immune systems coevolved, their function and coordination with other body systems, and their development from the embryo onwards including aging . At the same time, such an outline should provide an adequate background for easy application of cam ideas to the detail of practical cam work in public health, clinical and medical practice, and yet not stray far away from its essence, the very biology that under girds it . Cam is organismic, considers the whole individual and is inclusive, not reductionist nor exclusive . Concerning senescence and age - associated diseases, that accompany longer living populations, substantial attention is now focused on searching for: (i) mechanisms of aging and (ii) approaches to ameliorate or lessen the effects through the use of therapies, some of which utilize natural products from aquatic and terrestrial plants and animals . Clearly there are numerous treatments to explore and to understand long past the anecdotal information that has been passed on through centuries . There is evidence for treatment of diseases of an ever - aging society especially in developed countries . It is of great interest that these remedies now being refined by ecam approaches derives almost entirely from primitive societies where there are minimal facilities essential for analyses by an evidence - based approach . Massimo mori said: [] if the scientific research is free, the same is not valid for its technological applications, which have to serve an ecology of culture marked by wisdom, an ecology of the mind, as wrote gregory bateson, in harmony with nature; nature, where the uncertainty principle of werner heisenberg has scientific and philosophical value; harmony, comprising the clash and entropy of ilya prigogine as factors of transformation . A holistic view recomposing the divided self of ronald d. laing, giving deepness to the one - dimensional man of herbert marcuse. Prof . Angelo marzollo highlighted the importance of combining the art of to say with the art of to do, i.e., that the learned man, the intellectual is direct evidence of the ideas carried on by him, that the body does not contradict the mind, that the body - mind unit remains not only a theoretical statement . Giovanni sambin said: although the theme is science, i can speak only about my specialization . I am fascinated by oriental wisdom, but i know almost nothing about it because of lack of time . I am here only because i am trying to learn tai ji quan, with master roberto benetti. Mathematics is important in western science . Galilei was the first to propose that the laws of nature are written in the language of mathematics . This has been the base for inventions characterizing industrial revolution in xixth century . By using mathematics so mathematics is at the base of present - day western technological supremacy . In modern times, debate concerning foundations of mathematics has been most lively at the beginning of xxth century . One of its outcomes was the invention of programmable computers, which speaks of its importance . I am not a believer, but i find constructivism much more convincing than the now dominating theory, which is called classical and is considered by most scientists as an absolute truth . To do modern mathematics one needs an abstract notion of concept, or set . In the classical approach, one has classical logic (by which all propositions are either true or false, any third possibility like abstinence is excluded) and axiomatic set theory (all sets are there already, a static universe containing now, and ever, all possible concept - sets). The mental scheme looks as something like: classical mathematics is at the base of western science, which is the reason for technological superiority, which makes western people the owners of the world . Hence it must be that western mathematics, and all what follows from it, is an absolute truth, which can be imposed on others by force . Needless to say, this view can be extremely dangerous . Here and in all my life, my aim is to show that a different foundation of mathematics is possible, which has the same applications, but which avoids any form of fundamentalism . A way of doing mathematics is possible, in which the will of power is replaced by harmony with nature, control is replaced by knowledge, brute external force is replaced by internal energy, the search for the i want to emphasize that originally this was not motivated by some ethical or political principles, rather by the search for a better foundation of mathematics . Master flavio daniele emphasized as most advanced research of the last decades of the past century in the field of cognitive sciences and neurosciences led to a new theory, revolutionizing the traditional cartesian concept of mind . This theory, known as the santiago theory of cognition, claims that mind can no longer be regarded as a thing but as a process . Process, which is cognition to which belong perceptions, emotions and actions, the language, the conceptual thought and all the attributes of conscience, which is peculiar to man . This view, perfectly in line with the eastern traditional thought, entails that mind with its cognitive processes goes beyond the rational aspect as it includes the whole process of life . A further implication of this theory, which will show its vast potential, when it will be absorbed at the general cultural level, is that mind and matter are no longer regarded as separated dimensions, but as complementary aspects of the sole phenomenon of life: the process (the mind) and the structure (the brain). Mind and matter, process and structure are indivisibly connected at all levels of life: from the simplest cell to the most complex organism . This connection is so deep, as most recent studies in the field of cognitive sciences has demonstrated, that we can state, conceptual thought, on the whole, is physically incarnated in the body and in the brain. This goes beyond the simple consideration that to think we need a brain and leads to state that human reason does not transcend body, but it is structurally shaped by our physicality and body experience. A further discovery of cognitive sciences, consequent to it that: (1) mind is deeply incarnated in the body, and the other that, (2) thought is mainly unconscious, is that (3) abstract concepts are to a large extent metaphorical. For the time being, present researchers have not explained in detail the neurophysiological dynamics underlying the formation of abstract concepts; however, the scientists lakoff and johnson state that the neural and cognitive mechanisms enabling us to sense and move are the same to create also our conceptual structures and our ways of reasoning. This statement that conceptual structures and ways of reasoning come from the same neural and cognitive structures of perception and movement, is extremely important for the practitioners of the arts of movement (taiji quan, yoga, sacred dances, ritual gestures or mudra), as it demonstrates and confirms the creative power of movement, which no longer acts as simple instrument at the service of mind, to play the fundamental role of shaper of the cognitive capabilities (conceptual thought, speech, conscience) of the human mind . By paraphrasing maturana and varela cognition, the process of knowledge, and it is identical to the process of life self . Nevertheless, beyond the dialectics coming from the differences, it is more and more important to reintroduce a principle, acting as basis for the man universally, a principle which is internal and unconventional, an equal able to give the equal to all psycho - biological behaviors of the human being . In italy this kind of research has always taken place, and in recent years has made its way into the experimental evidence of a psychology able to touch this dimension.
Cardiovascular disease is the most common cause of mortality in dialysis patients that is responsible for about 60% of their mortality and is also 30 times more common than in general population . Different cardiovascular disorders, such as left ventricular hypertrophy (lvh), coronary artery diseases, congestive heart failure (chf), and arterial hypertension are commonly seen in these patients . In addition, calcification of cardiac valves are common and may cause valvular and annular thickening that in turn could lead to valvular stenosis or regurgitation . Some predisposing factors of cardiac disorders in dialysis patients are secondary hyperparathyroidism, long term hypertension, and anemia . Different electrocardiographic abnormality may be seen in dialysis patients, such as st, t change, ventricular and supra - ventricular arrhythmia and qt interval prolongation . In the electrocardiogram, qt interval is the time between the start of the q wave and the end of the t wave . When heart rate increased then qt interval was decreased, so qt interval could be corrected with heart rate . There are a number of different correction formulas, but the standard clinical correction is to use bazett's formula (qt/r - r, where r - r is the rr interval in seconds). The normal corrected qt (qtc) interval for males and females are 430 ms and 450 ms, respectively . The borderline qtc for a male is 431 - 450 and borderline qtc for a female is 451 - 470 and the abnormal qtc range for a male is> 450 and for a female is> 470 . The qtc interval could be prolonged due to electrolyte abnormalities (hypomagnesaemia and hypokalemia), drug consumption (antihistaminic, antiarrhythmic, and antibiotics), brain trauma and genetic abnormalities (long qt syndrome). Patients with chronic renal failure and dialysis patients had a greater qtc interval and qt dispersion (qtd) (qtd = qtmax qtmin) compared with the normal population . A single session of hd could be increased qtc in patients undergoing hd . Moreover, qt interval may be a predictor of ventricular arrhythmia and cardiovascular mortality in chronic kidney disease and dialysis patients . Although there are some studies about qtc and qtc dispersion in hemodialysis (hd) patients with different results; however, there are only a few studies concerning relationship of qtc internal and qtd with echocardiography findings, so the aim of this study was the evaluation of these relationship in dialysis patients . In a cross - sectional study, 60 hd patients with age> 18 years and the dialysis duration> 3 months were enrolled . Exclusion criteria were: antiarrhythmic drugs consumption, history of cardiac diseases such as arrhythmia, heart block or chf . Before dialysis session, electrocardiography (ecg) and echocardiography also, qtd was measured by mentioned formula (qtd = qtmax qtmin) in 12 leads ecg . The patients were on hd, by fresenius digital machine (4008b, germany) and gambro digital machine (ak95 and ak96, sweden), 2 - 3 times / week, as a regular method (4 - 4.5 h with blood flow of 250 - 350 ml / min, dialysate flow of 500 ml / min and ultra - filtration based on the patient's condition). The used buffer was bicarbonate powder, and the type of filters were intermediate and high efficient polysulfone membrane (r5, r6) made by soha factory under license of fresenius company . Serum creatinine, hemoglobin, ca, parathyroid hormone (pth), na, k, hco3, and ph were checked by ra1000 machine (made in italy). All laboratory tests were done before the dialysis session and were checked in a single laboratory . Echocardiography and ecg were done before the dialysis session and by single cardiologist and technician respectively . All data and information were confidential, and for ecg and echocardiography taking an informed consent was taken from each patient . In echocardiography, left ventricular ejection fraction (lvef), lvh, pulmonary artery pressure (pap) and valvular disorders were evaluated . At the end of the study, data were analyzed using spss software (version 19, ibm corporation). Pearson correlation coefficient, two - independent samples t - test and anova were used for statistical analysis . This study was approved by ethical committee of shahrekord university of medical sciences with the grant number of 904 . Mean body mass indexes (bmis) (post dialysis) and duration of dialysis were 21.77 3.6 mean qtc interval of the patients was 0.441 0.056 s; however, qtc interval in men and women were 0.43 0.04 s and 0.45 0.07 s, respectively (p> 0.05). Qtd in all of the patients was 64.17 25.93 ms, however in men and women were 62.70 28.05 ms and 66.52 22.48 ms respectively (p = 0.87). Mean urea reduction ratio in men and women were 68.14% 9.34% and 68.22% 3.88%; however, kt / v was 1.45 0.17 and 1.49 0.16 among men and women, respectively . Characteristics of patients and their association with qtc and qtd sd = standard deviation, bmi = body mass index, pth = parathyroid hormone, qtc = corrected qt, qtd = qt dispersion there was no statistically significant relationship between qtc interval or qtd with duration of dialysis, bmi, age, and gender (p> 0.05). In ecg, lvh was seen in 23 (38.3%) patients and st change in 14 patients (23.3%). In echocardiography, mitral regurgitation (mr), tricuspid regurgitation (tr), and aortic insufficiency (ai) were found in 54, 47, and 11 patients respectively [table 2]. In addition, no significant relation was found between qtc interval and qtd with mr, tr, ai, lvh, septal thickness (st) and pap [table 3]. Qtc interval and qtd has also no correlation with serum pth or ca, k or hco3 . Severity of valvular disorders in the patients mr = mitral regurgitation, tr = tricuspid regurgitation, ai = aortic insufficiency echocardiographic findings and their association with qtc and qtd qtc = corrected qt, qtd = qt dispersion, pap = pulmonary artery pressure, lvef = left ventricular ejection fraction, lvh = left ventricular hypertrophy, mr = mitral regurgitation, tr = tricuspid regurgitation, ai = aortic insufficiency, pe = pericardial effusion our findings revealed that, in hd patients, qtc interval and qtd had not any correlation with valvular disorders (mr, ai, and tr), lvh or other echocardiographic findings such as pap and st . Furthermore, there was no relationship between qtc interval and qtd with serum ca, k, hco3, pth . In hd patients, cardiac abnormalities such as vascular calcification are common and are associated with the development of lvh, that may cause increased cardiac arrhythmias and mortality . Elevation of calcium - phosphorus product can also cause valvular calcification and stenosis in these patients . Mitral and aortic calcification and stenosis are common in these patients; however, tricuspid and pulmonic valve calcification is rare . Qtc interval prolongation in chronic kidney disease and hd patients was shown in some studies such as covic et al . And ljutic et al . Studies which postdialysis qtc interval was 434 29 ms and 445.7 36.9 ms respectively, while in our patients qtc interval was 444.6 54.5 ms . Selby and mcintyre in a review article reported that hd can increase qtc interval and qtd and is also capable of inducing arrhythmias and increasing mortality especially in patients with ischemic heart disease . In nakamura study on 48 dialysis patients, throughout the follow - up period, there was a higher incidence of cardiovascular death in patients with prolongation of qtc dispersion after hd . Showed that the serum potassium was significantly higher in hd patients when compared to continuous ambulatory peritoneal dialysis (capd) patients and rate of qt interval dispersions was significantly higher in hd and capd patients when compared with healthy controls . They concluded that there is a tendency to cardiac arrhythmias in hd patients during the postdialysis period . Maule et al . Showed that after the hd session, qtc increased in 56% and decreased in 43% of the patients after dialysis session, cell - associated mg levels and qtd increased significantly in averbukh et al . Study, so he concluded that excess daily mg intake and increased concentrations of cell - associated mg could be responsible for qtc prolongation in these patients . Change in serum electrolytes during hd may also be responsible at least partially, to increase qtc or qtd . For example, increase of qtc interval after a dialysis session, and its correlation with plasma calcium level, postdialysis blood pressure, lvef and st was shown in covic et al . Showed that qtd increased during hd due to serum k depletion and then return to baseline 2 h after the end of dialysis . Qt prolongation during dialysis may also predispose to arrhythmia especially in the cardiac disease patients, nppi et al . Demonstrated that hd increases qtd if a low - calcium dialysate is used . Therefore, use of a low - calcium dialysate may predispose hd patients to ventricular arrhythmias . 16 hd patients showed that qtc interval has a reverse correlation with k and ca concentrations of dialysate . However, in hd children, ozdemir et al . Found that qtc interval has no correlation with the patients sex, age and presence of hypertension or lvh but patients with left ventricular systolic dysfunction had significantly greater qtc dispersion . Similar to our study, the changes in serum ca or k of the patients during dialysis were not associated with the qtc interval in this study . Moreover, in hekmat et al.s study on 49 hd patients, qtc interval had not correlation with serum electrolytes and blood gas findings . In voiculescu study, there was no statistically significant correlation between qt interval and serum concentrations of mg, po4 in hd patients too . In addition, qtc interval was not dependent from lvef, arrhythmias or sudden death . Our results had some similarities with some of above - mentioned studies as well as some differences with the others . The observed controversy between our study and some of these studies might be due to the discrepancy in the number of cases or racial differences of the patients, and more importantly, the fact that we did not compare predialysis and postdialysis qtc interval . We could not find any study on the correlation of qtc interval with lvh or valvular heart disease in hd patients; however, there are some studies related to the correlation of qtc and lvh with other diseases or conditions . For example, mayet et al . Reported that qtd has correlated with left ventricular mass index in hypertensive individuals . Found that in athletes, lvh induced by physical training activity is not associated with an increase in qtd, whereas pathological increase in lvh secondary to hypertension could increase qtd . The study of dimopoulos et al . On the evaluation of qtd and left ventricular mass index in elderly hypertensive and normotensive patients reported that hypertensive patients had greater left ventricular mass index and higher qtd . The study had some limitations such as small sample size and lack of qtc measurement after hd session and comparison of it before and after procedure . By our knowledge, this is one of the first studies on the correlation of qtc or qtd with valvular abnormality and lvh in hd patients . There were a few studies about the relationship of qtc and lvh in nonrenal patients, so we could not compare our results with other studies . Based on our results, in hd patients, qtc interval or qtd was not correlated with echocardiographic findings or laboratory exam results . Therefore, it can be concluded that qtc interval prolongation probably has not any correlation with cardiac mortality of the hd patients.
Asthma is a chronic inflammatory condition of the airways, characterized by limited airflow and punctuated by acute symptoms related to hyperresponsiveness to a variety of triggers, such as allergens or fumes (1). Chronic inflammation of the airways can lead to persistent alterations in airway structure, including sub - basement membrane fibrosis, mucus hypersecretion, injury to epithelial cells, smooth muscle hypertrophy, and angiogenesis (2). Asthma symptoms include wheezing, dyspnea, coughing, and chest tightness associated with broad but variable airflow obstruction in the lungs (3). During acute asthma events, symptom severity may range from mild to life - threatening, the latter being due to severe bronchospasm, airway edema, impaired gas exchange, and ultimately, respiratory failure . Worldwide, an estimated 300 million people suffer from the disease and prevalence is increasing (47). Of the 25 million americans with asthma, 12 million experience acute symptoms (8) and asthma was linked to 3447 deaths (about 9 per day) in 2007 (5). Asthma symptoms account for about 500,000 hospitalizations and nearly 2 million emergency room visits per year in the united states (6). Asthma - associated morbidity remains high despite improvements in diagnosis and the availability of comprehensive national and international clinical practice guidelines for managing the disease (2,3,9). Avoiding triggers is an important first step in asthma management, but may require lifestyle changes that patients find difficult or unacceptable (e.g., giving away a family pet), and adherence to lifestyle modifications is poor (1,5). If lifestyle changes do not successfully prevent and control asthma symptoms, pharmacologic therapy can reduce the frequency and severity of asthma exacerbations, and reverse airflow obstruction during acute attacks (2). Most asthma medications are delivered as orally inhaled products in order to achieve local effects in the lung and to minimize systemic adverse effects . Inhaled asthma medications are categorized into two general classes: long - term control medications (also known as preventive or maintenance medications), which are taken regularly to achieve and maintain control of persistent asthma, and rapid - acting drugs (also known as rescue medications) taken as needed to provide prompt reversal of acute airflow limitation and relieve bronchospasm . Onset of action of inhaled drugs for rescue from acute bronchospasm is approximately 510 minutes . Rescue medications are typically short - acting 2-adrenergic agonists (sabas), such as albuterol, but may be a long - acting beta agonist with rapid onset of action, such as formoterol (10). Bronchodilators provide relief of bronchoconstriction by relaxing bronchial smooth muscle and functionally enlarging the luminal diameter of the airways . Sabas are delivered via wet nebulization or metered dose inhaler (mdi) (1). Mdis may provide better clinical outcomes and fewer adverse effects compared with nebulizers (11); however, nebulizers are useful for young children, older adults, and for patients who are unable to use an mdi . Among other factors, patient nonadherence to therapy is an important contributor to poor asthma control . While often volitional, nonadherence can also be inadvertent when prescribed medications are taken improperly . For example, patients may think they are taking their asthma medication when they actually are activating a nearly empty or an altogether empty mdi to deliver orally inhaled asthma medication (12). Mdis deliver a limited number of effective medication doses, as listed in the prescribing information for each product . After the manufacturer - recommended number of doses is expelled, the mdi will continue to actuate many more times (13). Accurately assessing the doses in an mdi is critically important for bronchodilating medications used for rescue from acute asthma symptoms . And significantly overestimate (or less commonly, underestimate) the remaining amount of active asthma rescue medication in mdis without dose counters, that the techniques used to guesstimate whether a recue mdi is effectively empty are unreliable, and how having a rescue mdi with an integrated dose counter mechanism can improve the health and quality of life of patients with asthma . We searched embase and medline english literature up to july, 2012 with search terms dose counter and asthma and dose counter and metered dose inhaler . When pertinent articles were identified, we searched for relevant references used in those papers . For environmental reasons, inhalers with chlorofluorocarbon (cfc) propellant were no longer sold in the us from 2009, and the propellant in current mdis is hydrofluoroalkane (hfa); however, much of the research reviewed here was conducted on mdis with cfc propellant . Activating a nearly empty or an altogether empty mdi to deliver orally inhaled asthma medication appears to be a common occurrence when patients use an mdi without a dose counter (12,14,15). Mdis contain more drug formulation than the labeled number of drug doses to ensure dosing consistency in each actuation, up to the labeled number (16). Short of recording every actuation, there is no accurate and practical way to gauge the remaining number of effective doses in an mdi without the addition of a dose - counting mechanism . The invention of the metered - dose valve led to development of mdis in the 1950s (13). The dosing and performance and, consequently, drug efficacy may be directly dependent on the design of the mdi . Mdis have 3 main components: the canister containing the drug formulation, the metering valve, which determines the quantity of formulation dispensed upon actuation, and an actuator (mouthpiece) which directs the aerosol into the patient s lungs . In addition to the bronchodilating drug, the formulation contains a liquefied gas propellant (hfa) and may contain nonactive excipients . With current valve designs, it is not possible for an mdi to cease delivering drug doses completely at an exact point . Mdis continue to deliver a spray, which may not be within the labeled specifications for the active drug, for up to twice the nominal number of recommended doses . In one study, mdis with cfc propellant had, on average, 86% more actuations than the labeled dose number, and mdis with hfa propellant had 52% more (17). Importantly, the amount of drug in those additional actuations is variable; propellant and excipients form up to 99% of an asthma drug formulation (16). With continued use beyond the recommended number of doses, drug delivery per actuation becomes inconsistent and unpredictable, with the amount of active drug eventually becoming negligible, a phenomenon known as tail - off (13,18). Thus, after the recommended number of doses, the mdi may appear to be delivering a therapeutic spray when, in fact, it is not . Tail - off may be rapid (e.g., within 5 actuations), or erratic, requiring 1020 actuations before the canister is finally empty of its drug contents, depending on several factors, including the valve design (13). Mdis with valves that have formulation fill holes at the base of the retaining chamber have more rapid and less erratic tail - off (figure 1a) than mdis with valves with fill holes at higher levels (figure 1b). If the height of the liquid formulation falls below the fill hole, at the time of actuation there may be incomplete filling of the chamber (13). Tail - off is particularly problematic when the medication delivered by the mdi is formulated as a suspension rather than a solution . All short - acting asthma rescue medications currently available in an mdi are formulated as suspensions . If the drug substance adheres to the walls of the container or valve components, dose delivery and particle size distribution could be inconsistent (19). For this reason, shaking a rescue inhaler before actuation is an important part of correct mdi use . Panel a shows tail - off from 3 separate mdis with fill holes located at the base of the retaining cup that allow formulation to enter the valve (valve - down orientation). Panel b shows more erratic tail - off characteristics of 3 separate mdis with valve design in which the fill holes are located at higher levels relative to the base of the retaining cup (adapted from schultz (13)). Shown here is a proair metered - dose inhaler (manufactured by teva pharmaceutical industries, ltd . Panel a shows tail - off from 3 separate mdis with fill holes located at the base of the retaining cup that allow formulation to enter the valve (valve - down orientation). Panel b shows more erratic tail - off characteristics of 3 separate mdis with valve design in which the fill holes are located at higher levels relative to the base of the retaining cup (adapted from schultz (13)). Shown here is a proair metered - dose inhaler (manufactured by teva pharmaceutical industries, ltd . Studies conducted to assess the accuracy of asthma patients ability to gauge doses remaining in their mdis have consistently shown high error rates (14,15,20,21). As noted, the only way to reliably determine the number of remaining doses in an mdi with no dose counting feature is by carefully and consistently tracking each dose, then subtracting it from the labeled number of doses . However, many people are not even aware of the recommended number of drug doses in their mdis . In a study conducted to investigate how asthma sufferers determined when to replace their mdis, 54% did not know the maximum number of actuations recommended by the manufacturer for their particular inhaler (15). Even when recommended dosing is known, patient records or diary entries of doses taken are rarely kept . In a study by ogren et al ., only 8% of patients kept any record of their mdi actuations (15). Most patients did not receive instructions from their health care providers to keep a count of doses taken; in a survey conducted by sander et al ., only 36% of respondents reported having been instructed to keep track of the number of mdi - delivered doses of their asthma medications (14). Other methods by which patients estimate remaining medication in their mdis are not reliable (1315). Estimations of whether an mdi requires replacement based on the weight of the inhaler, or on the force, sounds, and taste of the actuation, are often made based on the volume of the remaining propellants and excipients and not on the remaining doses of active medication meeting therapeutic specifications (15,17). In a study by rubin and durotoye of pediatric asthma patients, 72% of the children (or their parents) reported using an mdi until they could no longer similarly, in the study by sander and colleagues, 1 in 5 surveyed patients reported that they assessed their albuterol inhaler to be empty when it stopped spraying, unaware that propellant continues to spray long after the albuterol has run out (14). Many patients report shaking their mdis to assess the amount of drug remaining in them (15,20,22). In one study, patients who adopted this method overestimated remaining medication by about 40 doses (21). Accordingly, 84% of mdis evaluated in this study had been used well past the recommended number of actuations . It has been suggested that floating an inhaler in water, known as a float test, can provide a measure of the inhaler s useful contents (20). However, no universal flotation status accurately reflects when a device has reached the maximum recommended number of actuations (15). Moreover, this test is not only unreliable since some inhalers will float when they are full, it may damage the mdi by obstructing the metering valve (1517). In practice, patients who are not tracking rescue medication doses often either discard an mdi that may still contain acceptable metered doses of drug, or continue to use a product that may no longer contain the labeled within - specification dosage . The former is wasteful and expensive and the latter is potentially dangerous (16). Holt and colleagues asked patients receiving asthma treatment to return their mdis, when deemed empty, to their physicians (21). Of 109 returned mdis, 11% of empty mdis still contained more than 20% (> 40 doses) of the recommended metered doses (table 1) (21). Sander et al . Reported that more than half (53%) of the asthma patients they surveyed who used an inhaled bronchodilator (n = 342) refilled their bronchodilator prescriptions more frequently than recommended in national guidelines (2,14). A bronchodilator prescription typically requires refill only a few times a year, yet almost 20% of patients reported refilling their inhaler at least once a month . The authors speculated that the excessive number of bronchodilator refills might be due, in part, to throwing away partially used inhalers to forestall the prospect of finding an inhaler empty during an acute asthma event (14). Table 1.discrepancies in patient perceptions of the number of doses of rescue medication remaining in an mdi with no dose counter . Seventeen patients with asthma who regularly use an mdi estimated the number of salbutamol doses remaining in mdis that had been partially emptied to different degrees (adapted from holt et al . (21)).actual number of remaining salbutamol doses in mdis which had been partially emptied40 doses30 doses20 doses10 doses0 dosesrange of patient estimations of the number of doses in the mdi (min max)0180019001400180080mean difference between the actual and the estimated number of doses in the mdi76.881.063.054.823.6proportionate overestimations of the remaining doses in the mdi+36.8%+51.0%+43.0%+44.8%+23.6% discrepancies in patient perceptions of the number of doses of rescue medication remaining in an mdi with no dose counter . Seventeen patients with asthma who regularly use an mdi estimated the number of salbutamol doses remaining in mdis that had been partially emptied to different degrees (adapted from holt et al . (21)). While discarding an mdi with effective doses remaining is both costly and wasteful, relying on rescue medication from a depleted mdi during acute bronchospasm can be life - threatening . In the study by holt and colleagues, collected mdis labeled to contain 200 salbutamol doses two separate studies found that more than 40% of asthma patients replaced their mdi when it was completely empty and over 20% said they could never tell when their mdi was running out of medication (22,23). In the sander et al . Study, 87 asthmatic patients25% of those surveyed discovered that their albuterol inhaler was empty when needed for relief from acute asthma symptoms, and of them, 8% had to call for emergency assistance (14). Most of these 87 patients (82%) considered their mdi empty only when nothing came out of it, making it likely that they were inhaling only propellant for many doses, thereby increasing their risk of prolonged bronchoconstriction and airflow limitation requiring urgent care . When asked, many patients report that not knowing how much medication is left in their mdis makes them anxious about receiving a subtherapeutic dose or no medication at all (22,24). Dose counters are, therefore, likely to alleviate patient anxiety about running out of asthma medication in emergency situations . Thus, it is not surprising that patient satisfaction studies consistently show wide acceptance and approval of dose counters on mdis (2224). Patients rate dose counters among the top 5 best features of their mdi (14,23). In addition to relieving anxiety, reliable knowledge of when to replace an mdi can improve asthma management and, in turn, improve patients quality of life (25). Because they are preferred by patients (22) moreover, physician inspection of dose counters at office visits may provide a means of evaluating patient adherence to treatment and present an opportunity to discuss appropriate use of the mdi and proper inhalation technique . The invention of the metered - dose valve led to development of mdis in the 1950s (13). The dosing and performance and, consequently, drug efficacy may be directly dependent on the design of the mdi . Mdis have 3 main components: the canister containing the drug formulation, the metering valve, which determines the quantity of formulation dispensed upon actuation, and an actuator (mouthpiece) which directs the aerosol into the patient s lungs . In addition to the bronchodilating drug, the formulation contains a liquefied gas propellant (hfa) and may contain nonactive excipients . With current valve designs, it is not possible for an mdi to cease delivering drug doses completely at an exact point . Mdis continue to deliver a spray, which may not be within the labeled specifications for the active drug, for up to twice the nominal number of recommended doses . In one study, mdis with cfc propellant had, on average, 86% more actuations than the labeled dose number, and mdis with hfa propellant had 52% more (17). Importantly, the amount of drug in those additional actuations is variable; propellant and excipients form up to 99% of an asthma drug formulation (16). With continued use beyond the recommended number of doses, drug delivery per actuation becomes inconsistent and unpredictable, with the amount of active drug eventually becoming negligible, a phenomenon known as tail - off thus, after the recommended number of doses, the mdi may appear to be delivering a therapeutic spray when, in fact, it is not . Tail - off may be rapid (e.g., within 5 actuations), or erratic, requiring 1020 actuations before the canister is finally empty of its drug contents, depending on several factors, including the valve design (13). Mdis with valves that have formulation fill holes at the base of the retaining chamber have more rapid and less erratic tail - off (figure 1a) than mdis with valves with fill holes at higher levels (figure 1b). If the height of the liquid formulation falls below the fill hole, at the time of actuation there may be incomplete filling of the chamber (13). Tail - off is particularly problematic when the medication delivered by the mdi is formulated as a suspension rather than a solution . All short - acting asthma rescue medications currently available in an mdi are formulated as suspensions . If the drug substance adheres to the walls of the container or valve components, dose delivery and particle size distribution could be inconsistent (19). For this reason, shaking a rescue inhaler before actuation is an important part of correct mdi use . Panel a shows tail - off from 3 separate mdis with fill holes located at the base of the retaining cup that allow formulation to enter the valve (valve - down orientation). Panel b shows more erratic tail - off characteristics of 3 separate mdis with valve design in which the fill holes are located at higher levels relative to the base of the retaining cup (adapted from schultz (13)). Shown here is a proair metered - dose inhaler (manufactured by teva pharmaceutical industries, ltd . Panel a shows tail - off from 3 separate mdis with fill holes located at the base of the retaining cup that allow formulation to enter the valve (valve - down orientation). Panel b shows more erratic tail - off characteristics of 3 separate mdis with valve design in which the fill holes are located at higher levels relative to the base of the retaining cup (adapted from schultz (13)). Shown here is a proair metered - dose inhaler (manufactured by teva pharmaceutical industries, ltd ., horsham, pa). Studies conducted to assess the accuracy of asthma patients ability to gauge doses remaining in their mdis have consistently shown high error rates (14,15,20,21). As noted, the only way to reliably determine the number of remaining doses in an mdi with no dose counting feature is by carefully and consistently tracking each dose, then subtracting it from the labeled number of doses . However, many people are not even aware of the recommended number of drug doses in their mdis . In a study conducted to investigate how asthma sufferers determined when to replace their mdis, 54% did not know the maximum number of actuations recommended by the manufacturer for their particular inhaler (15). Even when recommended dosing is known, patient records or diary entries of doses taken are rarely kept . In a study by ogren et al ., only 8% of patients kept any record of their mdi actuations (15). Most patients did not receive instructions from their health care providers to keep a count of doses taken; in a survey conducted by sander et al ., only 36% of respondents reported having been instructed to keep track of the number of mdi - delivered doses of their asthma medications (14). Other methods by which patients estimate remaining medication in their mdis are not reliable (1315). Estimations of whether an mdi requires replacement based on the weight of the inhaler, or on the force, sounds, and taste of the actuation, are often made based on the volume of the remaining propellants and excipients and not on the remaining doses of active medication meeting therapeutic specifications (15,17). In a study by rubin and durotoye of pediatric asthma patients, 72% of the children (or their parents) reported using an mdi until they could no longer similarly, in the study by sander and colleagues, 1 in 5 surveyed patients reported that they assessed their albuterol inhaler to be empty when it stopped spraying, unaware that propellant continues to spray long after the albuterol has run out (14). Many patients report shaking their mdis to assess the amount of drug remaining in them (15,20,22). In one study, patients who adopted this method overestimated remaining medication by about 40 doses (21). Accordingly, 84% of mdis evaluated in this study had been used well past the recommended number of actuations . It has been suggested that floating an inhaler in water, known as a float test, can provide a measure of the inhaler s useful contents (20). However, no universal flotation status accurately reflects when a device has reached the maximum recommended number of actuations (15). Moreover, this test is not only unreliable since some inhalers will float when they are full, it may damage the mdi by obstructing the metering valve (1517). In practice, patients who are not tracking rescue medication doses often either discard an mdi that may still contain acceptable metered doses of drug, or continue to use a product that may no longer contain the labeled within - specification dosage . The former is wasteful and expensive and the latter is potentially dangerous (16). Holt and colleagues asked patients receiving asthma treatment to return their mdis, when deemed empty, to their physicians (21). Of 109 returned mdis, 11% of empty mdis still contained more than 20% (> 40 doses) of the recommended metered doses (table 1) (21). Sander et al . Reported that more than half (53%) of the asthma patients they surveyed who used an inhaled bronchodilator (n = 342) refilled their bronchodilator prescriptions more frequently than recommended in national guidelines (2,14). A bronchodilator prescription typically requires refill only a few times a year, yet almost 20% of patients reported refilling their inhaler at least once a month . The authors speculated that the excessive number of bronchodilator refills might be due, in part, to throwing away partially used inhalers to forestall the prospect of finding an inhaler empty during an acute asthma event (14). Table 1.discrepancies in patient perceptions of the number of doses of rescue medication remaining in an mdi with no dose counter . Seventeen patients with asthma who regularly use an mdi estimated the number of salbutamol doses remaining in mdis that had been partially emptied to different degrees (adapted from holt et al . (21)).actual number of remaining salbutamol doses in mdis which had been partially emptied40 doses30 doses20 doses10 doses0 dosesrange of patient estimations of the number of doses in the mdi (min max)0180019001400180080mean difference between the actual and the estimated number of doses in the mdi76.881.063.054.823.6proportionate overestimations of the remaining doses in the mdi+36.8%+51.0%+43.0%+44.8%+23.6% discrepancies in patient perceptions of the number of doses of rescue medication remaining in an mdi with no dose counter . Seventeen patients with asthma who regularly use an mdi estimated the number of salbutamol doses remaining in mdis that had been partially emptied to different degrees (adapted from holt et al . (21)). While discarding an mdi with effective doses remaining is both costly and wasteful, relying on rescue medication from a depleted mdi during acute bronchospasm can be life - threatening . In the study by holt and colleagues, collected mdis labeled to contain 200 salbutamol doses two separate studies found that more than 40% of asthma patients replaced their mdi when it was completely empty and over 20% said they could never tell when their mdi was running out of medication (22,23). In the sander et al . Study, 87 asthmatic patients25% of those surveyed discovered that their albuterol inhaler was empty when needed for relief from acute asthma symptoms, and of them, 8% had to call for emergency assistance (14). Most of these 87 patients (82%) considered their mdi empty only when nothing came out of it, making it likely that they were inhaling only propellant for many doses, thereby increasing their risk of prolonged bronchoconstriction and airflow limitation requiring urgent care . When asked, many patients report that not knowing how much medication is left in their mdis makes them anxious about receiving a subtherapeutic dose or no medication at all (22,24). Dose counters are, therefore, likely to alleviate patient anxiety about running out of asthma medication in emergency situations . Thus, it is not surprising that patient satisfaction studies consistently show wide acceptance and approval of dose counters on mdis (2224). Patients rate dose counters among the top 5 best features of their mdi (14,23). In addition to relieving anxiety, reliable knowledge of when to replace an mdi can improve asthma management and, in turn, improve patients quality of life (25). Because they are preferred by patients (22) moreover, physician inspection of dose counters at office visits may provide a means of evaluating patient adherence to treatment and present an opportunity to discuss appropriate use of the mdi and proper inhalation technique . In 2003, the us food and drug administration issued a guidance to industry emphasizing the importance of (but not requiring) integrated dose counters or dose indicators on mdis for orally inhaled medications targeted to the lungs (16). Dose indicators that rely solely on a color coded display or indicator symbol to signal when the mdi is nearing the end of its recommended doses are less precise than dose counters that use a numeric display (figure 2). Typically, discarding an mdi is recommended when the dose counter reads 0 or by the expiration date, whichever comes first . The expiration date for a rescue inhaler is generally 1 year after the prescription is filled (26) or after the foil pouch is opened (27). At present, the only available albuterol rescue inhalers with integrated dose counting mechanisms are proair hfa (teva pharmaceutical industries, ltd .) (26) and ventolin hfa (glaxosmithkline) (27). If there is a down - side to integrated dose counting mechanisms on mdis, it may be that they are more expensive to produce than mdis without them . After 2008, generic albuterol inhalers stopped being made because they contained cfc propellant (it is unknown when generic albuterol inhalers may be available again). The estimated annual expenditure related to health care and lost productivity due to asthma was more than $20 billion according to the national heart, lung, and blood institute (28). Nevertheless, dose counters may reduce health care costs by decreasing asthma - related morbidity and attendant medical costs (14,17). For example, inhalation of rescue medication rather than propellant to control acute bronchospasm and quell acute asthma symptoms may avoid the cost of an emergency room visit (29). Although there have been relatively few studies conducted to assess the use of mdis, they consistently show patients frequently overestimate the amount of active asthma rescue medication in mdis without dose counters . Dose counters provide the only accurate and practical method of ascertaining the remaining number of effective doses in an mdi . By ensuring that patients do not use a rescue mdi beyond the recommended number of actuations and that they are receiving the appropriate metered dose of asthma medication, dose counters can improve asthma management and potentially decrease asthma - related morbidity and mortality, and improve patients quality of life . For these reasons, integrated dose counting mechanisms should be required on mdis that deliver asthma rescue medication.
From november 2008 through may 2010, m. tuberculosis isolates were cultured during routine care of adults> 18 years of age with primary tb and no history of treatment . The patients were from 2 regional referral centers, the irkutsk regional tb - prevention dispensary and the research practice center for phthisiatry (yakutia); the study was approved by the institutional review boards at the university of virginia and irkutsk state medical university . Initial pretreatment isolates were grown on lowenstein - jensen agar slants and identified to species in accordance with world health organization recommendations . Drug susceptibility was tested by absolute concentration method on agar slants; drugs tested were rifampin (critical concentration 40 g / ml), isoniazid (1 g / ml and 10 g / ml), ethambutol (2 g / ml), streptomycin (10 g / ml), ethionamide (30 g / ml), and kanamycin (30 g / ml). Dna extraction was performed on all isolates, followed by 12-loci mycobacterial interspersed repetitive unit variable number tandem repeat (miru - vntr) analysis (7) and further lineage definition by region of difference deletions, or for ural strains as described (5). Phylogenetic tree construction was based on the miruvntrplus database (8), and vntr international type numbers were confirmed on the sitvit database (9). Dna from mdr isolates was amplified and sequenced for the known drug - resistance determining regions katg, inha, rpob, embb, gyra, rrs, and eis by using methods described by the centers for disease control and prevention (10). Sequences were compared with published sequences for m. tuberculosis h37rv by using genedoc version 2.7.0 . Among 235 patients with primary tb (130 from yakutia, 105 from irkutsk), isoniazid monoresistance was found in isolates from 16 (12%) from yakutia and 19 (18%) from irkutsk (p = 0.27). Multidrug resistance was found for 61 patients (36 [28%] from yakutia and 25 [24%] from irkutsk) (p = 0.55). Mean age (sd) for these 61 patients was 33 (12) years, 40 (66%) were male, and these characteristics did not differ significantly between patients from irkutsk and from yakutia . However, no hiv - infected patients were identified from yakutia compared with 11 (44% with mdr tb) from irkutsk (p<0.001). Twelve mdr tb patients from irktutsk died (outcome unknown for the other 13 patients), including all with hiv, compared with 4 (11%) from yakutia who died (p = 0.002). Follow - up varied and was limited mostly to inpatients . Among all 235 patients with primary tb, strains of the beijing family were significantly more common among those from irkutsk (70 [67%]) than from yakutia (40 [31%]) (p<0.001). However, strains found in yakutia (s 256 [11%], t 8 [7%], and ural 171 [5%]) were not found in irkutsk (table 1). The cluster of s 256 (miru profile 233325153325) was the most common among primary mdr tb isolates from yakutia and was fully 86% mdr (table 1; technical appendix). * miru - vntr, mycobacterial interspersed repetitive unit variable number tandem repeat (original 12-loci profile). Included genotypes found in> 5 isolates only; mit, miru vntr international type; mdr, multidrug - resistant tuberculosis (conventional resistance to isoniazid and rifampin); na, not applicable . Significance determined by analysis with yates correction or fisher exact test when appropriate . Among isolates from patients with primary mdr tb, 51 (84%) were available for dna sequencing: 27 from yakutia and 24 from irkutsk (table 2). Among isoniazid - resistant isolates, the mutation in codon 315 of katg was present in 91% . Among rifampin - resistant isolates, mutations in the resistance - determining region of rpob (codons 511533) were present in only 79% . The pnca mutation was common across genotypes from both sites, occurring in 62% of isolates amplified . Notably, both isolates with mutation in eis from yakutia occurred in mdr strains with the s 256 genotype and without rrs mutation . * all inha mutations were associated with a ser315thr mutation in katg except for 1 isolate in which katg did not amplify . Excluding 25 silent pnca mutations (ser32ser most common, n = 14). For all 3 mutations of eis associated with kanamycin resistance, rrs was wild type . In eastern siberia,> 25% of primary tb was mdr, equivalent to the highest proportion reported from the russian federation (2). However, regionally specific genotypic patterns and resistance mutations were identified . As expected, in irkutsk primary mdr tb was driven by strains of beijing lineage (5,6). Yet in the more geographically isolated population of yakutia, a strain previously unidentified in the russian federation, s 256, had a miru profile recently found among canadian aboriginal populations (11). In yakutia, s 256 was highly drug resistant and was the most common genotype among patients with primary mdr tb . Although rpob mutations were found in only 79% of rifampin - resistant isolates, these findings are consistent with those in a recent report from novosibirsk oblast, which similarly included non - beijing and s - family strains and found a sensitivity of only 63% for the rpob mutation (12). Lack of phenotypic correlation can result from alternate mechanisms of resistance or imperfect conventional susceptibilities in lowenstein - jensen medium or from use of old drug stock . Such discrepancy necessitates urgent clarification because substitution of conventional susceptibility testing with molecular probe based methods such as genexpert mtb / rif (cepheid, ca, usa) has been strongly advocated but would lead to dramatically different results and treatment regimens (13). Of note, isolates of the s 256 strain accounted for a proportion of the cases in which mutation in the promoter region of eis was associated with kanamycin resistance, but rrs was wild type . Commercial assays have focused on the rrs locus, which has greater sensitivity for amikacin, as the sole target for the class of injectable agents (14), yet in eastern siberia, the injectable agent available is kanamycin . Furthermore, we found a range of reported and unreported mutations across the entire pnca gene; most were point mutations resulting in amino acid substitution, but some strains had mutations that resulted in deletion or frameshift . Phenotypic methods and assays of functional pyrazinamidase activity should be performed in this region because results might have major implications for novel mdr tb drugs that work best with pyrazinamide (15). We were unable to obtain detailed clinical information about all patients with primary tb, thus preventing adequate comparison of nongenotypic risk factors for mdr tb or establishment of definitive epidemiologic links among clustered isolates . Furthermore, lack of conventional fluoroquinolone or pyrazinamide susceptibility testing limited comparison with gyra and pnca mutations, respectively . Despite these limitations, this work characterizes severe isoniazid monoresistant and mdr tb in eastern siberia among patients with no history of tb treatment . The regionally distinct phylogenetic patterns and certain drug - resistance mutations necessitate careful application of novel diagnostics and empiric therapeutic strategies . Phylogenetic trees of mycobacterium tuberculosis from patients with primary tuberculosis, yakutia and irkutsk, russian federation.
Studies over the past four decades have shown that 3.9%-16% of patients with pancreatic cancer (paca) have a family history of the disease, which is referred to as familial pancreatic cancer (fpc). Fpc describes families with at least two first - degree relatives with confirmed exocrine paca that do not fulfil the criteria of other inherited tumor syndromes with increased risks of paca, such as peutz - jeghers syndrome, hereditary pancreatitis, and hereditary breast and ovarian cancer . The first studies to explore what is now referred to as fpc were case reports of families in which there were multiple cases of paca . In 1973, macdermott and kramer described a family, in which paca developed in four out of the six siblings . A small fraction of this aggregation can be accounted for inherited cancer syndromes, including familial atypical multiple - mole melanoma (famm), peutz - jeghers syndrome, hereditary breast - ovarian cancer (hboc), hereditary pancreatitis (hp), and hereditary nonpolyposis colorectal cancer (hnpcc). These syndromes arise as a result of germline mutations in the cdkn2a (famm), stk11 (peutz - jeghers syndrome), brca1, brca2, atm (hboc), prss1 (hp), mlh1, and msh2 (hnpcc) genes . In addition, heredity plays an important role in certain patients with apparently sporadic paca . Many patients with pacas caused by a germline mutation in a cancer - causing gene do not have a pedigree suggestive of a familial cancer syndrome . Although much has been learned over the past few decades regarding the aggregation of paca in some families, the genetic basis for most of this familial aggregation is still poorly understood . Ten founder mutations in four cancer predisposing genes have been identified in poland [4 - 6]. These include three founder mutations in brca1 (5382insc, 4153dela, and c61 g), four in chek2 (1100delc, ivs2 + 1g> a, del5395, and i157 t), one variant allele in nbs1 (657del5), and two in palb2 (509_510delga and 172_175delttgt) [4 - 6]. The results of our previous case / control studies showed that these mutations in brca1, chek2, nbs1, and palb2 are associated with the cancer risk at different sites of origin [4 - 12]. A recent study evaluated the prevalence of these founder mutations among sporadic paca patients and showed that the risk of sporadic paca is associated with the slavic founder mutation of nbs1 657del5 (odds ratio [or], 3.80; p=0.002). A hereditary predisposition to different cancers, which is characteristic of the aforementioned genes, suggests that there could be a potential link between polish founder mutations and a predisposition to fpc . Therefore, the aim of this study was to establish the prevalence of ten polish founder mutations in brca1, chek2, nbs1, and palb2 among individuals from families with familial paca syndrome and to assess their possible association with the fpc risk in poland . A total of 400 individuals from fpc syndrome families were genotyped and the frequencies of the founder mutations were compared with a set of healthy control subjects representative of the polish population . The recruitment of individuals for this study was conducted between 2002 and 2014 . The majority (80%) were patients of our center, living in north - western poland, as well as in other cities of the country: biaystok, krakw, ld, olsztyn, opole, rzeszw, widnica, zielona gra, kielce . The study involved 400 probands from families with a diagnosis of fpc syndrome, who were either healthy individuals (n=295) or fpc patients with cancer (n=105). Table 1 lists the characteristics of the individuals included in the study . Each of the 400 study participants came from a family, in which there were two or more cases of paca among the first - degree relatives . The mean age of the participants enrolled in this study was 49.44 years (range, 19 to 91 years). A family history of cancer was taken either by the construction of a family tree or the completion of a standardized questionnaire . This study was conducted in accordance with the declaration of helsinki and all participants signed an informed consent document prior to donating the blood samples . Furthermore, this study was approved by the ethics committee of the pomeranian medical university in szczecin, poland . The first consisted of 2,000 newborn children from 10 hospitals throughout poland (szczecin, biaystok, gorzw, katowice, wrocaw, pozna, opole, ld, and rzeszw) collected between 2003 and 2004 . Samples of cord blood from unselected infants were forwarded to the study center in szczecin . The second group was taken from adult patient lists of three family doctors practicing in the szczecin region . A total of 1,000 controls were selected at random from the patient lists of these family doctors . The third group consisted of 1,000 adults from szczecin who submitted blood for paternity testing . A sample of dna was forwarded to the reference laboratory without any identifying information . To ensure comparability of the control groups, the allele frequencies of the 10 alleles was computed separately for the adult and neonatal control groups and compared to determine if there were any differences in allele frequencies . Ten founder mutations in the brca1, chek2, nbs1, and palb2 genes were genotyped, as described previously . Briefly, brca1 mutations, 4153dela and 5382insc were detected using the allele - specific oligonucleotide polymerase chain reaction (pcr), and c61 g was detected using the restriction fragment length polymorphism pcr . The ivs2 + 1g> a and i157 t variants in chek2 were detected using restriction fragment length polymorphism pcr analysis, and the 1100delc mutation was analyzed using allelespecific oligonucleotide pcr . The two recurrent truncating mutations of palb2 (509_510delga and 172_175delttgt) were detected using a taqman assay (life technologies, carlsbad, ca) using a lightcycler 480 real - time pcr system (roche life science, mannheim, germany). Sanger direct sequencing was undertaken to confirm the presence of mutations, using a bigdye terminator v3.1 cycle sequencing kit (life technologies), according to the manufacturer s protocol . All ten mutations were analyzed successfully in 398 of the 400 fpc cases (99.5%). The two that failed to be analyzed due to the poor quality of dna were discarded from statistical analysis . The ors were generated from two - by - two tables and the statistical significance was assessed using a chi - square or fisher exact test with a yates correction . The recruitment of individuals for this study was conducted between 2002 and 2014 . The majority (80%) were patients of our center, living in north - western poland, as well as in other cities of the country: biaystok, krakw, ld, olsztyn, opole, rzeszw, widnica, zielona gra, kielce . The study involved 400 probands from families with a diagnosis of fpc syndrome, who were either healthy individuals (n=295) or fpc patients with cancer (n=105). Table 1 lists the characteristics of the individuals included in the study . Each of the 400 study participants came from a family, in which there were two or more cases of paca among the first - degree relatives . The mean age of the participants enrolled in this study was 49.44 years (range, 19 to 91 years). A family history of cancer was taken either by the construction of a family tree or the completion of a standardized questionnaire . This study was conducted in accordance with the declaration of helsinki and all participants signed an informed consent document prior to donating the blood samples . Furthermore, this study was approved by the ethics committee of the pomeranian medical university in szczecin, poland . The first consisted of 2,000 newborn children from 10 hospitals throughout poland (szczecin, biaystok, gorzw, katowice, wrocaw, pozna, opole, ld, and rzeszw) collected between 2003 and 2004 . Samples of cord blood from unselected infants were forwarded to the study center in szczecin . The second group was taken from adult patient lists of three family doctors practicing in the szczecin region . A total of 1,000 controls were selected at random from the patient lists of these family doctors . The third group consisted of 1,000 adults from szczecin who submitted blood for paternity testing . A sample of dna was forwarded to the reference laboratory without any identifying information . To ensure comparability of the control groups, the allele frequencies of the 10 alleles was computed separately for the adult and neonatal control groups and compared to determine if there were any differences in allele frequencies . Ten founder mutations in the brca1, chek2, nbs1, and palb2 genes were genotyped, as described previously . Briefly, brca1 mutations, 4153dela and 5382insc were detected using the allele - specific oligonucleotide polymerase chain reaction (pcr), and c61 g was detected using the restriction fragment length polymorphism pcr . The ivs2 + 1g> a and i157 t variants in chek2 were detected using restriction fragment length polymorphism pcr analysis, and the 1100delc mutation was analyzed using allelespecific oligonucleotide pcr . The two recurrent truncating mutations of palb2 (509_510delga and 172_175delttgt) were detected using a taqman assay (life technologies, carlsbad, ca) using a lightcycler 480 real - time pcr system (roche life science, mannheim, germany). Sanger direct sequencing was undertaken to confirm the presence of mutations, using a bigdye terminator v3.1 cycle sequencing kit (life technologies), according to the manufacturer s protocol . All ten mutations were analyzed successfully in 398 of the 400 fpc cases (99.5%). The two that failed to be analyzed due to the poor quality of dna were discarded from statistical analysis . The ors were generated from two - by - two tables and the statistical significance was assessed using a chi - square or fisher exact test with a yates correction . A founder mutation in one of the four genes studied was observed in 44 out of 398 fpc cases (11.06%) and in 283 out of 4,000 controls (7.08%) (p=0.011; or, 1.56; 95% confidence interval, 1.12 to 2.18). Table 2 provides details of the prevalence of founder mutations studied among individuals from fpc families and in the population control group . In the group of all fpc individuals, the largest and statistically significant difference compared to the population control group was observed for palb2 172_175delttgt (0.5%; or, 10.05; p=0.048). When the genotyping results were classified according to the health status of the probands, statistically significant differences were observed for the brca1, palb2 and chek2 genes only in the group of fpc cases with cancer . Two (5382insc, c61 g) of the three polish founder brca1 mutations were detected in three of the 105 fpc cases with cancer (2.86%) (or, 6.72; p=0.006) compared to 17 of the 4,000 controls (0.42%). Palb2 mutations were observed in two of the 105 fpc cases with cancer (1.90%) (or, 9.52; p=0.014) compared to eight of the 4,000 controls (0.20%). Chek2 mutations (all four mutations combined) were detected in 14 of the 105 fpc cases with cancer (13.33%) (or, 2.26; p=0.008) compared to 236 of the 4,000 controls (5.9%). The chek2 i157 t missense mutation contributed the largest and was present in 11 of the 105 fpc cases with cancer (or, 2.17; p=0.026) compared to 193 of the 4,000 controls . A single nbs1 mutation (657del5) was detected in three of the 389 unselected fpc cases (0.75%): two healthy fpc probands (0.68%), and one of the 105 fpc cases with cancer (0.95%), compared to 22 of the 4,000 controls (0.55%), but none of the associations were statistically significant . This study observed a significant correlation between the prevalence of the polish founder mutation 172_175delttgt of palb2 gene and the fpc . In addition, an increased incidence of changes was observed in genes brca1 (three founder mutation combined: p=0.006; or, 6.72) and chek2 (four chek2 mutations combined: p=0.008; or, 2.26), particularly the chek2 i157 t mutation (p=0.03; or, 2.17) among the cancer - affected individuals from the fpc syndrome families . The fact that brca1 and palb2 mutations might be causative of fpc in a small subset of families is relatively new [14 - 16]. Furthermore, the results are consistent with the observations that palb2 and brca1 mutations are reported in fpc, especially in the families with a history of breast cancer . Breast cancer was also observed among the first degree relatives of one of the palb2 172_175delttgt and one of the brca1 5382insc mutation carriers from fpc . The most valuable observation of the present study is the increased risk of fpc among carriers of chek2 mutations, particularly the chek2 i157 t missense variant . Chek2 is a human homologue of the yeast cds1 and rad53 genes, which encodes the cell cycle checkpoint kinase . Therefore, chek2 plays an important role in the network of cell cycle regulation and dna damage repair, which are crucial processes in preventing cancer development . Four founder alleles of chek2 are present in poland; three of these result in a truncated chek2 protein (1100delc, ivs2 + 1g> a and del5395) and the fourth (i157 t) is a missense substitution of an isoleucine by a threonine in exon 3 . The feature of the founder mutations in the chek2 gene is the multiorgan cancer risk among carriers . Positive associations with protein - truncating alleles in the chek2 gene are observed for cancers of the prostate and thyroid . The large germline deletion in chek2 the missense variant, i157 t, is associated with an increased risk of colon, kidney, prostate, thyroid, and stomach cancers . In light of existing research, it can be assumed that chek2 gene mutations are more specific to a certain percentage of cancers of the gastrointestinal tract . In a study conducted at our center including 463 individuals, it has been shown that the i157 t variant of chek2 increases the risk of colorectal cancer among mismatch repair negative hnpcc / hnpcc - related families from poland (or, 2.1; p=0.0004). Polish founder mutations in the chek2 gene were also observed in 57 out of 658 unselected patients with gastric cancer (8.7%) compared to 480 out of 8,302 controls (5.8%) (or, 1.6; p=0.004). Overall, it can be concluded that fpc is another malignant tumor from the spectrum of digestive system cancers associated with mutations of the chek2 gene . The results of a previous study conducted among 383 paca cases did not show this association for sporadic cancers of the pancreas . The current relationship between the chek2 mutation and the occurrence of fpc can be explained by the multiorgan nature of the familial cancer predisposition of chek2 mutations . Note that among the fpc cancer cases with detected chek2 mutations, there were cases not only with paca, but also with cancer of the breast, cervix, thyroid, and melanoma . Thus far, the possible relationship between chek2 mutations and fpc has not been evaluated extensively . In one existing german study based on the genotyping of 35 patients from families with fpc syndrome, the chek2 1100delc mutation was identified in one out of 35 fpc cases (3%). The authors of that study suggested a possible contribution of the chek2 mutation in a small subset of fpc . In addition, it is believed that mutations of the chek2 gene, similar to the mutations in the other aforementioned genes, cause only a small percentage of fpc cases . Therefore, it can be assumed, that the search for genes that predispose to fpc, will bring to light new genes, but none of them will be responsible for a large percentage of families with fpc . Note that mutation carriers with cancer in the present study were affected by cancers at different sites (supplementary table 1). Among those, chek2 mutation carriers had the largest variety of different neoplasms, i.e., multiple breast cancers, cervical cancers, and single cases of thyroid cancer, paca, and malignant melanoma . Three brca1 carriers with cancer were observed, two of which had breast cancer and one ovarian cancer . These three brca1 mutation carriers had no other family member with breast and/or ovarian cancer . The nature of the chek2 gene as a multiorgan cancer predisposition gene has been reported [4,5,8 - 11], which explains the occurrence of cancer at different sites among the chek2 mutation carriers in the present study . Despite the single cases of different cancers in these families with paca, they only fulfil the criteria for fpc and did not meet the criteria for any other defined inherited tumor syndrome except for three carriers of a brca1 mutation (hboc, brca1 type). Our previous study conducted among 383 paca patients reported that the polish founder mutation in nbs1 (657del5) is associated with an increased risk of paca (or, 3.80; p=0.002). While the results of the current study, including almost 400 cases from fpc families presented here, did not show a relationship between the nbs1 and fpc syndrome . It might mean that a nbs1 mutation is not associated with the risk of fpc . On the other hand, it is equally possible that a relationship between the nbs1 and fpc risk was not observed because of the relatively small number of cases with cancer (n=105) present in this study, in addition to the low penetrance of nbs1 mutation . Indeed, more cases of cancer from fpc families should be analyzed in the future to resolve this suspicion . The major limitation of this study was the small number of cases with cancer from fpc families subjected to genotyping . These results should be verified with a larger series of fpc cases from both the polish and other populations . This case control study indicated that mutations in palb2 (172_175delttgt) appear to increase the risk of fpc in heterozygous carriers from the polish population . In addition, the fpc risk appears to be increased in carriers of brca1 (two of the three occurring: 5382insc, c61 g) and chek2 founder mutations (particularly chek2-i157 t missense variant). Therefore, in the polish population, founder mutations in the genes, brca1, palb2, and chek2, cause a small percentage of familial paca . Studies in slavic and other populations should be performed to confirm this association and evaluate the utility of testing these genes for fpc in cancer genetic counselling.
The earliest mammals were shrew - like creatures who could search for food and hide in places that were too cool and dark for many of their lizard contemporaries (1). In addition to being warm blooded, mammals also were adept at performing adaptive thermogenesis, giving them the ability to further increase their metabolic rates when conditions would otherwise result in hypothermia . From the perspective of humans in 2015, it is easy to forget that thermoregulation, in all of its dimensions, was a critical part of mammalian history thermogenesis comes from chemical reactions in which the liberated free energy is not captured in other molecules (e.g., atp or creatine phosphate) or used for work . The best known example of a heat - generating pathway is the futile cycle of proton pumping in brown fat and beige fat through the actions of uncoupling protein 1 (ucp1) (2,3). In cells expressing ucp1, the oxidation of lipids and carbohydrates results in the extraction of high - energy electrons, which flow down the electron transport chain (etc) as protons are pumped across the inner mitochondrial membrane (fig . The resulting electrochemical gradient across the inner mitochondrial membrane, which is usually coupled to atp synthesis via complex v of the etc, is dissipated by a leak of protons back across the inner membrane by ucp1 . Thus, much of the chemical energy generated by fuel oxidation in brown fat cells powers a futile proton cycle, which does no work and is instead liberated as heat . This process is typically thought of as being indirectly activated by cold, via the sympathetic nervous system (sns). Adaptive thermogenesis can also be activated directly by cold in beige adipocytes and by other stimuli that may signal independently from the -adrenergic receptors . The reducing equivalents generated by the tricarboxylic acid (tca) cycle enter the etc . This generates a proton gradient across the inner mitochondrial membrane . Instead of linking this gradient to atp synthesis via complex v, it is also worth noting that, from a thermodynamic perspective, there is nothing special about proton cycling, and such a thermogenic machine could, in theory, be built from other components of cellular metabolism . Indeed, some deep - diving fish have a heater organ, which is a specialized muscle that entirely lacks a myofibrillar apparatus . This organ, found between the eye and brain in deep - diving marlin and certain tuna, uses a futile cycle of calcium leaking from the sarcoplasmic reticulum and atp - dependent calcium uptake to raise the local temperature near the brain of the fish (4). In this case, the physiological goal is not to warm the whole body of the marlin, but to just make it a bit more astute than the other fish around it! Is ucp1 the only thermogenic pathway of importance in mammals? While no other pathway of thermogenesis except shivering has been convincingly demonstrated, suggestive data from the scientific literature hints that such pathways probably exist . If mice lacking ucp1 are abruptly switched from ambient temperatures to cold (4c), they develop life - threatening hypothermia (5). On the other hand, if they are gradually exposed to lower temperatures, they can survive quite normally at 4c (6). This suggests that there are compensatory thermogenic programs that can be activated as long as thermal stress is applied more gradually . In addition, there are examples of experiments in mice where certain mutations have caused very robust increases in energy expenditure and thermogenesis, but where the levels of ucp1 mrna and protein are unchanged . For example, deletion of par-1b / mark2 in mice results in increased energy expenditure and brown fat activation, although ucp1 protein levels were unchanged (7). Ucp1 brown fat has long been known to occur in two distinct anatomical locations in rodents . Developmentally formed depots, best typified by the interscapular and perirenal regions, are composed of tightly packed brown fat cells of relatively uniform appearance . These depots exist under most physiological conditions, although they may change in color and lipid content . On the other hand, ucp1 cells can accumulate in small pockets in white fat depots, especially in the subcutaneous adipose tissues, particularly when mice are exposed to long - term cold or stimuli with hormones such as catecholamines and other -adrenergic agonists (8). This pathway of adaptive thermogenesis is robustly activated by cold via an indirect pathway mediated by the sympathetic nervous system . However, fat cells are also able to directly sense cold, though the mechanism is not well understood (9). In both anatomical situations, brown fat cells have multiple small lipid droplets, numerous mitochondria, and rich innervation and vascularization . Despite these similarities, it is now clear that the classical brown fat cells and the inducible beige fat cells come from different developmental lineages and are, in fact, distinct cell types . The key finding in this regard was the observation that the classical developmentally formed brown fat arises from a myf5 lineage shared with skeletal muscle (10). Consistent with this, primary cultures of classical brown fat express low levels of certain genes characteristic of skeletal muscle (11). Subsequent work indicated that the brown fat / skeletal muscle decision occurs between 9.5 and 12.5 days of gestation in mice (12). Conversely, the ucp1 cells induced in subcutaneous white fat depots by cold or -adrenergic agonists come from a myf5 lineage (10). These beige fat cells have now been cloned and express a relatively low level of thermogenic genes, such as ucp1 and type 2 deiodinase in the basal state, but can induce these genes to levels essentially equivalent to classical brown fat cells when given hormonal stimuli (13). Moreover, the beige and brown fat cells have gene signatures that allow for distinction of these cells types; however, their comparative physiological properties and overall roles in metabolism are not completely understood . It is now clear that thermogenic adipocytes, taken as a whole, contribute very importantly to metabolic homeostasis in rodents . The partial ablation of ucp1 cells through the transgenic expression of a toxigene led to these animals being more susceptible to obesity and diabetes (14). Similarly, mice lacking ucp1 through targeted mutation had an increased body weight and fat content, at least when raised at thermoneutrality (15). Excessive shivering apparently prevented obesity when these experiments were performed at ambient temperatures . The first transgenic model with increased brown and beige fat was the adipose - selective expression of foxc2, a transcription factor that activated camp metabolism in recipient cells (16). These mice, which were resistant to diet - induced obesity and diabetes, had expansions of both the classical brown and beige fat . Deciphering the individual roles of brown and beige fat cells prd1-bf-1-riz1 homologous domain - containing protein-16 (prdm16) is an important transcriptional coregulator in both brown and beige fat, but when its expression was elevated through a promoter expressed in all fat depots (ap2), phenotypic changes were observed in the erstwhile white fat depots, which developed copious pockets of beige fat cells (17,18). The classical brown fat, which expresses very high levels of prdm16 in the basal state, showed few or no changes with a further elevation in prdm16 expression . But this occurred only when the transgenic mice also received stimulation with a -adrenergic agonist . While studies in cultured cells had shown that classical brown fat cells require prdm16 to develop and maintain a thermogenic gene program, ablation of prdm16 from all fat cells in vivo was not sufficient to significantly alter the function of the classical brown fat . On the other hand, the development of beige fat cells was severely reduced, at both the histological and molecular level (19). These mice developed a moderate obesity, compared with control animals, with an unusual expansion of the subcutaneous fat depots . These beige fat deficient mice also showed a rather profound hepatic insulin resistance that was associated with liver steatosis . One last point of interest is that the subcutaneous fat from the prdm16-ablated mice showed the presence of more inflammatory macrophages and increased the expression of proinflammatory genes . The latter aspect was also observed with cultured subcutaneous cells lacking prdm16, indicating that this transcriptional coregulator plays a role in the relative resistance of subcutaneous fat to inflammation and is an important determinant of subcutaneous versus visceral fat phenotype . The role of prdm16 in the classical brown fat in vivo is interesting and somewhat more complex . Ablation of this factor with a myf5-driven cre recombinase causes a loss of brown fat function and gene expression, but only at 6 months of age (20). However, ablation of prdm16 along with its closest homolog, prdm3, causes an early and severe loss of brown fat thermogenic gene expression and normal histology . Thus, it appears that both thermogenic cell types depend on prdm16, but, at least in early life, the classical brown fat has a second factor that can support development, prdm3 . Harnessing the therapeutic potential of brown and beige fat requires a detailed understanding of the molecular mechanisms responsible for the determination and maintenance of each cell type . The nuclear hormone receptor peroxisome proliferator activated receptor (ppar) is necessary and sufficient for the development of all fat cells (21). However, white and brown adipocytes have drastically different phenotypes, indicating that other transcriptional regulators must be involved . A yeast two - hybrid screen identified ppar coactivator-1 (pgc-1) as a cold - inducible binding partner of ppar (22). In brown fat pgc-1 is now appreciated to regulate numerous physiological processes in a variety of metabolically important tissues . While brown and beige fat cells lacking pgc-1 have significantly blunted thermogenic gene expression, these cells still retain the molecular signature of brown fat cells (23,24). The transcriptional regulator prdm16 was found to be highly enriched in brown fat, whereas it is virtually undetectable in visceral white fat cells . Forced expression of prdm16 in cultured white fat cells induces pgc-1 and thermogenic genes, as well as mitochondrial genes and brown fat identity genes (17). In addition to activating these pathways, prdm16 also binds ccaat / enhancer binding protein (c / ebp) and recruits the corepressor proteins ctbp1 and ctbp2 to repress white fat or muscle gene expression (25). Importantly, the expression of prdm16 and its binding partner c / ebp in fibroblasts is sufficient to promote differentiation into functional brown fat cells, which can be detected by [f]-2-fluoro-2-deoxy - d - glucose ([f]-fdg) positron emission tomography (pet) when transplanted into mouse models (26). An increasing number of transcriptional regulators has been identified as important in brown and beige fat biology . The transcription factor early b - cell factor-2 (ebf2) is enriched in brown relative to beige adipocytes . It functions upstream of prdm16 to promote binding of ppar to the promoters of brown - selective genes (27). Ehmt1 is a histone lysine methyltransferase that purifies with the prdm16 transcriptional complex in brown fat . Ehmt1 is required for brown fat lineage specification and for thermogenesis (28). Presumably, there are also beige - selective factors upstream of prdm16 and beige - selective epigenetic regulators that modulate the phenotype of these cells . It is able to compete with prdm16 for binding to ppar, and thereby modulates white versus brown / beige phenotype (29,30). Forced expression of tle3 in adipocytes results in impaired thermogenesis, while deletion enhances thermogenesis in brown and beige fat . A number of other factors have also been shown to play roles in this biology, but a comprehensive review is beyond the scope of this article . Until fairly recently, brown fat was thought to be present in meaningful amounts only in human infants and small mammals . The combination of insufficient hair, fur, and insulation, along with a high body surface area - to - mass ratio make babies and small mammals particularly susceptible to hypothermia . As a result, they have developed significant interscapular brown fat, which defends body temperature by adaptive thermogenesis . This brown fat was thought to regress by adulthood, unless exposed to catecholamine excess (as in pheochromocytoma) or long - term cold exposure (as in outdoor workers in cold climates). In 2009, these studies made use of [f]-fdg pet to confirm that adults have glucose - avid tissue with imaging characteristics of adipose in deposits in the supraclavicular and spinal regions . As assayed by [f]-fdg pet, the amount of active tissue is inversely associated with bmi, and this activity is increased by cold exposure . Follow - up studies (34) have shown that this tissue has the histological appearance of adipose tissue and expresses several molecular markers of brown fat including ucp1 . Current efforts are focused on developing optimal imaging modalities to detect this tissue and to quantify its absolute amounts and activity . This is particularly important since brown and beige fat also oxidize lipids, which would not be detectable by [f]-fdg pet technology . Research has increasingly suggested that adult human brown fat shares features of the inducible beige fat described in rodents . Brown and beige fat cell lines have now been cloned from mice, allowing for the identification of unique molecular markers of each cell type . Multiple studies (13,35,36) have shown that beige - selective markers are expressed more in human brown fat samples than are markers of the classical brown fat of rodents . A very recent study (37) characterized a human brown fat cell line and showed that it actually shares more molecular features with beige than brown adipocytes . However, the brown fat in human infants, located in the interscapular region, seems most similar to the interscapular classical brown fat in rodents . In some adult humans, cells with both brown and beige characteristics have been identified, with the depth in the neck seeming to be a determinant of relative brown versus beige character (36,38,39). While further studies will clarify this question, this tissue may well contain both brown and beige cells in varying amounts in each individual . As a deeper understanding of brown and beige fat has emerged, researchers have turned to strategies to induce the activity of these tissues as a possible therapy for obesity and metabolic diseases . Calculations have suggested that maximal cold - induced brown fat thermogenesis would be between 25 and 400 kcal / day in lean healthy volunteers (40). Given the inducibility of beige fat, activating these cells could result in even more substantial effects . However, the greatest benefits may not relate to increased energy expenditure per se, but rather might result from enhanced glucose and lipid disposal . In addition to taking up and consuming glucose (3133), brown adipose tissue takes up free fatty acids from triglyceride - rich lipoproteins . Cold exposure in mice upregulates this pathway, resulting in accelerated plasma triglyceride clearance (41). While compounds like dinitrophenol can result in impressive weight loss, they have also been associated with complications, including fatal hyperthermia . While this has understandably resulted in caution, it is possible that more specific uncouplers or targeted activators of ucp1 or other futile cycles may provide metabolic benefit with an acceptable safety profile . In that regard, a recent publication (42) described the development of controlled - release mitochondrial protonophores that can apparently safely uncouple in the liver, and in rats these compounds can improve insulin resistance, diabetes, hypertriglyceridemia, and hepatic steatosis . Brown and beige fat activity is robustly induced by thyroid hormone and catecholamines, but side effects would almost certainly limit their use . The 3-selective adrenergic agonist cl 316,243 potently activates brown and beige fat in rodents (43). This drug was recently shown to activate human brown fat, which may renew interest in this pathway (44). Since cold robustly activates brown and beige fat, some investigators have suggested that moderate cold exposure could be used as a therapeutic approach . At least in healthy subjects, daily exposure to 19c for 2 h was sufficient to activate brown fat, resulting in weight loss (45). In another small human study (46) while these studies suggest the potential of cold as a treatment modality, our societal preference for thermal comfort may make this unfeasible as a broad approach . Thiazolidinediones (tzds) cause browning of the white fat and may do so by stabilizing prdm16, resulting in its accumulation (47). However, these drugs have been associated with weight gain, fluid retention, and cardiovascular events, which have diminished enthusiasm for their use . Deacetylation of ppar by sirt1 promotes browning of the white fat, suggesting the possibility of developing compounds that selectively modulate this pathway (48). Other regulators of interest include bone morphogenetic protein 7 (bmp7) and bmp8b, cyclooxygenase-2 (cox-2), and natriuretic peptides, though their pleiotropic actions might limit their potential as drug targets . Fibroblast growth factor 21 (fgf21) is being examined as a therapeutic agent, but it also has diverse actions and may be associated with bone loss . Translating these discoveries into drug candidates will ultimately require a more complete understanding of brown / beige fat biology in humans . Mouse studies suggest that prdm16 is a key control point in brown and beige fat phenotype . An increasing understanding of the relevant modifications of prdm16 and the upstream signaling pathways involved may provide new opportunities for therapeutic targeting of brown and beige fat.
In a recent issue of critical care, meyer and colleagues report interesting surveillance data from icus in 30 german hospitals, based on a large amount of microbiology and pharmacy data gathered between 2001 and 2008 . One of the main study findings is a decreasing rate of methicillin - resistant staphylococcus aureus (mrsa) and a dramatic increase of third generation cephalosporin (3gc) resistant enterobacteriaceae over the study period . In recent years, most european countries have succeeded in reducing the burden of disease caused by mrsa . This progress has, however, been partly off set by the increase across europe in the prevalence of multiresistant gram - negative pathogens producing extended - spectrum betalactamases (esbls) or carbapenemases . The current study confirms this worrisome finding and also shows an increase in carbapenem, 3gc and fluoroquinolone use in german icus with a stable volume of overall antibiotic use . The latter two antibiotic classes have been repeatedly identified as risk factors for carriage of multidrug resistant gram - negatives . Carbapenems on the other hand are first - line drugs for the treatment of infections due to esbl - producing bacteria . It is tempting to assume that overuse of fluoroquinolones and 3gc antibiotics contributed to the observed increase in esbl producers, which subsequently increased carbapenem use . The ecological nature of the data, however, makes it difficult to infer clear cause - and - effect relationships, as does the failure to differentiate between hospital- and community - acquired isolates and clinical versus surveillance cultures . In addition, the analysis of trends is hampered by variation in the number of participating icus over time . Nevertheless, the increased burden of multidrug - resistant gram - negative bacteria is a real phenomenon . In contrast to mrsa, esbl producers - notably strains of escherichia coli carrying plasmids of the ctx - m family - are mostly imported from the community into the hospital . Assuming a relationship with antibiotic use in animals and subsequent transmission of antibiotic resistant e. coli via the food chain is alluring, but strong epidemiological evidence is still lacking . The recently started european union - funded saturn project (impact of specific antibiotic therapies on the prevalence of human host resistant bacteria) will gather more information on these risk factors . As mentioned above, antibiotic overuse in humans probably plays a central role in the spread of esbl producers . In the context of this study it is noteworthy that germany has a relatively high level of fluoroquinolone use in the community . As to antibiotic use in hospitals, the overall quantity of antibiotic use in german icus is comparable to that of other countries . Although there is important heterogeneity between icus, inappropriate antibiotic use is still common in germany (as in many other countries), where infectious diseases as a stand - alone speciality and antibiotic stewardship programmes are still underdeveloped . Compared to other european countries or highly publicised health threats, such as bioterrorism and swine flu, public awareness and political commitment to control multidrug - resistant microorganisms has been slow to rise in germany . Only recently (2009) has a german reference centre for surveillance of gram - negative bacteria been established, which focuses, among other things, on detection of carbapenemase producing bacteria . A first outbreak of carbapenemase producing klebsiella pneumoniae in germany has recently been reported, probably linked to an index patient with previous healthcare contact in greece . The fact that two icus in 2008 reported carbapenem resistant k. pneumoniae in the current study raises the concern that these strains might already be more common in central europe than previously assumed since detection of these strains may be difficult with routine laboratory techniques . With regard to esbl - producers the growing community reservoir makes it unlikely that we will be able to control the spread by conventional measures targeted at nosocomial infection control . The promotion of prudent antibiotic use in the community and animal husbandry should therefore be a key priority . As to carbapenemase - producing enterobacteriaceae, early identification of these strains and aggressive infection control measures seem essential . Examin ing novel decolonization strategies for gram - negative enterobacteriacae might be a further strategy worth evaluating . If we manage to enforce all these measures, we will hopefully be able to think positive again - even with regard to gram - negatives . 3gc: third generation cephalosporin; esbl: extended - spectrum betalactamase; mrsa: methicillin - resistant staphylococcus aureus . Preparation of this commentary was supported in part by the 6th and 7th framework programme of the european community in the context of the projects' changing behaviour of health care professionals and the general public towards a more prudent use of anti - microbial agents' (champ, contract sp5a - ct-2007 - 044317) and' impact of specific antibiotic therapies on the prevalence of human host resistant bacteria' (acronym saturn, agreement fp7-health-2009-n241796).
Primary malignant tumors of the hypopharynx are predominantly squamous cell carcinomas (sqccs), as primary small - cell carcinoma (smcc) of the hypopharynx is rare . Combined primary smcc and sqcc of the hypopharynx, referred to as composite tumor of the hypopharynx, is even more rare, with only 3 cases previously reported (13). We herein report a the case of a patient with primary combined smcc with an sqcc element and investigate the expression of specific proteins for molecular - targeted therapy . A 74-year - old man, with a ~50-year history of excessive alcohol consumption and smoking, presented with a 3-month history of throat pain and hoarseness . On hypopharyngoscopy, a tumor was identified in the right anterior wall of the piriform sinus (fig . A pathologist analyzed the biopsy sample and diagnosed the lesion as sqcc, as no smcc component was identified in the biopsy specimen . The right vocal cord was not fixed . A contrast computed tomography (ct) scan of the neck revealed a heterogeneously enhanced tumor sized 251738 mm extending through the right piriform sinus (fig . Moreover, fluorodeoxyglucose positron emission tomography (fdg - pet) revealed high - level accumulation in the primary tumor, with a maximum standardized uptake value of 14.6 . There was no evidence of cervical lymph node metastasis, primary lung tumor, or distant metastasis . The patient was diagnosed with hypopharyngeal cancer classified as t2n0m0, according to the 2009 union for international cancer control staging system (4). We performed a tracheostomy, total laryngectomy, right hemithyroidectomy and bilateral lateral neck dissection (level ii microscopically, two components were identified in the lesion, namely a poorly differentiated sqcc and an smcc containing chromatin - rich nuclei with scanty cytoplasm (fig . Pathologically, multiple metastatic cervical lymph nodes were identifed bilaterally (right side, levels ii and iii; and left side, level iii). However, after the first course, adjuvant chemotherapy was discontinued due to prolonged bone marrow suppression, although the therapy was effective . However, despite treatment, distant metastases in the thoracic vertebrae (t5, 9 and 10) were confirmed by ct 6 months later . The patient's condition started to deteriorate and he succumbed to the disease 7 months after the initial treatment . On gross examination of the laryngectomy specimen, the tumor extended superiorly to involve the right supraglottis and exhibited variable gross morphology, ranging from ulcerated, to nodular, to plaque - like growth . The tumor infiltrated 1.3 cm in depth, but did not involve the thyroid cartilage, vocal cord, or trachea . Microscopic sections from the larynx revealed a combined tumor, composed predominantly of smcc localized in the upper side and sqcc localized in the lower side (figs . 1 and 2a). We observed 2 components in the lesion: a poorly differentiated sqcc and a small - sized smcc with finely granular, hyperchromatic nuclei, inconspicuous nucleoli and scanty cytoplasm . The metastatic potential of the lesion was further supported by the smcc component found to infiltrate lymphatic vessels (fig . Diffuse cd56 positivity in the smcc component in the cervical lymph nodes supported the histological diagnosis (fig . The sqcc component was negative for neuroendocrine markers, including cd56, synaptophysin and chromogranin a, although it was positive for ck 34e12 (fig . Immunoreactivity to ki-67 was seen in the nuclei of tumor cells . In the smcc component, the ki-67 labeling index was 50.2%, while that in the sqcc component was 47.5% (fig . To predict the prognosis of this patient, we evaluated the expression of epidermal growth factor receptor (egfr). Furthermore, to evaluate the possibility of using molecular - targeted therapy for combined smcc and sqcc, the expression of proteins such as platelet - derived growth factor receptor (pdgfr), vascular endothelial growth factor receptor 2 (vegfr2) and kit was examined . On immunohistochemical analysis, the smcc element was strongly positive for kit and pdgfr; however, egfr and vegfr2 were not expressed (fig . The sqcc element was mildly positive for pdgfr and egfr; however, kit and vegfr2 were not expressed (fig . Genomic dna was extracted from frozen tumor specimens using the qiaamp dna mini kit (qiagen, hilden, germany), according to the manufacturer's protocol . A molecular genetic analysis of kit (exons 9, 11, 13 and 17) and pdgfr (exons 12, 14 and 18) was performed using the polymerase chain reaction (pcr) direct sequencing method, as previously reported (58). Pcr products were extracted and subjected to a computed automatic dna sequencing (abi prism 3100 genetic analyzer; applied biosystems, carlsbad, ca, usa). Analysis of exon 12 of pdgfr from other normal tonsillar tissues revealed a similar ag change at the 1849 nucleotide position . Comparison of this sequence variation with the single - nucleotide polymorphism (snp) database revealed the presence of a known snp at codon 567 of pdgfr (9). The hpv status was determined using the hpv typing set (takara bio ., tokyo, japan), a pcr primer set specifically designed to identify hpv genotypes 16, 18, 31, 33, 35, 52 and 58 in genomic dna . The pcr products were separated by electrophoresis through a 9% polyacrylamide gel and stained with ethidium bromide . Our case was negative and not considered as a high - risk hpv status (fig . On gross examination of the laryngectomy specimen, a mass involving the right piriform sinus was palpable . The tumor extended superiorly to involve the right supraglottis and exhibited variable gross morphology, ranging from ulcerated, to nodular, to plaque - like growth . The tumor infiltrated 1.3 cm in depth, but did not involve the thyroid cartilage, vocal cord, or trachea . Microscopic sections from the larynx revealed a combined tumor, composed predominantly of smcc localized in the upper side and sqcc localized in the lower side (figs . 1 and 2a). We observed 2 components in the lesion: a poorly differentiated sqcc and a small - sized smcc with finely granular, hyperchromatic nuclei, inconspicuous nucleoli and scanty cytoplasm . The metastatic potential of the lesion was further supported by the smcc component found to infiltrate lymphatic vessels (fig . Diffuse cd56 positivity in the smcc component in the cervical lymph nodes supported the histological diagnosis (fig . The sqcc component was negative for neuroendocrine markers, including cd56, synaptophysin and chromogranin a, although it was positive for ck 34e12 (fig . Immunoreactivity to ki-67 was seen in the nuclei of tumor cells . In the smcc component, the ki-67 labeling index was 50.2%, while that in the sqcc component was 47.5% (fig . Furthermore, to evaluate the possibility of using molecular - targeted therapy for combined smcc and sqcc, the expression of proteins such as platelet - derived growth factor receptor (pdgfr), vascular endothelial growth factor receptor 2 (vegfr2) and kit was examined . On immunohistochemical analysis, the smcc element was strongly positive for kit and pdgfr; however, egfr and vegfr2 were not expressed (fig . The sqcc element was mildly positive for pdgfr and egfr; however, kit and vegfr2 were not expressed (fig . Genomic dna was extracted from frozen tumor specimens using the qiaamp dna mini kit (qiagen, hilden, germany), according to the manufacturer's protocol . A molecular genetic analysis of kit (exons 9, 11, 13 and 17) and pdgfr (exons 12, 14 and 18) was performed using the polymerase chain reaction (pcr) direct sequencing method, as previously reported (58). Pcr products were extracted and subjected to a computed automatic dna sequencing (abi prism 3100 genetic analyzer; applied biosystems, carlsbad, ca, usa). Analysis of exon 12 of pdgfr from other normal tonsillar tissues revealed a similar ag change at the 1849 nucleotide position . Comparison of this sequence variation with the single - nucleotide polymorphism (snp) database revealed the presence of a known snp at codon 567 of pdgfr (9). Tokyo, japan), a pcr primer set specifically designed to identify hpv genotypes 16, 18, 31, 33, 35, 52 and 58 in genomic dna . The pcr products were separated by electrophoresis through a 9% polyacrylamide gel and stained with ethidium bromide . Our case was negative and not considered as a high - risk hpv status (fig . On gross examination of the laryngectomy specimen, a mass involving the right piriform sinus was palpable . The tumor extended superiorly to involve the right supraglottis and exhibited variable gross morphology, ranging from ulcerated, to nodular, to plaque - like growth . The tumor infiltrated 1.3 cm in depth, but did not involve the thyroid cartilage, vocal cord, or trachea . Microscopic sections from the larynx revealed a combined tumor, composed predominantly of smcc localized in the upper side and sqcc localized in the lower side (figs . 1 and 2a). We observed 2 components in the lesion: a poorly differentiated sqcc and a small - sized smcc with finely granular, hyperchromatic nuclei, inconspicuous nucleoli and scanty cytoplasm . The metastatic potential of the lesion was further supported by the smcc component found to infiltrate lymphatic vessels (fig . Diffuse cd56 positivity in the smcc component in the cervical lymph nodes supported the histological diagnosis (fig . The sqcc component was negative for neuroendocrine markers, including cd56, synaptophysin and chromogranin a, although it was positive for ck 34e12 (fig . Immunoreactivity to ki-67 was seen in the nuclei of tumor cells . In the smcc component, the ki-67 labeling index was 50.2%, while that in the sqcc component was 47.5% (fig . Furthermore, to evaluate the possibility of using molecular - targeted therapy for combined smcc and sqcc, the expression of proteins such as platelet - derived growth factor receptor (pdgfr), vascular endothelial growth factor receptor 2 (vegfr2) and kit was examined . On immunohistochemical analysis, the smcc element was strongly positive for kit and pdgfr; however, egfr and vegfr2 were not expressed (fig . The sqcc element was mildly positive for pdgfr and egfr; however, kit and vegfr2 were not expressed (fig . Genomic dna was extracted from frozen tumor specimens using the qiaamp dna mini kit (qiagen, hilden, germany), according to the manufacturer's protocol . A molecular genetic analysis of kit (exons 9, 11, 13 and 17) and pdgfr (exons 12, 14 and 18) was performed using the polymerase chain reaction (pcr) direct sequencing method, as previously reported (58). Pcr products were extracted and subjected to a computed automatic dna sequencing (abi prism 3100 genetic analyzer; applied biosystems, carlsbad, ca, usa). Analysis of exon 12 of pdgfr from other normal tonsillar tissues revealed a similar ag change at the 1849 nucleotide position . Comparison of this sequence variation with the single - nucleotide polymorphism (snp) database revealed the presence of a known snp at codon 567 of pdgfr (9). Tokyo, japan), a pcr primer set specifically designed to identify hpv genotypes 16, 18, 31, 33, 35, 52 and 58 in genomic dna . The pcr products were separated by electrophoresis through a 9% polyacrylamide gel and stained with ethidium bromide . Our case was negative and not considered as a high - risk hpv status (fig . In the head and neck, smcc most commonly arises in the larynx, but has also been reported in the sinonasal tract and salivary glands (10,11). Combined smcc of the larynx has only been reported in 17 cases in the literature to date . Moreover, the majority of the patients succumbed to the disease within 2 years of diagnosis, even with adjuvant radiation and chemotherapy (1214). Despite the rarity of these tumors, the clinical behavior of combined smcc of the larynx appears to be similar to that of the hypopharynx . Metastasis from a primary lung smcc must be carefully distinguished from a primary hypopharyngeal or laryngeal smcc by imaging studies of the lung (15). In this case, fdg - pet revealed high - level accumulation in the hypopharynx, without evidence of a lung tumor or distant metastasis . A chest contrast ct scan confirmed the absence of a primary lung tumor . According to the world health organization classification of head and neck tumors in 2005, neuroendocrine carcinomas may be classified as typical carcinoid, atypical carcinoid, and smcc (16). Neuroendocrine types of smcc associated with an sqcc component are referred to as combined carcinomas . Neuroendocrine neoplasms of the hypopharynx and larynx constitute a morphologicaly heterogeneous group of tumors, with considerable differences in clinical behavior and very different treatment strategies (12). Combined smcc of the hypopharynx is extremely rare and is often difficult to pathologically diagnose prior to surgical treatment . Moreover, the prognosis is very poor due to early metastasis, without established treatment regimen (3). Of the 4 reported cases of combined smcc of the hypopharynx, including our case, 3 succumbed to the disease within 1 year (table i). Multi - modality therapy should be performed for combined smcc of the head and neck region . The present case reports new findings: positive expression of kit and pdgfr in the smcc element and positive expression of egfr and pdgfr in the sqcc element . Furthermore, to the best of our knowledge, the present study is the first to report a kit and pdgfr mutation analysis in combined hypopharyngeal smcc . Kit and pdgfr, both mapped to chromosome 4q12, have structural similarities with other pdgfr family members . A majority of gastrointestinal stromal tumors (gists) display a gain - of - function mutation in the kit proto - oncogene that encodes the kit protein . The majority of mutations occur in kit exon 11, but mutations may also be found in exon 9 and rarely in exons 13 and 17 . The pdgfr gene is also very similar to the kit gene, and pdgfr mutations have been found in exons 12, 14 and 18 . Terada et al (18) reported kit protein expression in 100% and pdgfr expression in 65% of small - cell lung carcinoma cases; however, there were no genetic mutations of kit and pdgfr in small - cell lung carcinoma (18). In a previously reported case of esophageal combined smcc, kit and pdgfr were expressed in the smcc component, but not in the sqcc component (5). It was found that smcc of the lung and extrapulmonary organs expressed kit and pdgfr, but there were no mutations in these genes within the hot spots of gists . No biomarker has been yet proven to predict response to targeted therapy in either smcc or sqcc . Moreover, a predictive biomarker in one cancer type may not be helpful in another cancer type, suggesting that different mechanisms may be involved in combined smcc . To the best of our knowledge, a molecular - targeted therapy for treating combined smcc of the hypopharynx and larynx has not yet been reported . Our case is the first to report kit and pdgfr expression in combined smcc in the hypopharynx and larynx; if similar results are obtained from larger series, these data may have management and prognostic implications through the possible use of targeted biological therapy in these tumors . At present, smcc treatment remains a significant challenge for the oncologists, with several targeted agents under evaluation . However, there is no effective molecular - targeted therapy for smcc . Evaluating the expression of the kit and pdgfr genes within hypopharyngeal smcc may facilitate the development of novel agents for the molecular - targeted therapy of this tumor . However, further investigations are necessary regarding these proteins expression and clinical application of molecular targeted therapy for hypophageal combined smcc.
Cells and viruses: bovine testicle (bt) cells were grown in eagle s minimum essential medium (emem) (nissui pharmaceutical, tokyo, japan) supplemented with 0.295% tryptose phosphate broth (tpb) (becton dickinson, san jose, ca, u.s.a .) And 5% fetal bovine serum (fbs) (mitsubishi chemical, tokyo, japan). Bovine kidney cell line mdbk - hs and porcine kidney cell line sk - l were grown in emem supplemented with 0.295% tpb, 10% horse serum (hs) (life technologies, carlsbad, ca, u.s.a .) And 10 mm n, n - bis (2-hydroxyethyl)-2-aminoethanesulfonic acid (bes) (sigma - aldrich, st . Bovine fetal muscle (bfm) cells were grown in emem supplemented with 0.295% tpb, 5% fbs, 5% hs and 10 mm bes . Cells were confirmed to be free from bvdv, and fbs was confirmed to be free from both bvdv and anti - bvdv neutralizing antibodies . The bvdv gbk strain is an adventitious bvdv2 isolated from cells of the bovine kidney cell line gbk . The ndv miyadera strain was propagated in 10-day - old embryonated hens eggs . The new jersey serotype strain of vsv was grown in sk - l cells . Cloning of a pair of end and end viruses from viral stocks: a reverse plaque formation technique was used to obtain a pair of end and end viruses from viral stocks as described previously . Sequencing: bvdv gbk_e and gbk_e strains, the full - length cdna clones and the in vitro - rescued viruses were completely sequenced as described previously . In brief, the nucleotide sequences of cdna clones and pcr fragments from viral rna were determined using the bigdye terminator v3.1 cycle sequencing kit (life technologies) and a 3130 genetic analyzer or a 3500 genetic analyzer (life technologies) according to the manufacturer s protocol . Sequencing data were analyzed using genetyx version 10 software (genetyx, tokyo, japan). Plasmid constructs: the cdna fragments from the gbk_e strain, obtained by reverse transcription polymerase chain reaction (rt - pcr), were cloned into plasmid pcr - blunt ii - topo (life technologies) using the zero blunt topo cloning kit (life technologies). The cdna sequence was flanked by a modified t7 promoter sequence at the 5 end and a pst i restriction site at the 3 end . Subclones were assembled into a full - length cdna clone, termed pgbk_e, by replacing the csfv genome of the full - length cdna clone of the csfv alfort187 - 1 strain pa187 - 1 with the genome of the gbk_e strain using appropriate restriction enzymes and the in - fusion hd cloning kit (clontech, mountain view, ca, u.s.a .) According to the manufacturer s protocol . The full - length cdna clone pgbk_e was transformed and propagated in competent cell stbl3 cells (life technologies) and purified using the qiagen plasmid plus midi kit (qiagen, hilden, germany) according to the manufacturer s protocol . G, i2623v, d3148 g and d3502y were constructed in the pgbk_e backbone using the quickchange lightning multi mutagenesis kit (agilent technologies, santa clara, ca, u.s.a .) And the in - fusion hd cloning kit . The plasmid pgbk_e / d136 g has a single amino acid substitution at position 136 of gbk_e, whereas the plasmids pgbk_e / i2623v; d3148 g; d3502y and pgbk_e / d136 g; i2623v; d3148 g; d3502y have multiple amino acid substitutions at positions 2623, 3148 and 3502 of gbk_e and at positions 136, 2623, 3148 and 3502 of gbk_e, respectively . Full - length pcr amplification, in vitro rna transcription, transfection and viral recovery: the cdna - derived viruses were rescued as described previously with some modifications . The full - length genome amplification strategy was employed to obtain a full - length pcr amplicon for in vitro rna transcription . The cdna clones were amplified using primers 5gbke_t7 (5-taa tac gac tca cta ta gta tac gag att agc taa agt act cg 3, t7 promoter sequence underlined) and 3gbke (5- ggg gct gtt aga ggc atc ctc tag tc 3) with accuprime taq dna polymerase high fidelity (life technologies). Viral rna was transcribed in vitro from the purified full - length pcr amplicon using the megascript t7 kit (life technologies), the remaining pcr amplicon was digested using dnase (life technologies), and the viral rna was then purified with a microspin s-400 column (ge healthcare, buckinghamshire, u.k . ). Mdbk - hs cells (3 10 cells) were transfected with 10 g of viral rna in a 0.4 cm cuvette using the gene pulser xcell electroporation system (bio - rad, hercules, ca, u.s.a .) The cells were incubated at 37c in 5% co2 for 3 days, and the supernatants were then transferred onto naive mdbk - hs cells to obtain infectious viruses . The viruses were named according to the plasmid from which they were rescued, replacing the cdna - derived viruses generated in the present study are shown in fig . 1.schematic representation of the genomic differences between gbk_e virus and gbk_e virus, and mutant viruses derived from cdna clones generated in the present study . (a) four amino acid substitutions, d136 g, i2623v, d3148 g and d3502y, were found in the gbk_e virus in comparison with the gbk_e virus . (b) three recombinant viruses with combinations of amino acid substitutions were generated in the gbk_e backbone by site - directed mutagenesis and in - fusion techniques . The white and gray boxes indicate the nonstructural and structural proteins, respectively .. schematic representation of the genomic differences between gbk_e virus and gbk_e virus, and mutant viruses derived from cdna clones generated in the present study . (a) four amino acid substitutions, d136 g, i2623v, d3148 g and d3502y, were found in the gbk_e virus in comparison with the gbk_e virus . (b) three recombinant viruses with combinations of amino acid substitutions were generated in the gbk_e backbone by site - directed mutagenesis and in - fusion techniques . End assay and measurements of type i interferon production: the end assay was conducted using bfm cells as described previously . In brief, bfm cells grown in 96-well plates were infected with bvdv and incubated for 5 days at 37c in 5% co2 . The end phenomenon was regarded as positive, if strong cpe was observed in ndv - infected cells . Measurements of type i interferon production in bvdv - infected cells were performed using a previously described plaque reduction method with vsv as a challenge virus, with some modifications . In brief, the supernatants from bvdv - infected cells were inactivated by exposure to uv light (254 nm) in the uv crosslinker (atto corporation, tokyo, japan) under the condition of 500 mj / cm . Viral inactivation of samples was confirmed using indirect fa techniques with anti - bvdv ns3 monoclonal antibody #46/1 after inoculation of samples onto mdbk - hs cells and incubation for 2 days at 37c in 5% co2 . Then, monolayers of sk - l cells in 12-well plates were inoculated with 1 ml of a four - fold dilution of uv - inactivated supernatant and incubated for 24 hr at 37c in 5% co2 . Interferon titers were expressed as reciprocals of dilutions that reduced the number of challenge viral plaques by 50% . Detection of irf-3 in bvdv - infected cells by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (sds - page) and western blotting: sds - page and western blotting were performed as described previously . Bfm cells were infected with bvdv (m.o.i . Of 1) in six - well plates and incubated for 5 days at 37c in 5% co2 . After transferring the proteins from the gels to the immunobilion - p transfer membrane (millipore, billerica, ma, u.s.a . ), the membranes were treated with anti - human irf-3 rabbit polyclonal antibodies (genetex, hsinchu city, taiwan), goat anti - rabbit igg - hrp conjugate (bio - rad) and immunobilion western detection reagents (millipore), in that order . The membranes were read using lumi vision pro (aishin seiki, kariya, japan), and specific bands for irf-3 were detected . Cloning of a pair of end/ end viruses from the bvdv gbk strain and determining their whole genome sequences: a pair of end and end viruses (gbk_e and gbk_e, respectively) was cloned from the bvdv gbk strain by means of reverse plaque formation techniques and limited dilution . The genomes of gbk_e and gbk_e viruses were both 12,284 nucleotides in length and encoded 3,897 deduced amino acids . Comparison of the complete genome sequences of both viruses revealed only six nucleotide and four amino acid differences (d136 g, i2623v, d3148 g and d3502y, the numbers refer to the amino acid position in gbk_e) between the two viruses (table 1table 1.differences of amino acid sequences in the genome of gbk_e and gbk_eviral proteinnns4bns5ans5bamino acid position1362,6233,1483,502gbk_ediddgbk_egvgya) d: aspartic acid, i: isoleucine, g: glycine, v: valine, y: tyrosine . ). The complete genome sequences of the gbk_e and gbk_e strains were deposited in the ddbj / embl / genbank databases under accession numbers ab894423 and ab894424, respectively . A) d: aspartic acid, i: isoleucine, g: glycine, v: valine, y: tyrosine . Generation and characterization of in vitro - rescued viruses: the infectious bvdv vgbk_e was successfully rescued by electroporation of mdbk - hs cells with viral rna transcribed in vitro from a full - length pcr amplicon obtained from a cdna clone pgbk_e . The mutant viruses vgbk_e / d136 g, vgbk_e / i2623v; d3148 g; d3502y and vgbk_e / d136 g; i2623v; d3148 g; d3502y were also recovered from full - length cdna clones . Sequencing of the complete genomes of these three viruses confirmed the mutations at the desired amino acid positions and demonstrated the lack of any other mutations in comparison with the parental gbk_e virus . To investigate the biological properties of the mutant viruses, bfm cells were inoculated with both the parental gbk_e and the in vitro - rescued vgbk_e viruses . The results revealed that both gbk_e and vgbk_e were ncp (data not shown) and exhibited the end phenomenon (end) (fig . Bfm cells infected with gbk_e, gbk_e or the in vitro - rescued viruses were superinfected with newcastle disease virus (ndv). Cells infected with gbk_e, vgbk_e or vgbk_e / i2623v; d3148 g; d3502y exhibited distinguishable cpe after superinfection with ndv (end), whereas those infected with gbk_e, vgbk_e / d136 g or vgbk_e / d136 g; i2623v; d3148 g; d3502y did not exhibit cpe (end) in addition, titration of both viruses in mdbk - hs cells revealed that vgbk_e exhibited the same growth characteristics as wild - type gbk_e (data not shown). Moreover, compared with the wild - type gbk_e strain, other in vitro - rescued viruses grew equally in mdbk - hs cells (data not shown). Bfm cells infected with gbk_e, gbk_e or the in vitro - rescued viruses were superinfected with newcastle disease virus (ndv). Cells infected with gbk_e, vgbk_e or vgbk_e / i2623v; d3148 g; d3502y exhibited distinguishable cpe after superinfection with ndv (end), whereas those infected with gbk_e, vgbk_e / d136 g or vgbk_e / d136 g; i2623v; d3148 g; d3502y did not exhibit cpe (end) identification of the amino acid determinants responsible for inhibition of type i interferon production: as expected, the mutant viruses vgbk_e / d136 g, vgbk_e / i2623v; d3148 g; d3502y and vgbk_e / d136 g; i2623v; d3148 g; d3502y remained ncp (data not shown). An end assay revealed that the vgbk_e / d136 g and vgbk_e / d136 g; i2623v; d3148 g; d3502y viruses were changed to end; bfm cells infected with these viruses did not exhibit cpe after ndv superinfection . In contrast, the vgbk_e / i2623v; d3148 g; d3502y virus remained end; bfm cells infected with this virus exhibited clear cpe after superinfection with ndv, as observed in bfm cells infected with gbk_e (fig . The amount of type i interferon in supernatants from bfm cells infected with the parent or one of the mutant viruses gbk_e, gbk_e, vgbk_e, vgbk_e / d136 g, vgbk_e / i2623v; d3148 g; d3502y or vgbk_e / d136 g; i2623v; d3148 g; d3502y was measured in sk - l cells . Secretion of type i interferon was inhibited in bfm cells infected with gbk_e, vgbk_e or the virus carrying the mutations without npro (vgbk_e / i2623v; d3148 g; d3502y), which is in consistent with the results from the end assay . Cells infected with gbk_e or viruses carrying mutations in n(vgbk_e / d136 g and vgbk_e / d136 g; i2623v; d3148 g; d3502y) did induce type i interferon (table 2table 2.measurements of type i interferon in cells infected with wild - type and in - vitro rescued virusesvirusestype i interferongbk_e<4vgbk_e<4vgbk_e / d136g4vgbk_e / i2623v; d3148 g; d3502y<4vgbk_e / d136 g; i2623v; d3148 g; d3502y4gbk_e4). These results clearly indicate that the aspartic acid of n(at position 136 of the gbk_e strain) is the key amino acid residue that determines the capacity to inhibit the production of type i interferon and induce the end phenomenon . Detection of irf-3 in cells infected with the parental or mutant viruses: to investigate the expression levels of irf-3 in bvdv - infected cells, irf-3 in the lysates of bfm cells infected with gbk_e, gbk_e, vgbk_e or one of the three mutant viruses was detected by western blotting . Irf-3 was not detected in the lysates of cells infected with gbk_e, vgbk_e or vgbk_e / i2623v; d3148 g; d3502y, whereas clear bands of irf-3 appeared in the lysates of cells infected with the end gbk_e virus or one of the n mutant viruses (vgbk_e / d136 g and vgbk_e / d136 g; i2623v; d3148 g; d3502y) (fig . 3fig . Irf-3 was not detected in western blots of the lysates of cells infected with gbk_e, vgbk_e or vgbk_e / i2623v; d3148 g; d3502y, whereas clear bands of irf-3 appeared in western blots of the lysates of cells infected with gbk_e, vgbk_e / d136 g or vgbk_e / d136 g; i2623v; d3148 g; d3502y . ). Irf-3 was not detected in western blots of the lysates of cells infected with gbk_e, vgbk_e or vgbk_e / i2623v; d3148 g; d3502y, whereas clear bands of irf-3 appeared in western blots of the lysates of cells infected with gbk_e, vgbk_e / d136 g or vgbk_e / d136 g; i2623v; d3148 g; d3502y . Comparison of amino acid sequences of zinc - binding trash motif in n: the amino acid sequences of the zinc - binding trash motifs in the n proteins from various bvdv strains deposited in the ddbj / embl / genbank databases were compared with those of the gbk_e and gbk_e strains . At least one strain per subgenotype [1a1o (except for 1l) and 2a2c] [11, 18] was chosen in the present study . As a result, bvdv strains, except for the g strain, contain amino acid residues cys112-cys134-asp136-cys138 in the zinc - binding trash motif of n, as observed in gbk_e (fig . 4.comparison of amino acid sequences of the zinc - binding trash motifs in n. the amino acid sequences of the zinc - binding trash motifs in the n proteins of various bvdv strains deposited in the ddbj / embl / genbank databases were compared with those of gbk_e and gbk_e . At least one strain per subgenotype [1a1o (except for 1l) and 2a2c] was chosen . The cysteines (cs) at positions 112, 134 and 138 of the zinc - binding trash motif are highlighted in bold and underlined . The accession numbers of strains from the ddbj / embl / genbank are as follows: nadl (m31182), sd-1 (m96751), osloss (m96687), cp7 (u63479), bega (af049221), 721 (af144463), f (af287284), 3186v6 (af287282), w (af287290), a (af287283), g (af287285), 23 - 15 (af287279), ks86 - 1ncp (ab078950), suwancp (kc853440), zm-95 (af526381), shitara/02/06 (ab359930), is25cp/01 (ab359931), 890 (u18059), hokudai - lab/09 (ab567658) and nrw 14 - 13_dup () (hg426485).). Comparison of amino acid sequences of the zinc - binding trash motifs in n. the amino acid sequences of the zinc - binding trash motifs in the n proteins of various bvdv strains deposited in the ddbj / embl / genbank databases were compared with those of gbk_e and gbk_e . At least one strain per subgenotype [1a1o (except for 1l) and 2a2c] was chosen . The cysteines (cs) at positions 112, 134 and 138 of the zinc - binding trash motif are highlighted in bold and underlined . The accession numbers of strains from the ddbj / embl / genbank are as follows: nadl (m31182), sd-1 (m96751), osloss (m96687), cp7 (u63479), bega (af049221), 721 (af144463), f (af287284), 3186v6 (af287282), w (af287290), a (af287283), g (af287285), 23 - 15 (af287279), ks86 - 1ncp (ab078950), suwancp (kc853440), zm-95 (af526381), shitara/02/06 (ab359930), is25cp/01 (ab359931), 890 (u18059), hokudai - lab/09 (ab567658) and nrw 14 - 13_dup () (hg426485). Both bvdv and csfv inhibit the production of type i interferon in vitro through proteasomal degradation of cellular irf-3 [1, 4, 8, 28]. Cells infected with these (end) viruses exhibit strong cpe after superinfection with ndv or some orbiviruses [15, 20]. One of the two domains of pestivirus n, the c - terminal domain containing a zinc - binding trash motif consisting of cys112-cys134-asp136-cys138, is required for irf-3 binding and prevention of type i interferon production [7, 32]. For csfv, it has been reported that mutations at positions 112, 134, 136 or 138 in n of end viruses abolish the inhibition of type i interferon production, while mutations at positions 112 or 136 in n of end viruses restore this function [24, 32, 33]. For bvdv, previous studies have revealed that amino acid substitutions l8p, e22v or h49l in n abolish its capacity to interfere with type i interferon production [2, 6]. However, to the best of our knowledge, there were no studies with bvdv that had approached the basic mechanism of the inhibition of type i interferon production using the amino acid differences between a pair of end and end viruses as well as viruses genetically engineered on the basis of these differences . Pestiviruses are quasispecies; single strains consist of populations with various characteristics, such as end and end . We cloned a pair of end and end viruses (gbk_e and gbk_e) from the bvdv gbk strain using reverse plaque formation techniques [5, 19]. Determination of complete genome sequences of these viruses revealed that there were only four amino acid differences between them (table 1). To clarify the molecular mechanism of the inhibition of type i interferon production and the end phenomenon, we generated a full - length cdna clone of gbk_e (pgbk_e) as well as three other full - length cdna clones with single (d136 g) or multiple (i2623v; d3148 g; d3502y and d136 g; i2623v; d3148 g; d3502y) mutations . The ifn bioassay of vgbk_e and three mutant viruses revealed that vgbk_e and vgbk_e / i2623v; d3148 g; d3502y exhibited the end phenomenon and inhibited the production of type i interferon, whereas the n mutant viruses vgbk_e / d136 g and vgbk_e / d136 g; i2623v; d3148 g; d3502y were end and did not inhibit the production of type i interferon (fig . This result demonstrates that the single aspartic acid residue in the zinc - binding trash motif of n, which occurs at position 136 of the genome of the gbk_e virus, is the key to determine viral capacity to inhibit type i interferon production and display the end phenomenon . Because it has been reported that the n proteins of both bvdv and csfv inhibit the production of type i interferon by proteasomal degradation of cellular irf-3 [1, 4, 8, 28], we assessed whether n of wild - type gbk_e, wild - type gbk_e, vgbk_e and the three mutant viruses reduced the amount of cellular irf-3 in infected cells by western blotting . An apparent reduction in cellular irf-3 was observed in cells infected with wild - type gbk_e, vgbk_e or vgbk_e / i2623v; d3148 g; d3502y, whereas wild - type gbk_e and mutant viruses with mutations in n showed no reduction in cellular irf-3 (fig . The results indicate that the inhibition of type i interferon production and the end phenomenon displayed by gbk_e occurs by the degradation of cellular irf-3 caused by combining with the zinc - binding trash motif . Comparison of vgbk_e / d136 g; i2623v; d3148 g; d3502y (the same amino acid sequence as gbk_e) with vgbk_e / i2623v; d3148 g; d3502y revealed that the function of n was restored by a single g136d mutation in n of the gbk_e virus . In the present study, a comparison of the amino acid sequences of gbk_e, gbk_e and viruses from ddbj / embl / genbank databases revealed that amino acid residues cys112-cys134-asp136-cys138 in the zinc - binding trash motif of n were well conserved among field bvdv isolates (fig . It is reported that these four residues are required for the inhibition of type i interferon production [7, 32]. Taken together, the above - mentioned results suggest that these field isolates inhibit the type i interferon production in infected cells, although there remains a possibility that amino acid residues other than those of trash motif contribute to this phenomenon . The g strain contains the amino acid residue glu136 in the trash motif of n (fig . 4), and this may affect the structure of n and result in the production of type i interferon in vitro . It was reported that 35 out of 45 (77.8%) field isolates of bvdv in japan contained end virus as the predominant virus population compared with end virus and seven isolates (15.6%) contained similar titers of end and end viruses, whereas two isolates (4.4%) contained only end virus . Therefore, further studies are needed to investigate how quasispecies of bvdv (end and end) contribute in vivo . In conclusion, our results indicate that a single mutation in the trash motif at position 136 of bvdv n abolishes its interaction with bovine irf-3 and halts the degradation of irf-3 . Moreover, the mutation of the amino acid residue at position 136 of gbk_e restores its function as an interferon antagonist, as shown for csfv . However, how n interacts with irf-3 and the nature of the cascade after interaction with n and irf-3 are hardly understood . In addition, it is unknown how the inhibition of type i interferon production contributes to the viral infection strategy when cells are infected with bvdv in vivo . Therefore, an additional study is also required to reveal the fundamental mechanism by which pestiviruses inhibit the production of type i interferon and how this mechanism functions in vivo.
In central and northern europe as well as in north america a significant proportion of patients who suffer from birch pollen allergy develop intolerance to certain kinds of fruits and vegetables . Such birch pollen - related food allergies are the result of initial sensitization to the major birch pollen allergen, bet v 1, and subsequent immunologic cross - reactivity of the bet v 1-specific ige antibodies with structurally homologous food proteins . Among the most frequent triggers of birch pollen - related food allergies are apples, with> 70% of all individuals that are sensitized to birch pollen developing allergic reactions when consuming apples . Symptoms typically occur locally at the site of food contact and within minutes after apple consumption, including itching and swelling of the lips, tongue, and throat (oral allergic syndromes, oas). Frequently, allergic patients can also exhibit symptoms of food - induced rhinoconjunctivitis and dyspnea . In apples (malus domestica), the major allergen that is responsible for birch pollen - related food allergies is the 17.5 kda protein mal d 1 . Mal d 1 belongs to group 10 of pathogenesis - related (pr) proteins that are activated in plants in response to different kinds of stress . The concentration of mal d 1 in apples is highly dependent on the cultivar and also influenced by various biotic and abiotic factors, storage conditions, and storage duration . Typically, 130 g of mal d 1 per gram of fresh apple (accounting for up to 7% of total soluble protein) is present directly after harvest . After storage, these values can rise to values exceeding 100 g mal d 1 per gram of apple . Although mal d 1 has been found in both the pulp and peel of apples, higher concentrations are present in the peel . On the basis of this observation, and because mal d 1 appears to be up - regulated upon biotic stress, it has been speculated that this protein may play a role in plant defense response to pathological situations . Mal d 1 is encoded by a multigene family, and a number of isoforms of mal d 1 have been identified to date, which are clustered into four groups on the basis of their dna sequence similarities, that is, mal d 1.01, mal d 1.02, mal d 1.03, and mal d 1.04 . Pcr screening and mass spectrometric studies showed that mal d 1 isoforms are not cultivar specific and that mixtures of isoforms are present in apple fruits . Along with mal d 1.02, and depending on the cultivar, isoforms from the mal d 1.01 cluster are by far the most abundant isoforms found in apples . Within the mal d 1.01 cluster, immunologic investigations of naturally occurring mal d 1 isoforms revealed only small differences of their ige binding capacities and it appears that divergent allergenicities of apple strains are predominantly determined by different mal d 1 expression levels . Whereas the immunological properties of mal d 1 suggest that this protein has a three - dimensional structure and ige binding epitopes that are similar to those of bet v 1 and other members of the pr-10 protein family, experimental structural data for mal d 1 have not been available to date . As a first step toward structural characterization, we recently assigned the nmr backbone and side chain h, c, and n chemical shifts of the isoform mal d 1.0101 . Golden delicious, were the first isoforms for which the dna sequence was determined and are identical at the amino acid level . Here the dna of mal d 1.0101 (genbank nucleotide code x83672, protein code caa58646) was cloned into the expression vector pet28b by using the restriction sites ncoi and xhoi . Construct integrity was ensured by dna sequencing (microsynth ag, balgach, switzerland), and the protein was expressed in escherichia coli bl21 star (de3). Mal d 1.0101 was purified by anion exchange and size exclusion chromatography as described in detail elsewhere . The mass and the amino acid sequence of purified mal d 1.0101 were confirmed by mass spectrometry using a 7 t fourier transform ion cyclotron resonance (ft - icr) mass spectrometer (bruker daltonics, bremen, germany) with an attached electrospray ionization (esi) source . Protein concentrations for nmr spectroscopic experiments for structure determination were 0.5 mm for n / c - labeled and 0.8 mm for n - labeled samples in 91% h2o/9% d2o (v / v) at ph 6.9, 10 mm sodium phosphate, and 7 or 11.2 mm l - ascorbic acid, respectively . All nmr experiments were carried out at 298 k, using either a 500 mhz agilent directdrive spectrometer (agilent technologies, santa clara, ca, usa) equipped with a room temperature probe or a 600 mhz bruker avance ii+ spectrometer (bruker biospin, karlsruhe, germany) equipped with a prodigy cryoprobe . Nmr resonance assignments of mal d 1.0101 were made using standard triple - resonance methods and were deposited at the biological magnetic resonance data bank (bmrb) under accession no . Three - dimensional n and c edited noesy - hsqc experiments (mixing times of 150 ms) were recorded for derivation of distance restraints . Nmr data were processed using nmrpipe and analyzed with ccpnmr . For measuring protein translational diffusion, we employed a stimulated echo pulsed field gradient nmr experiment . Experimental details were identical to those reported for bet v 1 . For the determination of the hydrodynamic radius of mal d 1.0101, we used dioxane as a standard reference under identical buffer conditions, assuming a hydrodynamic radius of 2.12 . Structure calculations were performed with the program xplor - nih 2.42 using a simulated annealing protocol . An initial structural model was generated with cs - rosetta using the bmrb cs - rosetta server . A total of 2079 distance restraints were obtained from 3d n and c edited noesy - hsqc spectra . Noe values were converted on the basis of peak intensities into distances with upper limits of 3.0 (strong), 4.0 (medium), 5.0 (weak), and 6.0 (very weak). Dihedral angle restraints were predicted using talos+ and cs - rosetta . In all regular secondary structure elements hydrogen bonds were included for backbone amide protons, if the n edited noesy - hsqc spectra did not show a water exchange cross peak . Of 100 generated structures, the 20 lowest energy structures were picked and further refined in explicit solvent with the amber14 simulation package using pmemd.cuda and the amber force field 99sb - ildn . Each structure was soaked into a truncated octahedral solvent box of tip3p water molecules with a minimum wall distance of 10 . For the refinement, hydrogen atoms and water molecules were minimized with fixed heavy atoms . The temperature was increased from 0 to 300 k, where the structures were simulated using the noe distance restraints, minimized again, and validated using the protein structure validation software (psvs) suite (table 1). The coordinates of the mal d 1.0101 structures were deposited in the protein data bank under the accession code number 5mmu . Calculated for all residues, using sum over r. largest violation among all 20 reported structures . The three - dimensional structure of mal d 1.0101 consists of a curved, seven - stranded antiparallel -sheet (1-7) embracing a long helix at the c - terminus of the protein (3) and two consecutive short helices (1, 2) (figure 1). The edges of the -sheet are formed by strands 1 and 2, which are connected by helices 1 and 2 that form a v - shaped support for the c - terminal part of helix 3 . In total, 35% -sheet and ca . 25% helical structure, agreeing well with secondary structure estimates from infrared and circular dichroism . As in other proteins from the pr-10 family, strands 2 and 3 are connected by a glycine - rich loop motif (gly46-asn47-gly48-gly49-pro50-gly51). Together, these structural elements create the large internal cavity that is typical for the canonical pr-10 fold . From figure 1b it is evident that in our nmr structural ensemble of mal d 1, secondary structure elements are very well - defined and conformationally homogeneous in all 20 structural models . Only slightly elevated levels of conformational heterogeneity are observed for some of the solvent - exposed loops that connect secondary structure elements and the c - terminus of the protein . Nmr solution structure of the major apple allergen mal d 1.0101 (pdb accession code 5mmu). Secondary structure elements are labeled 1 (val2ser11), 2 (gln40glu45), 3 (ile53thr57), 4 (tyr66ile74), 5 (ser80gly88), 6 (glu96val105), 7 (ser111thr121), 1 (pro15val23), 2 (ala26ile33), 3 (lys128asp152). (b) backbone overlay of the ensemble of the 20 lowest energy structures of mal d 1.0101 . Secondary structure elements are colored from red (n - terminus) to purple (c - terminus). A peculiar feature of the pr-10 fold is the large internal cavity . In mal d 1.0101, the volume of this cavity is ca . 2230, which is comparable in size to those of other pr-10 proteins . As found in the birch pollen allergen bet v 1 and other homologous food allergens, in mal d 1 the majority of amino acids that form the surface of the cavity are hydrophobic (figure 2). A large proportion of the inner cavity surface is formed by amino acid residues in the -sheet whose hydrophobic side chains are located at the protein interior (ile56 (3), val67 (4), ile71 (4), tyr81 (5), tyr83 (5), leu85 (5), ile98 (6), tyr100 (6), ile113 (7)) along with inward - pointing residues in the long amphiphilic helix 3 (val132, val134, ala139, leu142, phe143, ile146), the two short helices 1 (phe22, val23) and 3 (ala26, ile30), and loop regions (ile38, phe58, tyr64, ala90). In addition, a few polar and charged side chains are located at the inside of the molecule and form part of the cavity surface, such as asp27 (2), his69 (4), ser115 (7), and lys138 (3), so that the cavity itself is actually amphiphilic, as noted before for the major birch pollen allergen, bet v 1 . In crystal structures of other pr-10 proteins the cavity is occupied by water, amphiphilic ligand molecules, or components of the crystallization buffer . For mal d 1, it is currently not known whether ligands bind specifically to the cavity or what the biological function of ligand binding could be . (a) internal cavity of mal d 1.0101, colored according to the lipophilic potential as implemented in moe, where hydrophilic regions are colored in blue and lipophilic regions are colored in yellow . (b) surface representation of the lowest energy solution structure of mal d 1.0101 . The two amphiphilic entrances to the internal cavity are indicated as 1 (between the n - terminal end of helix 3 and the loops connecting strands 34 and 56) and 2 (between the edge of the -sheet and the c - terminal end of helix 3). The internal cavity in mal d 1 can be reached by two openings (figure 2). One entrance to the protein interior, 1, is shaped by residues in the n - terminal half of helix 3 (his131, val134) along with the loops connecting strands 34 (gln63, tyr64) and strands 56 (asp89). Together, these amino acids create an amphiphilic access route to the protein interior . A second amphiphilic entrance, 2, is present at the edge of the -sheet between helix 3 (lys136, his140, lys144, and glu147) and strand 1 (asn7, phe9, and ser 11). In the nmr solution structures of mal d 1 this access route entries to the internal cavity at similar locations have also been described for other members of the pr-10 protein family . Figure 3 shows a comparison of mal d 1 with bet v 1 and birch pollen - related food allergens from the pr-10 family whose structures have been determined so far . Despite the fact that sequence identities between these proteins are only slightly higher than 50% in some cases, their three - dimensional structures are generally very similar, with backbone rmsd values for secondary structures typically below 2 . In light of the observed immunologic cross - reactivity between mal d 1 and the major birch pollen allergen, bet v 1 the backbone rmsd between mal d 1.0101 and the hyperallergenic isoform bet v 1.0101 (61% sequence identity) of the birch pollen allergen is 2.13 (1.70 for secondary structure elements). Of note, mal d 1 and bet v 1 differ in length by one amino acid, and divergent presumptions have been made about the location of the gap in mal d 1 . On the basis of sequence alignments of pr-10 food allergens it has been proposed that either the loop right before or right after strand 7 is one residue shorter in mal d 1 . Our solution structure shows that the loop right before strand 7 is the one that is shorter in mal d 1.0101 (glu96val105 in both mal d 1.0101 and bet v 1.0101) and 7 (ser111thr121 in mal d 1.0101 and ser112thr122 in bet v 1.0101) occupy identical positions and have equal hydrogen bonding patterns in the antiparallel -sheets of these proteins . They are connected via loops consisting of four residues (cys - gly - ser - gly in mal d 1) and five residues (thr - pro - asp - gly - gly in bet v 1), respectively, which produces a small structural difference in these loop segments between the two proteins . (a) overlay of the lowest energy structure of mal d 1.0101 (green, pdb accession code 5mmu) with the structures of the major birch pollen allergen bet v 1.0101 (blue, 4a88), the carrot allergen dau c 1.0103 (orange, 2wql), the celery allergen api g 1.0101 (gray, 2bk0), the soybean allergen gly m 4.0101 (yellow, 2k7h), the strawberry allergen fra a 1e (red, 2lpx), and the cherry allergen pru av 1.0101 (purple, 1e09). Amino acids are marked with asterisks (identical), colons (conserved), and dots (semiconserved). Mal d 1 is known to have a tendency for cysteine - mediated dimerization, as shown for the isoform mal d 1.0108 by nonreducing gel electrophoresis and size exclusion chromatography . Like mal d 1.0108, the isoform mal d 1.0101 contains a single cysteine residue, cys107 . In the three - dimensional solution structure of mal d 1.0101 cys107 is located at the c - terminal tip of strand 7, with its side chain oriented toward the protein surface . To probe the oligomerization state of mal d 1.0101 under the conditions that we employed for nmr structure determination (ph 6.9, 10 mm sodium phosphate, 14 mol equiv of l - ascorbic acid, 298 k) we performed pulsed - field - gradient nmr diffusion experiments . We obtained a value of 21.6 0.8 for the hydrodynamic radius of mal d 1.0101, which is comparable to the hydrodynamic radius of monomeric bet v 1.0101 (20.1) under similar experimental conditions . This is consistent with our observation that, using the same buffer, mal d 1.0101 elutes from a size exclusion column with a retention time that is virtually identical to that of bet v 1.0101 . These results were further verified by ft - icr mass spectrometry, which shows that mal d 1.0101 does not form dimers or higher order aggregates . The nmr solution structure of mal d 1 shows that this protein consists of a highly curved antiparallel -sheet and three -helices forming a large internal cavity, very similar in fashion to other pr-10 proteins . This is in agreement with the observed immunologic cross - reactivity between mal d 1 and the major birch pollen allergen, bet v 1, as well as other food allergens from the pr-10 protein family . In most patients bet v 1 is the sensitizing agent, whereas bet v 1-specific ige antibodies subsequently cross - react with mal d 1 and elicit an allergic response, as reflected by the clinical observation that apple allergy develops only after the onset of birch pollinosis . Along these lines, cross - inhibition experiments of mal d 1 using sera from apple - allergic patients showed that mal d 1 shares ige epitopes with the major birch pollen allergen, bet v 1 . From a structural perspective, limited information about the exact nature of binding epitopes of mal d 1 and bet v 1 is available . Detailed structural information about a sequentially discontinuous (i.e., conformational) b - cell epitope in bet v 1 was obtained by cocrystallizing the particular isoform bet v 1.0112 with an antigen - binding fragment (fab) derived from the murine monoclonal igg antibody bv16 . This epitope is formed by the segment between glu42 and thr52 (including the glycine - rich loop motif between strands 2 and 3), along with arg70, asp72, his76, ile86, and lys97 of bet v 1, covering approximately 10% (900) of the entire protein surface . Binding of bv16 to this epitope measurably reduces serum ige interactions, indicating that ige and monoclonal igg bv16 compete for overlapping binding surfaces on bet v 1 . Moreover, mutation of a central residue (glu45ser) significantly reduced the ige binding capacity of bet v 1, confirming the significance of this particular epitope for interactions with ige . Figure 4a shows the molecular interaction surface that corresponds with the bv16 epitope in the apple allergen . In mal d 1 these residues form a contiguous surface patch along with a somewhat distal residue (glu76), similar in shape and size to the bv16 epitope of bet v 1 . Moreover, the contributing amino acids are largely conserved between mal d 1 and bet v 1 . Thirteen of the 16 amino acids in the bv16 epitope are identical, whereas only 3 residues are different in mal d 1.0101 and bet v 1.0101 (figure 4b). These data thus provide a structural rationale for the observed allergic cross - reactivity between birch pollen and apple allergens . Interestingly, mutational studies indicate that the ability of mal d 1 to bind serum ige from patients with birch pollen allergies can be increased by increasing the similarity of the bv16 epitope in mal d 1 to that of bet v 1, indicating that these amino acids are indeed involved in binding of bet v 1 specific to mal d 1 . Amino acid residues that correspond to the molecular interaction surface between monoclonal igg bv16 and bet v 1.0112 (residues glu42thr52, arg70, asp72, glu76, ile86, and lys97 in mal d 1.0101) are colored in blue . Amino acid positions that were shown to be crucial for ige recognition of mal d 1 in mutational analyses (thr10, ile30, thr57, ser111, thr112, and ile113) are shown in green (ile30 and ile113 are located in the protein interior and do not contribute to the surface). (b) amino acid similarities between bet v 1.0101 and mal d 1.0101 using a color gradient from lilac (highly similar) to teal (highly dissimilar). Epitope residues that are different between bet v 1.0101 and mal d 1.0101 are labeled . Similarities were calculated on the basis of substitution matrix scores (blosum62) as implemented in moe . It is likely that mal d 1 contains more than a single conformational epitope . A number of amino acid positions that are relevant for ige recognition have been identified by mutational analysis . For a five - point mutant of mal d 1.0108 (thr10pro, ile30val, thr57 asn, thr112cys, and ile113val) a markedly reduced capacity for binding mal d 1-specific ige was found in vitro . Skin prick tests in apple - allergic patients comparing wild - type mal d 1 with the five - point mutant further showed a significantly lower ability of the mutant protein to induce skin reactions in vivo . Further experiments showed that the t - cell recognition level of wild - type mal d 1 is conserved in the five - point mutant . Because these five amino acids are likely involved in ige interactions not only in mal d 1 but also in bet v 1, they could well be part of common cross - reacting epitopes in these two allergens . This is corroborated by mutational studies, which showed that peptide stretches encompassing these residues are indeed involved in immunological cross - reactivity between mal d 1 and bet v 1 . In addition, in an independent study, ser111 was identified as being essential for ige binding to mal d 1, and a ser111cys mutation resulted in significantly reduced affinity for ige in immunoblotting experiments . Figure 4a shows that these six residues are fairly dispersed on the protein surface of mal d 1 and that neither of these amino acids overlaps with the bv16 epitope . Amino acids thr10, ser111, and thr112 form a common patch on the protein surface, whereas thr57 is located approximately 3739 away and close to the bv16 epitope . Considering that an epitope of typical size (600900) and circular shape would have an arc length of 2834 on the mal d 1 surface, residues thr10, ser111, and thr112 are probably too far away from thr57 to be part of a common binding epitope . The remaining two residues, ile30 and ile113, do not reach the protein surface in mal d 1 . Whereas ile113 is close in space to the thr10-ser111-thr112 patch, its hydrophobic side - chain is pointing toward the interior of the protein, where it participates in a small hydrophobic core located at the inner end of the proteins cavity (between helices 1 and 3 and the -sheet). Residue ile30 is also located in the protein interior with its aliphatic side chain forming part of the internal cavity and does not contribute to the protein surface . Of note, because the loop between strands 6 and 7 is shorter by one residue in mal d 1 than in bet v 1, ser111 and thr112 of 7 in mal d 1 occupy the 7 positions of ser112 and ile113 in bet v 1 . The surface patch formed by thr10, ser111, and thr112 in mal d 1 thus appears to be less hydrophobic than the corresponding surface patch in bet v 1 (thr10, ser112, and ile113). As a matter of fact, also the protein surface surrounding these three residues displays a considerably lower level of similarity between mal d 1.0101 and bet v 1.0101 than other parts of the protein surface, as can be seen in figure 4b . This might in part be responsible for the different ige binding properties of these allergens . It has been noted, on the other hand, that epitope coincidence between bet v 1 and mal d 1 may be limited, as exemplified by a recent study describing the isolation of human ige binding to bet v 1 but not to mal d 1 . Moreover, different mal d 1 isoforms contain amino acid substitutions within potential ige interaction surfaces, suggesting that they may influence the immunologic reaction . It is clear that high - resolution structural data provide the basis to determine and compare structural details of (cross - reactive) binding epitopes in allergenic proteins . In addition, grafting of conformational epitopes by transferring stretches of residues between homologous allergens has become a valuable experimental tool . Epitope grafting was used to characterize the role of the bv16 epitope in mal d 1 by recreating this epitope on the mal d 1 surface, confirming its importance for ige binding and cross - reactivity with bet v 1 . In an orthogonal approach, several mal d 1 stretches encompassing residues that are crucial for ige binding were transferred to bet v 1 to investigate the role of these structural segments for cross - reactivity, and chimeras of bet v 1 and mal d 1 were created to map the epitope of a human monoclonal ige, which was isolated from a phage library, to the c - terminus of bet v 1 . In addition, epitope grafting provides access to chimeric allergens with fine - tuned antigenic properties, such as reduced ige binding capacitites, for molecule - based allergy diagnosis and specific immunotherapy . Knowledge of the structural details of these allergens elements is required to generate correctly folded chimeras, because transfer of (partly) mismatching stretches of secondary structure between different allergens may well be the reason for a loss of protein fold and, consequently, reduction of ige - binding capacities . The three - dimensional structure of mal d 1.0101 presented here provides the biophysical basis for elucidating the molecular details of immunological cross - reactivity in great detail.
Head and neck oncological resections may result in composite oro - mandibular defects involving the oral mucosa (lining), mandibular bone and the skin (cover). Anatomically, the fibula bone has a triangular cross section, it has a lateral surface where peroneal muscles are attached, this is a broad flat surface and is ideal for fixing the reconstruction plate during mandibular reconstruction, at the lower end of this peroneal surface is the posterior crural septum through which the peroneal artery perforators supplying the skin paddle run, this septum has variable length and mobility, which determines the movement of the skin paddle . On the posterior and medial facet of this triangle, originates the flexor hallucis longus (fhl) muscle and on the supero - medial aspect is the origin of the tibialis posterior muscle and between these two run the vascular pedicle . Due to this unique three - dimensional anatomy there is always a possibility of using the bone flap in a more efficient fashion for the reconstructive need . Existing literature, however, provides conflicting views about the use of a particular side and orientation of the fibula flap for achieving the optimal outcome . The purpose of this study is to confirm anatomically the effect of bone, soft tissue and vessel orientation on the ease of doing reconstruction . We also propose an algorithm for choosing the correct side of the fibula for a particular reconstructive need and at the same time minimising the donor side morbidity . Composite fibula flaps of the same dimension were harvested from both legs of a fresh cadaver . The harvested flaps were used to reconstruct the mandibular defect in different orientations, and the best configuration for each reconstructive requirement was assessed . Following variables were considered: placement of the vascular pedicle the pedicle needs to be placed anteriorly if the microvascular anastomosis is to be done on the opposite side due to vessel paucity in the ipsilateral neck.type of associated soft tissue defect the defect could be a mucosal defect (lining), the skin defect (cover) or a combined mucosa and skin defect . The pedicle needs to be placed anteriorly if the microvascular anastomosis is to be done on the opposite side due to vessel paucity in the ipsilateral neck . The defect could be a mucosal defect (lining), the skin defect (cover) or a combined mucosa and skin defect . Keeping the peroneal surface for plating, that is, facing outwards, four different configurations of the fibula flap are possible for a given mandibular defect . Implications of each one for reconstruction are discussed below . The skin paddle comes out from the lower and outer border of the reconstructed mandible because of the anatomical position of the posterior crural septum [figure 1]. If this skin paddle has to be taken inside the oral cavity, it has to travel over the plated surface of the fibula, which wastes about 3 - 4 cm of skin paddle width . The length of the posterior crural septum being variable also restricts the inside movement of the skin paddle . Ipsilateral fibula with pedicle posterior the fhl muscle lies at the submandibular region with the vascular pedicle lying on its superior aspect and therefore pulling the muscle inside for intraoral reconstruction kinks the vascular pedicle . In this situation, the septum location becomes superior and medial in relation to the bone . Hence, the skin paddle does not have to go around the bone to come inside and thus we do not lose skin paddle width when we turn it inside . It can thus be used for providing mucosal lining more easily . For moving the skin paddle outside to provide skin cover, it has to travel over the plated surface of the fibula although the wastage of skin paddle width is not much [figure 2]. Ipsilateral fibula with pedicle anterior the fhl muscle is supero - medial to the bone and lies within the oral cavity thus it can be easily utilized for intraoral lining . This configuration leads to a tissue orientation similar to that discussed in point 2 (ipsilateral fibula flap placing the vascular pedicle anterior and thus is suited for intra oral reconstruction [figure 3]. Contralateral fibula with pedicle posterior this configuration leads to a tissue orientation similar to that discussed in point 1 (ipsilateral fibula flap placing vascular pedicle posterior and thus is not particularly suited for intra oral reconstruction and skin paddle is best utilised outside [figure 4]. The skin paddle comes out from the lower and outer border of the reconstructed mandible because of the anatomical position of the posterior crural septum [figure 1]. If this skin paddle has to be taken inside the oral cavity, it has to travel over the plated surface of the fibula, which wastes about 3 - 4 cm of skin paddle width . The length of the posterior crural septum being variable also restricts the inside movement of the skin paddle . Ipsilateral fibula with pedicle posterior the fhl muscle lies at the submandibular region with the vascular pedicle lying on its superior aspect and therefore pulling the muscle inside for intraoral reconstruction kinks the vascular pedicle . In this situation, the septum location becomes superior and medial in relation to the bone . Hence, the skin paddle does not have to go around the bone to come inside and thus we do not lose skin paddle width when we turn it inside . It can thus be used for providing mucosal lining more easily . For moving the skin paddle outside to provide skin cover, it has to travel over the plated surface of the fibula although the wastage of skin paddle width is not much [figure 2]. Ipsilateral fibula with pedicle anterior the fhl muscle is supero - medial to the bone and lies within the oral cavity thus it can be easily utilized for intraoral lining . This configuration leads to a tissue orientation similar to that discussed in point 2 (ipsilateral fibula flap placing the vascular pedicle anterior and thus is suited for intra oral reconstruction [figure 3]. This configuration leads to a tissue orientation similar to that discussed in point 1 (ipsilateral fibula flap placing vascular pedicle posterior and thus is not particularly suited for intra oral reconstruction and skin paddle is best utilised outside [figure 4]. Of all the reconstructive options available for the complex mandibular defect, the free fibula osteo - cutaneous flap is the most widely used one . However, there is conflicting literature regarding the ideal donor side of the fibula . Hidalgo advocated selection of the donor area based on the quality of the recipient vessels in the neck, that is, ipsilateral fibula for anastomosis with the same sided neck vessels and contralateral otherwise . In all the cases, he prefers the skin paddle orientation of situation 1 and 4 (the skin paddle should cover the fibula from outside). The advantage cited is that the posterior crural septum covers the plate and acts as a second line of defence, preventing the plate exposure in the event of skin breakdown . However in this configuration if one attempts to reconstruct intraoral defects, one has to add at least 3 cm of extra width to the skin paddle because it has to go around the fibula before it could enter the oral cavity . This configuration also has the potential to kink the delicate peroneal perforators as they go around the bone, which could lead to a loss of the skin paddle . As the fhl muscle lies in the submandibular region, it will not be utilised as an intraoral seal . Yagi et al . Suggested considering the location of the pedicle in the neo - mandible, requirement of a skin paddle and orientation of the fibula to the remnant mandible in determining the ideal choice of side for the mandibular reconstruction, but he has also not emphasised the additional width of the skin paddle required if one does not choose the correct side and orientation of the fibula . Put forth that there is no donor side specificity of fibula free flap for complex oro - mandibular reconstruction . However, it has been noted by author that if one takes a very large skin paddle for composite tissue defects, mere need to add 3 cm of width does not make much difference and one could use any side and orientation of fibula for any defect reconstruction . The question of choosing the side and orientation arises only if one considers minimising the donor site deformity by harvesting only the exact amount of skin required . This would be possible only if one plans precisely, and the algorithm, which authors propose [algorithm 1], helps one to achieve better management of the donor, as well as the recipient site . Algorithm for ideal donor side selection the skin paddle of the fibula flap is usually harvested from its lower portion to ensure adequate pedicle length . Being the lower end of the leg, only small sized skin defects can be primarily closed . Moderate sized defects up to 8 cm in width in the lower leg may be closed using a soleal musculocutaneous perforator based propeller flap . A large skin defect arising from harvesting a big skin paddle needs to be covered with a split skin graft with its potential donor site morbidity . The basis for selection of the side of the donor fibula are the placement of the pedicle (i.e., anterior or posterior) and the reconstructive requirements (i.e., bone only, bone and lining, bone and skin cover or bone, lining and cover). An anteriorly placed pedicle may be considered in case of a vessel depleted neck or for a ramus reconstruction, when one needs to reconstruct ramus of the mandible and plans to keep the vascular pedicle posteriorly, there is a possibility of acute kink of the vessels if we choose to anastomose the vessels in the neck and if we choose to anastomose the vessels with superficial temporal vessels, one has to grapple with the problem of size mismatch between the donor and recipient vessels with an increased potential of failure . In other situations, based on our results, we can give our recommendations for the different composite defects as under: if the pedicle is positioned anteriorly, such a defect would be adequately reconstructed with an ipsilateral fibula with the fhl muscle (as described in point 2). The donor area skin can be closed primarily because no skin is harvested . In a posteriorly placed pedicle, when the pedicle is positioned anteriorly, such a defect would need an ipsilateral fibula with optimal sized skin paddle (as described in point 2). The donor area skin may be closed primarily, with a soleal propeller flap or skin graft . In a posteriorly placed pedicle, buccal mucosal defect extending to the upper alveolus and secondary reconstructions of the mandible would necessitate a contralateral neck anastomosis . These defects are most optimally reconstructed with the ipsilateral fibula if the pedicle has to be brought posteriorly (point 1) or with the contralateral fibula if the pedicle has to be brought out anteriorly (point 4). The skin paddle may be used to cover the skin defect and the fhl muscle for reconstructing the mucosal defect . However, if the size of the defect is beyond the dimension which could be covered by fibula, such defects are reconstructed with double flaps using the fibula skin paddle for one defect and a second free / regional flap for the other defect . This concept enables us to reduce the donor site defect by choosing the side of fibula flap, which has the skin paddle that will fall into an anatomically correct position to reconstruct the soft tissue defect . Closure of smaller defects primarily or with propeller flaps result in superior aesthetic outcome [figures 5 and 6]. If the pedicle is positioned anteriorly, such a defect would be adequately reconstructed with an ipsilateral fibula with the fhl muscle (as described in point 2). The donor area skin can be closed primarily because no skin is harvested . In a posteriorly placed pedicle, when the pedicle is positioned anteriorly, such a defect would need an ipsilateral fibula with optimal sized skin paddle (as described in point 2). The donor area skin may be closed primarily, with a soleal propeller flap or skin graft . In a posteriorly placed pedicle, buccal mucosal defect extending to the upper alveolus and secondary reconstructions of the mandible would necessitate a contralateral neck anastomosis . These defects are most optimally reconstructed with the ipsilateral fibula if the pedicle has to be brought posteriorly (point 1) or with the contralateral fibula if the pedicle has to be brought out anteriorly (point 4). The skin paddle may be used to cover the skin defect and the fhl muscle for reconstructing the mucosal defect . However, if the size of the defect is beyond the dimension which could be covered by fibula, such defects are reconstructed with double flaps using the fibula skin paddle for one defect and a second free / regional flap for the other defect . This concept enables us to reduce the donor site defect by choosing the side of fibula flap, which has the skin paddle that will fall into an anatomically correct position to reconstruct the soft tissue defect . Closure of smaller defects primarily or with propeller flaps result in superior aesthetic outcome [figures 5 and 6]. These defects are most optimally reconstructed with the ipsilateral fibula if the pedicle has to be brought posteriorly (point 1) or with the contralateral fibula if the pedicle has to be brought out anteriorly (point 4). Such defects can be reconstructed with a configuration described in points 2 and 3 . The skin paddle may be used to cover the skin defect and the fhl muscle for reconstructing the mucosal defect . However, if the size of the defect is beyond the dimension which could be covered by fibula, such defects are reconstructed with double flaps using the fibula skin paddle for one defect and a second free / regional flap for the other defect . This concept enables us to reduce the donor site defect by choosing the side of fibula flap, which has the skin paddle that will fall into an anatomically correct position to reconstruct the soft tissue defect . Closure of smaller defects primarily or with propeller flaps result in superior aesthetic outcome [figures 5 and 6]. The algorithm based selection of appropriate sided fibula flap facilitates complex mandibular reconstruction by placing the right kind of tissue at the right place and helps in reducing the donor site morbidity by allowing the surgeon to harvest only the required amount of skin.
Mesial temporal lobe epilepsy with hippocampal sclerosis (mtle - hs) is the most common type of partial or localization - related seizure disorder in humans . Mesial temporal sclerosis (mts) with prominent neuronal loss and gliosis in the hippocampus is the most common pathology in temporal lobe epilepsy (tle). Although the pathogenesis and epileptogenesis of hippocampal sclerosis (hs) has been studied for years, the information available so far still does not fully explain the situation . Patients with mtle - hs usually have a history of febrile seizures, status epilepticus, trauma or, in some cases, a mixture of all these symptoms . Recently, some studies have implicated inflammatory mechanisms that contribute or predispose to the occurrence of seizures in mtle - hs . Cytokines such as interleukins (il) are involved in inflammation, immune activation, cell differentiation and apoptosis . In seizures, there is increasing evidence to support the role of il-1 in reducing seizure threshold and epileptogenesis in the pilocarpine status epilepticus model of epilepsy . Found a strong association between the homozygotes for allele t at position -511 of the il-1 gene promoter region (il-1-511 t / t) and genetic predisposition to the development of hs in japanese tle patients . Stated that increased il-1-511 t - allele frequency proved to be a potent determinant of prolonged febrile seizure, thus a discrepancy in prolonged febrile seizure incidence could explain recent conflicting results . Three polymorphisms within the il-1 gene promoter region located at positions -1470, -511 and -31 basepairs from the transcription start site were found to be in strong linkage disequilibrium . Additionally, when analyzed in combination, interestingly, however, these two publications have conflicting results in terms of transcriptional activity of il-1 promoter . While chen et al . Concluded that the il-1-511 t allele was associated with higher il-1 promoter activity, wen et al . Demonstrated that haplotype combinations carrying -511 c allele cause higher levels of secretion of il-1 from the lipopolysaccharide (lps)-stimulated lymphocytes . In both of these studies, therefore, we intended to analyze the effect of -511 c / t polymorphism on il - i secretion from lps - induced lymphocytes in an attempt to reveal the possible role of this particular polymorhism in the etiopathogenesis of mtle - hs . Thirty patients with mtle - hs admitted to the akdeniz university hospital neurology department, turkey, were enrolled in this study . A control group of 32 healthy volunteers with matching age, gender ratio and ethnic origins was also included . Diagnosis of mtlehs was based on clinical history, electroencephalography, magnetic resonance imaging, and, in some patients, on histopathological findings after the selective amgydalahippocampectomy and anterior temporal lobectomy . The control subjects had no history of seizures or neurological disorders and no central nervous system or other infections at the time of blood sample collection . Peripheral venous blood samples (3 ml) were collected from each subject into vacutainer tubes containing edta . Dna was extracted from blood samples using the robotic system magnapure (roche) and the amount of dna was measured by spectrophotometer . The -511 region of il-1 gene has been genotyped with polymerase chain reaction - restriction fragment length polymorphism (pcr - rflp) using 2 mg of genomic dna as template . By using the primers for -511 c / t polymorphism described below, a 304 bp of dna region was amplified by pcr (total 35 cycles, each cycle 30 s at 95c, 45 s at 50c, and 45 s at 72c). Each pcr product was then cleaved by ava i enzyme and the digested pcr products were subjected to agarose gel electrophoresis . The gels were stained with ethidium bromide and the dna fragments were visualized by an uv transluminator . Pcr products acquired from those with a cc genotype were cleaved into two fragments of 190 and 114 bp by ava i enzyme, whereas the pcr products of people with tt genotype was not digested at all, yielding a product of 304 bp . Oligonucleotides used for pcr: forward primer: 5-tggcattgatctggttcatc reverse primer: 5-gtttaggaatcttcccactt peripheral blood of patients and healthy individuals was drawn into heparinized tubes, and lymphocytes were isolated with a density gradient method and pipetted into plates (210 cells each well) and incubated for 24 h (5% co2, 37c) in an rpmi 1640 culture medium including 10% fcs with (0.5 ng / ml) or without lps . The cells were precipitated by centrifuge at 1600 rpm for 10 min after 4 and 24 h. the supernatants were transferred to another tube and stored at 80c until elisa analyses . Il1 levels in cell culture supernatants were studied according to the manufacturer s protocol (ebioscience, cat . N. 88 - 7010). All statistical analysis was carried out using statistical package for social sciences (spss) windows version 15.0 (spss inc ., tests for differences in allelic and genotypic frequencies were performed using 2 test and fisher s exact test . Thirty patients with mtle - hs admitted to the akdeniz university hospital neurology department, turkey, were enrolled in this study . A control group of 32 healthy volunteers with matching age, gender ratio and ethnic origins was also included . Diagnosis of mtlehs was based on clinical history, electroencephalography, magnetic resonance imaging, and, in some patients, on histopathological findings after the selective amgydalahippocampectomy and anterior temporal lobectomy . The control subjects had no history of seizures or neurological disorders and no central nervous system or other infections at the time of blood sample collection . Peripheral venous blood samples (3 ml) were collected from each subject into vacutainer tubes containing edta . Dna was extracted from blood samples using the robotic system magnapure (roche) and the amount of dna was measured by spectrophotometer . The -511 region of il-1 gene has been genotyped with polymerase chain reaction - restriction fragment length polymorphism (pcr - rflp) using 2 mg of genomic dna as template . By using the primers for -511 c / t polymorphism described below, a 304 bp of dna region was amplified by pcr (total 35 cycles, each cycle 30 s at 95c, 45 s at 50c, and 45 s at 72c). Each pcr product was then cleaved by ava i enzyme and the digested pcr products were subjected to agarose gel electrophoresis . The gels were stained with ethidium bromide and the dna fragments were visualized by an uv transluminator . Pcr products acquired from those with a cc genotype were cleaved into two fragments of 190 and 114 bp by ava i enzyme, whereas the pcr products of people with tt genotype was not digested at all, yielding a product of 304 bp . Peripheral blood of patients and healthy individuals was drawn into heparinized tubes, and lymphocytes were isolated with a density gradient method and pipetted into plates (210 cells each well) and incubated for 24 h (5% co2, 37c) in an rpmi 1640 culture medium including 10% fcs with (0.5 ng / ml) or without lps . The cells were precipitated by centrifuge at 1600 rpm for 10 min after 4 and 24 h. the supernatants were transferred to another tube and stored at 80c until elisa analyses . Il1 levels in cell culture supernatants were studied according to the manufacturer s protocol (ebioscience, cat . N. 88 - 7010). All statistical analysis was carried out using statistical package for social sciences (spss) windows version 15.0 (spss inc ., tests for differences in allelic and genotypic frequencies were performed using 2 test and fisher s exact test . There was no significant difference between the mtle - hs patients and controls according to genotype and allele frequencies of il-1-511 . Levels of secreted il-1 from the cultured lymphocytes incubated either with or without lps were measured at three different time frames during the culture period (basal, 4 h and 24 h). However, no statistically significant difference was observed between the groups (table 3). Since il-1-511 t allele has been suggested to be associated with increased in vitro production of il-1 and that il-1 is involved in hs pathogenesis, we also analyzed the il-1 secretion between the patients and control individuals who carry the t allele at position -511 of the il-1 gene . However, although spontaneous secretion levels of il-1 were higher in mtle - hs patients, there was no significant difference between the groups (figure 1). In addition, despite the fact that both spontaneous and stimulated secretion levels of il-1 were higher in mtle - hs patients who carry the t allele with respect to those patients who do not, the difference was not statistically significant (figure 2). In this study, we found that neither the il-1 -511 c / t polymorphism nor the secreted il-1 from the cultured lymphocytes were associated with mtle - hs in a group of turkish patients . In our study, the average age of onset of seizures and gender were similar to those presented in other studies . On the other hand, the incidence of history of febrile seizures was found to be 76.7%, which was higher than the values mentioned in several previous reports . Cytokine proteins have both pro - inflammatory and anti - inflammatory forms . Among proinflammatory cytokines, three biallelic polymorphisms in il-1, all of which result from c to t transitions at positions -511, -31 or + 3954 from the transcriptional start site, have been most frequently evaluated for their association with diverse conditions besides epilepsy . Chen et al . Demonstrated that the il-1-511 t allele strongly enhanced the transcription of the il-1 gene in the context of the il-1-31 c allele . Il-1-511 t single nucleotide polymorphism has been thought to be associated with mtlehs and febrile seizures . Previously showed no association between il-1 gene polymorphism and tle with hs in turkish ancestry in a larger group . Recently, a meta - analysis and a review was published by kauffman et al . Demonstrating a modest association between the il-1-511 t polymorphism and tle with hs . In the current study, we did not find any significant difference between mtlehs patients and control group with respect to the genotype and allele frequencies of il-1-511 (table 2). In this study, the frequency of t / t genotype was found to be 15.6% whereas, it was reported to be 18% and 20.5% in european and japanese populations, respectively . During the acute phases of status epilepticus, upregulation of il-1 occurred in activated microglia and astrocytes and sustained inflammation was only evident in astrocytes during epileptogenesis . In chronic epileptic tissue, il-1 was still expressed by astrocytes, and it resumed in microglia when rats displayed a high frequency of spontaneous seizures . These findings demonstrated that il-1 is chronically over - expressed in astrocytes after status epilepticus, and il-1 expression in microglia is related to ongoing and severe epileptic activity . Together with il-1, tnf- and il-6 levels increase rapidly after the induction of seizures, then decline to basal levels within 48 - 72 h of the onset of seizures . But il-1 is still up - regulated in the brain 60 days after status epilepticus in rats with spontaneous seizures . The recent data suggested that lipopolysaccharide mimics the actions of an endogenously released protein called high mobility group box 1 that interacts with tolllike receptor 4 to promote seizures . Il-1 and high mobility group box 1 activate pathophysiological cascade of inflammatory events in epilepsy through binding to il-1r1 and tolllike receptor 4, respectively . Besides these experimental studies, some clinical studies have measured il-1 secretion from the lymphocytes and serum in febrile seizure and epilepsy patients . Showed increased plasma levels of il-6 and levels of il-1 unchanged after seizures in chronic localization - related epilepsy patients . Found no difference between febrile seizure patients and controls with respect to serum il-1 levels . Reported increased serum levels of il-6 in tle that supported chronic overproduction of cytokines in refractory focal epilepsy . On the other hand, measurement of the secreted il-1 levels from stimulated lymphocytes can differ from serum levels . Demonstrated that il-1 secretion from the lymphocytes was significantly higher in febrile seizure patients . In another study (haspolat, unpublished data, 2008), significantly increased levels of il-1 from lps - stimulated mononuclear cells at 24 h were observed in complicated febrile seizure patients compared to simple ones . Although these findings suggest that the increased levels of il-1 in mtle - hs patients may lead to hs and explain epileptogenesis and ictogenesis, in this study we were not able to detect any statistically significant differences in secreted il-1 levels from cultured lymphocytes between the patient and control groups . In addition, comparison of il-1 levels secreted from lymphocytes obtained from individuals carrying a t allele at the -511 position of the il-1 gene to those obtained from individuals who do not carry the t allele did not reveal any significant difference either . Interestingly, however, the spontaneous and stimulated levels of il-1 secretion were higher in mtle - hs patients carrying the t allele with respect to the patients who do not . The relatively small number of patients and controls could explain the reason why a statistically significant difference was not observed in this study . In addition, it should be kept in mind that promoter regulation is a complex mechanism involving a large variety of transcription factors acting together . Thus, besides (-511) polymorphism, a combination of different haplotypes and many other transcription factors binding to different sites should impinge on the transcriptional activity . In conclusion, in experimental studies, activation of cytokines has been observed especially in areas of onset of seizures and spreading of discharge . In clinical studies, cytokine levels in peripheral blood seem to reflect the central nervous system s production of cytokines, but these are not always correlated.
The presence of high levels of dental anxiety amongst dental care seekers yielded patients with negative attitudes towards dental treatment and rendered dental treatment more difficult to accomplish successfully4,16 . Assessment of anxiety in such patients is a crucial factor for the success of their management . Avoidance of dental care could be attributed to dental fear and anxiety in many patients7,9,23 . Dental anxiety might also affect patient - dentist relationship and obscure proper diagnosis of the actual dental problem8,25 . Taani15 (2002) showed that the levels of dental anxiety were higher among jordanian public school children than those from the private schools . Yet, dental fear and anxiety were found among the most reasons that underlie the irregular attendance in two thirds of the public school children and half of those from the private schools . It is a simple, easy to score, short, valid and reliable test for dental visit - associated anxiety5,13,19,22 . Humphris, morrison and lindsay21 (1995) provided a modified scale from the original corah dental anxiety scale . The modified dental anxiety scale was shown to be more comprehensive, highly valid and reliable, with a simpler and more consistent answering system . The modified dental anxiety scale will be, therefore, used to measure dental anxiety in the current study . Lack of educational courses specialized in increasing dental awareness amongst non - dental university students in jordan, in addition to shortage of information about correlation between field of study and dental anxiety levels in the dental literature have raised the idea of investigating the level of dental anxiety among the different student populations . This study was therefore designed to investigate the subjective ratings of dental anxiety levels among dental, medical and engineering jordanian university students . In addition, the present study aimed to explore the sources of dental anxiety and the impact of gender on the perceived dental anxiety . Anxiety related to dental treatment was assessed by means of corah's dental anxiety scale (das). However, the modified version of das was used21 where an extra item has been included referring to the respondent's feelings toward local anesthetic injection with especial reference to the site of the injection, because the pain experienced with local anesthetic injections varies according to its location in the mouth14 . Moreover, a simplified 5-point scale - answering scheme was devised ranging from not anxious to extremely anxious . The modified dental anxiety scale (mdas) contains 5 multiple - choice items including the followings: = if you went to your dentist for treatment tomorrow, how would you feel? If you were about to have your teeth scaled and polished, how would you feel? If you were about to have a local anesthetic injection in your gum, how would you feel? The scores for each of the 5 item responses were summed up to give an estimated value of dental anxiety . The questionnaire was distributed to the third to fifth year undergraduate dental, medical and engineering students at the jordan university of science and technology . Students were personally approached in the classrooms by the authors at the end of their class . The students were informed about the study and all the points in the questionnaire were explained and clarified . Descriptive statistics were obtained and the means, standard deviation and frequency distribution were calculated . Group comparisons were analyzed using twotailed student's t tests as well as one - way analysis of variance (anova) test . Statistical significance was based on the probability values of p= 0.05 . Furthermore, as a total score of 15 or more almost indicates highly anxious patient19, the frequencies of subjects with a score of 15 or more in the 3 student groups were also reported . The total number of the participants in the current study was therefore 535, which accounts for a response rate of 89.2 percent . The distribution of the participants according to gender and field of study is presented in table 1 . Table 2 presents the means and standard deviations of individual items and total scores of the modified corah dental anxiety scale with the results of one - way anova analysis comparing the various groups based on the field of study . Out of the several anxiety scale items, the highest anxiety score (3.4) was given for tooth drilling (item 3) and scored by the engineering students . The next most anxiety - producing item was the local anesthetic injection (3.32) which was scored by the medical students . However, for the items 1, 2, and 5, the only statistically significant differences were found between the dental and the medical students (f=7.92, p = 0.00; f=4.69, p= 0.01, f=3.39, p=0.03, respectively). Dental students were significantly less anxious about scaling and polishing of teeth (f = 8.99, p = 0.00) and about tooth drilling (f = 19.58, p = 0.00) than other groups . However, there were no statistically significant differences between medical and engineering students in relation to these items (table 2). As expected, dental students scored the lowest total dental anxiety scores (table 2) which were significantly lower than those scored by either medical or engineering students (f = 14.13, p = 0.00). Generally, in terms of total anxiety scores, women were relatively more anxious than men (t = -2.21, p = 0.03) (table 3). Women were particularly more anxious than men concerning items related to waiting in the dentist's sitting room (t = -2.56, p = 0.01) and local anesthetic injection (t = -2.62, p = 0.01). However, such gender variation was not significantly different when items related to going for dental visit tomorrow, having teeth drilled or having teeth scaled or polished were considered . Sd = standard deviation the numbers and percentages of subjects who had a total score of 15 or more are shown in table 4 . Surprisingly, medical students were found to be the most frequent among those who scored 15 or above . This study revealed that dental students do have lower levels of dental anxiety than their engineering and medical counterparts (p <0.01). The mean total scores for the mdas showed that severe dental anxiety was mostly associated with drilling and intraoral local anesthetic injection . Surprisingly, the medical students showed the highest total anxiety scores and the greatest percentage of subjects scoring 15 or more . The engineering students were already expected to score the highest anxiety scores as they do not receive health or dental awareness education, whilst the medical students are supposed to be more familiar with stress management related to health measures . However, some of the highly anxious patients may avoid showing their anxiety in order not to interfere with the dentist's procedure . Thus, the dentist should be aware of the patient's possible adverse reaction or distress19,21 . However, when the data are critically appraised, statistically significant difference between groups related to total anxiety scores or the scores of individual items of the anxiety scale could be seriously misleading and possibly do not always reflect an actual clinical significance . This could be attributed to the fact that the sample size is large, which added to the power of the study and revealed even the minor statistical differences between the groups . Thus conclusions made on actual differences in numbers and on absolute statistical significance should be drawn with caution . Lack of dental health education might result in patients' fear and anxiety which in turn might end with poor patient compliance and attitudes . This will make it more difficult to manipulate patients and yield difficult patients and thus increase the levels of dental profession - related stress1,12 . Assessing the level of patient anxiety before commencing the dental treatment may offer invaluable insight into the probable patient attitudes and behavior towards the dental treatment . This information will be further utilized in developing the best strategies to manage patient anxiety . Health - related behavior depends on oral health knowledge14 . In jordan, only students related to the dental field receive adequate dental health education as it is an integral part of their curriculum . Jordanian schools and universities pay little attention to the dental health education of their students . Students not related to the dental field receive little, if any, dental health education and their curricula lack such courses . Since the dental health education is generally ignored in the pre - university stage, medical and engineering students still possess the same ideas about dentistry and dental care at the university . On the other hand, dental field - related students do have the chance to formulate new and better ideas and understanding of the dental health care and for the above mentioned reasons, the dental treatment will still be considered mysterious and stressful for the non - dental students while the dental students will feel better during the dental treatment . This could partly explain the relatively higher scores of dental anxiety among the non - dental students compared to their dental peers . Irregular dental attendance may play a major role in increasing the levels of dental anxiety15,23 . If this is added to the lack of dental health education in jordan, the high levels of dental anxiety could be explained and clearly demonstrated among the non - dental students . This might form the bases for explaining the presence of higher levels of dental anxiety among women as well as medical students in this study . Female students were found to have higher levels of dental anxiety in all groups and they were more anxious about waiting in the sitting room and taking anesthetic injections . This corroborated the results from previous studies that showed higher levels of dental anxiety among women,11,21,24 . This finding might be explained on the basis that women have higher levels of neuroticism than men and that anxiety is positively associated with neuroticism9,10,20 . However, in the current study, the statistically significant difference between men and women was marginal and the difference in anxiety scores for both genders was minimal (men 12.29%, women 13.17%). Therefore, it could be inferred that statistical significance might not be necessarily interpreted as a clinical one . Medical students might be prone to have higher levels of neuroticism, due to their courses and stressful field of study2 and thus demonstrate higher levels of dental anxiety than engineering students . On the other hand, although dental students are leading stressful courses and profession they are yet still exposed to better dental health education and knowledge and thus develop favorable dental behavior . Presence of suitable dental health education and knowledge seems to be capable, to some extent, of overruling the effect of stress and personality factors on dental anxiety among dental students . The control of dental anxiety might be aided via good dental health education, regular dental visits, good patient - dentist relationship and suitable communication with the patients6 . For a successful dental treatment, a gentle, supportive, professional, sympathetic, quiet and more considerate approach should be followed when managing patients with dental anxiety . On their first visit, patients should be dealt with more sensitively in order to avoid increasing their anxiety and thus avoid their repulsion to the dental care9 . In view of the current available data, it appears that further dental health education measures are required to be applied among the jordanian non - dental university students and the population in general in order to control the levels of dental anxiety and thus improve patient dental attitudes and compliance . Suitable standards of dental health knowledge and education could overcome the negative effects of personality and reduce dental anxiety . Interception of dental anxiety at early stages will reduce the chance of resistant dental anxiety and fear which are difficult to deal with in the dental clinic . The importance of dental health education cannot be overemphasized in the reduction and control of dental anxiety . Pre - university as well as non - dental university curricula should include dental health education in order to help reduction of dental anxiety among the population . Although the current study utilized the modified anxiety scale and investigated the levels of dental anxiety among university students from different fields of study and the sample size was representative and large, further studies are required to investigate the effect of various correlates on dental anxiety.
Toxic epidermal necrolysis (ten) is a rare life threatening idiosyncratic mucocutaneous drug reaction characterized by widespread epidermal necrosis followed by epidermal detachment . Drug - induced ten is the commonest cause and antiepileptics, antibiotics, nonsteroidal anti - inflammatory drugs (nsaids) and allopurinol are the most commonly implicated agents . Here we report a case of ten which was induced by multiple drugs some of which have been rarely implicated in the causation of ten . A 42-year - old woman was referred to medical emergency with extensive epidermal detachment and ocular, genital, and oral lesions of 5 days duration leading to a diagnosis of toxic epidermal necrolysis (ten). Seven days prior to this admission, she had presented to a general practitioner with symptoms of vertigo, anxiety, and restlessness . Although no provisional or differential diagnosis was found in the documents provided by the patient, she was prescribed antihistamines (cinnarizine and dimenhydrinate), paracetamol / metoclopramide combination and multivitamins . After 8 - 12 hours, she developed fever, malaise, sore throat, and erythematous maculopapular rash in the perioral region . She discontinued all medications and was admitted to a general hospital where she was administered piperacillin / tazobactam and dexamethasone . It is unknown whether paracetamol or antihistamines were restarted in the hospital . Her condition deteriorated and she was referred to our hospital . On presentation, she was conscious and afebrile; her heart rate was 110/min and blood pressure was 190/80 mm hg . Cutaneous examination showed involvement of about 70% total body surface area, with skin necrosis, tenderness, and a positive nikolsky's sign . She had a history of hypertension and was on some antihypertensive medication (unknown) for a few years . Test reports at three time - points she was administered intravenous fluids, antihypertensive treatment (amlodipine 5 mg / day), injection hydrocortisone (50 mg iv twice daily), and continued on the same antibiotics . Wound care was given by applying topical antiseptics on eroded areas along with nonadherent dressings . Biochemical reports showed a decrease in blood urea (from 68 mg / dl on admission to 57.6 mg / dl on the second day after admission) and a decrease in serum creatinine (from 1.1 meq / l on admission to 0.78 parenteral therapy was gradually replaced by oral drugs . On the fourth day, she developed herpetic lesions on the oral mucosa and for that tablet acyclovir was advised thrice a day along with chlorhexidine mouthwash, syrup mucaine gel (antacid), and white petroleum jelly for lips . A tzanck smear, done on fifth day after admission, failed to show any giant cells or acantholytic cells (acc) [table 1]. This test was done to rule out pemphigus . The patient showed steady improvement with the therapy given and was discharged, after 8 days, without any sequelae . This is most likely a case of drug - induced ten as other causes like upper respiratory tract infection, viral infection, or malignancy were not present . As this was an acute presentation, which occurred right after the drugs were administered, systemic lupus erythematosus (sle) and rheumatological disorders were ruled out . Ten was diagnosed from the history and typical clinical features and biopsy was not needed . The prodrome was shorter (8 - 12 hrs) than the usual 1 - 3 days . This might have been due to the use of multiple drugs causing an unknown interaction or more likely due to a previous exposure to any or all of these drugs . Causality assessment was done for each of the drugs separately by the method of kramer et al . Paracetamol and metoclopramide were categorized as probable (score 5) and antihistamines and multivitamins as possible (score 3) causal drugs for this adverse drug reaction . Since this drug intake was uneventful for a long time, the chances of this medication causing this episode of ten are minimal . There have been two case reports of ten caused by paracetamol and a case control study showing increased risk of sjs or ten with paracetamol use . There have been no case reports of ten due to a combination of both the drugs, nor have there been any reports regarding ten being caused by antihistamines or multivitamins . However, the probability of other drugs being responsible could not be ruled out completely . Ten in this patient was probably caused by paracetamol and metoclopramide, but a possibility of an interaction as well as the role of other drugs cannot be ruled out.
After hyping deep - etch electron microscopy (em) for my whole career (heuser, 2011), i'll take this invitation to write an ascb award essay to talk it up some more! Replicas are not only impervious to beam damage in the electron microscope, forever the big problem, because the electron beam heats up the sample so terribly during viewing, but their electron - scattering power is also excellent, so they are simple to image and give super high - contrast . And the key thing to remember is that replicas are utterly faithful to whatever they are replicating they're just surface renderings, copying exactly the contours of the sample and displaying these contours in the electron microscope image . So the whole approach boils down to worrying about how to prepare your biological samples for replication . It takes the right equipment and some practice to make a proper replica, but, once mastered, it's utterly routine and simple to learn . When mark kirschner first watched me do it while helping me to put it on the map by providing gorgeous cytoskeletons [heuser and kirschner, 1980]he got bored right away and asked me, can't you teach a monkey to do that?) Anyway, replicas have a glorious history, because in the early days of em, way before thin - sectioning techniques had been developed, they were the only way to go the only way to get any sort of biological sample into the electron microscope . Thus the em pioneers in the 1940s used metal replicas to discover viruses and phages and to make the first halting characterizations of macromolecular assemblies like collagen and neurofilaments . What they lacked back then was a way to see inside cells, which keith porter achieved for the first time in 1945 by simply growing cells flat enough to see through in the electron microscope really, really flat and then fixing and staining them properly for em (his other huge contribution). People not familiar with em should be reminded that porter's 1945 images opened the door to cell biology, and his development of thin - sectioning techniques for cells in the following 10 years really put cell biology on the map . But back to replicas . The whole field of scanning electron microscopy (sem) was totally dependent on them because everything had to be coated with metal in order to be seen in the scanning electron microscope . Likewise, the exciting field of freeze - fracture em took off after hans moor teamed up with a swiss company that made replicating machines (balzers of lichtenstein) and mounted a microtome inside one, so that frozen cells could be fractured open (not quite thin - sectioned, the microtome wasn't that good). This made it possible for people to make metal replicas of frozen cells without melting them even a little bit some sort of miracle! Deep - etch em is a variant of what moor introduced (heuser and salpeter, 1979) and deserves special attention only because its purpose has been to avoid all of the fixation and staining and dehydrating procedures that had accompanied previous approaches to em and essentially to get living cells replicated after they were frozen (figure 1). We found that freeze fracture works just as well or better on unfixed cells and molecules, and therefore made a huge effort to devise a really good way to freeze living cells, tissues, and cell extracts without introducing such artifacts as ice - crystal damage . A platinum replica of the inside surface of a hela cell prepared by unroofing it in culture before quick - freezing and freeze - drying it in the usual way (heuser, 2000). Three - dimensional view was used for the publicity and table cards for our department's centennial celebration three years ago . Clathrin lattices found on all cell membranes and illustrates the various stages in their evolution, from totally flat to fully curved and ready to pinch off during endocytosis . Such three - dimensional deep - etch images were the first to illustrate that f - actin filaments (highlighted in purple) often become involved in the later stages of such clathrin coated pit formation and stay behind as circular scars after coated vesicles have left the surface (above the wash in washington university). As explained in this essay, the swell opportunity to view such expanses of the plasma membrane at such a high resolution was a lucky outcome of our being able to freeze samples fast enough to avoid ice - crystal formation and then, miraculously, to platinum - replicate such frozen membranes without melting them . The best way to freeze everything turned out to be a spruced - up version of an approach anthonie van harreveld had used in the 1960s at caltech to freeze brains in preparation for classical thin - section em . Van harreveld wanted to maintain the proper distribution of electrolytes in the brain and had reason to believe that the classical fixation techniques being used on brain were distorting this distribution . He reasoned that the freeze - substitution technique that ned feder and richard sidman had put on the map in the late 1950s would give him more realistic views . With this technique, a frozen sample is fixed and prepared for embedding in plastic by dissolving the ice out of it at subzero temperatures, using acetone or the like . Van reasoned, quite correctly, that this should prevent artifacts from occurring during fixation, because nothing ever melted; but how he came up with the idea to freeze the brain by slamming it onto an ultracold block of copper remains a mystery to this day . (it's fun to mention here that van harreveld didn't start developing this technique until he was already 60 years old!) Anyway, it sure worked for van, and it also worked for tom reese and me when we copied his slammer, even though we had to spend years ironing out the bugs and making a freezing machine that was mechanically sound and gave reproducible results (heuser et al ., 1979). The result was our so - called liquid helium cooled cryopress (renamed to avoid the distressing idea of a delicate piece of tissue being slammed against anything albeit, it's the abruptness of contact and the superfast extraction of heat from the sample by the copper block that gives such good freezing in the first place). Fast - forward to today, and we find that freeze substitution is still the backbone of modern efforts to image cells in the electron microscope, and indeed preserves the structure of cells far better than the techniques of fixation and plastic embedding developed by the pioneers of thin - section em . When combined with thicker sections, higher em voltages, and modern tomographic reconstruction techniques, it yields really outstanding images . So why aren't there more than 10 labs in the world using our (or van harreveld's) cryopress to get the quality of freezing our lab has depended on for decades? The answer lies in part with another advance that hans moor spearheaded in switzerland, again with the same enlightened balzers company producing vacuum evaporators, namely, high - pressure freezing . At the time, phase diagrams of water indicated that water could be frozen into an amorphous glass without the induction of any damaging ice - crystal formation by putting it under extreme pressure (> 2000 atm). Today, theories about how water turns into vitreous (noncrystalline) ice are much more complex, but moor went ahead and developed ways to put a biological sample under huge pressures and only then freeze it by spraying liquid nitrogen at it rather than slamming it against a liquid nitrogen cooled copper block . (the rapidity of freezing, he reasoned, should no longer be important if the pressure trick works as apparently it does .) Today, most em labs have a high - pressure freezer, and most of the em papers that are published on freeze - substituted cells have availed themselves of these devices . So why not use our slammer (or cryopress) for freezing before freeze substitution, since it's cheaper, faster, more reliable, and handles larger samples? Frankly, we don't get it! Not only that, but high - pressure frozen samples cannot be freeze - fractured at all at least no one has yet devised a way to do so because the samples end up encased in various sorts of metal pressure chambers, whereas our quick - frozen or cryopressed samples are spread out and open to the world (mandatory for freeze fracture, but also good for freeze substitution). And for that matter, why aren't more labs making good old replicas of quick - frozen, deep - etched molecules (heuser, 1983; goodenough and heuser, 1984; hanson et al ., 1997)? That is, of course, the ultimate mystery to us . Probably it's just because people don't realize that there are still good replicating machines available for purchase, and people don't realize that these machines aren't so expensive and are easy to operate . Well, as i said at the outset, i've been hyping our technique for decades and can't stop now . I believe that an opportunity is being missed and that simplifying techniques so that even a monkey could do it will attract not monkeys to the field, but serious young investigators who want to get their hands on electron microscopes and want to get the most true to life i'm a photographer at heart and love sharing images, all sorts of images, with people who appreciate them and can learn from them i love that more than anything . What fun it was, to be able to interact on a daily basis with the mark kirschners, tom pollards, ron vales, bernie gilulas, and ira mellmans of cell biology (and sorry to all those whom i didn't mention you know who you are!). Plus, a handful of people really fired me up: tom reese, my boss as a postdoc at the national institutes of health, with whom i became so intertwined for so many years that he and i will never know who did what or who deserves what credit in the original development of quick - freeze, deep - etch em (heuser and reese, 1973; heuser et al ., 1979); and then nobutaka hirokawa, who came to my lab as a postdoc, and immediately orchestrated a host of collaborations with leading cell biologists around the world that put deep etching on the map (before leaving for the university of tokyo to become chairman of the department of cell biology, and then dean of the medical school, and now head of the whole human frontier science program); and finally, my ex ursula goodenough, who absorbed my images and simply took off, making huge advances in several fields, thanks to her deep grasp of all aspects of cell biology . Finally, i'd like to simply add this: biological em was terribly interesting for me in the early days, back when it first allowed people to zoom in on the structures that light microscopists had been studying for so long and show what they actually were i used to wait with eager anticipation for each new issue of the journal of cell biology to arrive in the mail and then would devote a whole evening (maybe with a glass of wine) to carefully examining every new electron micrograph published that month . But em became even more captivating for me as people began more and more to systematically manipulate cells by physical and pharmacological (and eventually genetic) methods and then to look in the microscope to see how this altered the fine structures of their cells . This opened the door to true structure / function correlations at least when the effects of these experimental manipulations of cell physiology and biochemistry were properly determined, along with the microscopy . This era of em was the most fun for me, personally, but as it happened, this heyday was cut short by an overwhelming urge in some quarters to improve the methods of em, in an attempt to make the imaging of cells more this trend particularly captivated the equipment manufacturers and led to an arms race of microscope development that ended up making electron microscopes so very costly that only a few centers could support them anymore . The result was actually a curtailment of general, everyday em as it had been practiced by individual investigators in command of their own microscopes and published every month in the journal of cell biology . And as a consequence, over the past 15 years or so, em has gradually been relegated to a service status, carried out largely by em cores in most major institutions . Electron microscopists, and gone also is the use of em for all sorts of fun structure / function correlations . And helping to eclipse the routine em that i enjoyed so much have been all the tremendous advances in light microscopy, coupled with all the advances in digital camera recording of live - cell dynamics (not to mention the burgeoning field of superresolution light microscopy, crowned this year with the nobel awards). These huge advances have captivated nearly everyone still interested in functional correlations of cell structure and have left traditional em sort of out in the cold, an outcome i find most unfortunate . I feel strongly that seeing cell structures at the em level still is the only way to fully grasp their molecular architecture, and that seeing changes in their molecular architecture at this level is the only way to truly understand their function . I'm permanently stuck with the founding fathers' view that cell ultrastructure will ultimately display and explain all of cell function! George palade was my greatest hero, and his fun explanation in his nobel lecture of why he chose to study the pancreatic acinar cell is my favorite quote: perhaps the most important factor in this choice was the appeal of the amazing organization of the pancreatic acinar cell, whose cytoplasm is packed with stacked er cisternae studded with ribosomes . Its pictures had for me the effect of the song of a mermaid: irresistible and half transparent . Its meaning seemed to be buried under only a few years of work, and reasonable working hypotheses were already suggested by the structural organization itself . Irresistible and half transparent, indeed! Thanks, george . And thanks to all of you who cared to look at my images and all the institutions and funding agencies that made it possible for me to generate them! Every picture i take, i already have an audience for it right as i take it . (of course, they're not actually there, they may be continents away, but i'm imagining them being there and already planning how i will get that picture to them and what i'll tell them about it as soon as it's in the computer .) It's for showing to someone who immediately comes to mind as soon as that field pops into view in the electron microscope . Oh, pietro will love that huge neuromuscular junction; fulvio will be amazed by that quality of membrane preservation in freeze - substituted yeast; ursula will be psyched by that run of axonemal dynein; tom will be impressed with such a clear view of actin branchpoints . Only rarely am i lucky enough to have someone actually sitting next to me and to be able to talk to him or her right then, person to person maybe a new postdoc or a close collaborator who really needs to look over my shoulder to see how his or her prep came out . Anyway, i want each of my real or imaginary viewers to like that picture, to think it's a good picture attractive, clear, understandable, useful, illuminating, that is, illuminating something about the subject (be it a personal portrait or a picture of a cell interior or a molecule). I want my audience's appreciation! My whole drive of focusing all my work on improving techniques of preparation for em has come from wanting to take better pictures and get more of that appreciation . Besides that, there's just that darn old curiosity: what does it actually look like, what does it look like exactly? How good a picture of it can i take? How good - looking can i make it (or him or her, with my personal portraits)? (nic spitzer once irritably dubbed the latter my thin sections of life as i was clicking away while canoeing with him down a rapids on the allagash river, but not paddling .) Always on my mind is what's the most expressive or most characteristic or attractive attire or decoration i can outfit it (them) with? Osmium or platinum or gold or furs and silks? Capturing that best picture will help me to get to know my subject better, to really see it for what it is . Even artifacts can be extremely beautiful and informative, if one knows how one got them and what they say about what the structure was, before it got altered . All these aspects of photography i can appreciate by myself, all alone, but never as much as when there is just one other person with me, with the same inclination and proclivity . Sharing, mutual appreciation, communion that has been the whole name of the game for me in my research career . My advisor don fawcett, one of the great masters of em of all times, told me when i graduated from medical school, don't become an electron microscopist, you'll become everybody's slave . Actually, i think i can say that it turned out just the opposite: everyone else turned out to be my audience, my source of appreciation and self - worth, my foils, my mentors, and, most important of all, my best source for interesting things to look at in the electron microscope!
In women, cervical cancer is the second most common malignant tumor worldwide and is one of the leading causes of cancer - related death in developing countries . Although the incidence of cervical cancer is generally decreasing, it is still a serious public health problem worldwide, especially in developing countries, and the average age of cervical cancer patients is decreasing . Human papillomavirus (hpv) is considered the major cause of cervical cancer, but viral infection alone is not sufficient for its development . The pathogenesis of cervical cancer is still unclear and probably involves aberrant expression of numerous oncogenes and anti - oncogenes . Many distinct advances have been made in the prevention, surgical resection, radiotherapy, and chemotherapy of cervical cancer, but the prognosis of cervical cancer patients remains poor . Therefore, it is important to better understand molecular events in the invasion and metastasis of cervical cancer and to develop novel prognostic markers and therapeutic strategies . Stromal cell - derived factor-1 (sdf-1), which is a chemoattractant cytokine, is involved in a variety of physiological and pathological activities . With regard to cancer, substantial evidence indicated that sdf-1 plays crucial roles in the cell growth, apoptosis, invasion, and metastasis of many kinds of cancers [79]. It is well acknowledged that chronic inflammation is widely connected with the initiation and progression of cervical cancer . Inflammatory cytokines such as chemokines, inflammatory proteins, and adhesion molecules are known as the main causes of chronic inflammation . Numerous studies have revealed that nf-b, as a transcription factor, is cheifly responsible for the activity of these cytokines and is also implicated in the development of a wide range of cancers . However, to the best of our knowledge, the final role of sdf-1and nf-b in cervical cancer remains unclear . In the present study, we used tissue microarray and the immunohistochemistry method to detect the expression of sdf-1and nf-b in 105 cases of human cervical cancer tissues and their paired adjacent tissues . The purpose of this study was to investigate the association of sdf-1and nf-b expression with clinicopathological parameters of cervical cancer and to evaluate the prognostic value of sdf-1and nf-b expression in cervical cancer patients . We analyzed a total of 105 formalin - fixed, paraffin - embedded cervical cancer tissues and the adjacent tissues at the time of operation from january 2002 to november 2013 at east hospital, tongji university . All these cancer tissues were confirmed as cervical cancer by medical examination and hematoxylin and eosin staining after surgical resection . None of the patients received adjuvant chemotherapy, radiation therapy, or other anti - tumor therapies . Important related clinicopathological parameters of the patients, such as age, tumor size, figo stage, lymphatic metastasis, stromal invasion, differentiation, and survival time, were obtained from each patient s medical records and are shown in tables 1 and 2 . The survival time was calculated from the date of surgery to the date of death, or the last known follow - up . All cervical cancer tissue samples included in this investigation were obtained with patients written informed consent . A standard immunohistochemistry method was used to study sdf-1and nf-b expression in cervical cancer tissues and the adjacent tissues . Briefly, the tumor tissues and adjacent tissues were fixed in 10% formaldehyde and embedded in paraffin and then we cut the paraffin sections into 4-m sections . All the 4-m tissue sections were dewaxed and rehydrated with xylene and graded alcohol, respectively . We washed the sections with buffer solution for 5 min and then added the primary antibody into them at 4c overnight . After that, we washed the sections again and added the second antibody (r&d systems inc ., minneapolis, mn; dilution 1:50) into the sections . Afterwards, we washed the sections with phosphate - buffered saline (pbs) and developed them with 3, 3-diaminobenzidine (dab) for 5 min and counterstained them with hematoxylin . Results are presented as the percentage of the staining cells (0 to 100%) in tissues . Staining under 20% of the tissue cells or no staining was included in the negative group (), while the others belonged to the positive group (+). All the sections were assessed under an optical microscope by 2 independent investigators, and any discrepancy in immunohistochemistry was resolved by consensus . The expression of sdf-1 and nf-b in cervical cancer and adjacent cancer tissues was compared with the paired wilcoxon test . Chi - square test and fisher s exact test were used to assess the association between clinical characteristics of cervical cancer patients and sdf-1and nf-b expression . The prognosis of cervical cancer and sdf-1and nf-b expression were determined using kaplan - meier survival analysis and log - rank test for univariate analysis . All statistical analyses were performed using spss version 18.0 software (spss inc ., chicago, il). We analyzed a total of 105 formalin - fixed, paraffin - embedded cervical cancer tissues and the adjacent tissues at the time of operation from january 2002 to november 2013 at east hospital, tongji university . All these cancer tissues were confirmed as cervical cancer by medical examination and hematoxylin and eosin staining after surgical resection . None of the patients received adjuvant chemotherapy, radiation therapy, or other anti - tumor therapies . Important related clinicopathological parameters of the patients, such as age, tumor size, figo stage, lymphatic metastasis, stromal invasion, differentiation, and survival time, were obtained from each patient s medical records and are shown in tables 1 and 2 . The survival time was calculated from the date of surgery to the date of death, or the last known follow - up . All cervical cancer tissue samples included in this investigation were obtained with patients written informed consent . A standard immunohistochemistry method was used to study sdf-1and nf-b expression in cervical cancer tissues and the adjacent tissues . Briefly, the tumor tissues and adjacent tissues were fixed in 10% formaldehyde and embedded in paraffin and then we cut the paraffin sections into 4-m sections . All the 4-m tissue sections were dewaxed and rehydrated with xylene and graded alcohol, respectively . We washed the sections with buffer solution for 5 min and then added the primary antibody into them at 4c overnight . After that, we washed the sections again and added the second antibody (r&d systems inc ., minneapolis, mn; dilution 1:50) into the sections . Afterwards, we washed the sections with phosphate - buffered saline (pbs) and developed them with 3, 3-diaminobenzidine (dab) for 5 min and counterstained them with hematoxylin . Results are presented as the percentage of the staining cells (0 to 100%) in tissues . Staining under 20% of the tissue cells or no staining was included in the negative group (), while the others belonged to the positive group (+). All the sections were assessed under an optical microscope by 2 independent investigators, and any discrepancy in immunohistochemistry was resolved by consensus . The expression of sdf-1 and nf-b in cervical cancer and adjacent cancer tissues was compared with the paired wilcoxon test . Chi - square test and fisher s exact test were used to assess the association between clinical characteristics of cervical cancer patients and sdf-1and nf-b expression . The prognosis of cervical cancer and sdf-1and nf-b expression were determined using kaplan - meier survival analysis and log - rank test for univariate analysis . All statistical analyses were performed using spss version 18.0 software (spss inc ., chicago, il). Typical immunohistochemistry images of sdf-1 and nf-b in cervical cancer tissues and paired adjacent non - tumor tissues are shown in figures 1 and 2 . The positive percentage of sdf-1 expression in cervical cancer and adjacent non - tumor tissues were 68.6% (72/105) and 8.6% (9/105), respectively . The positive percentage of nf-b expression in cervical cancer and adjacent non - tumor tissues were 60.0% (63/105) and 5.7% (6/105), respectively . The chi - square test was used to confirmed that the difference in the expression level of sdf-1and nf-b between cervical cancer tissues and paired adjacent non - tumor tissues was statistically significant (p<0.05). The relationship between patients clinical parameters and the expression of sdf-1and nf-b is shown in tables 1 and 2, respectively, showing that the expression of sdf-1 in the cervical cancer tissues is significantly correlated with tumor size (=10.794, p=0.001) and figo histology grade (=7.000, p=0.008). However, there was no statistical correlation between sdf-1 expression and patient age, lymph node metastasis (lnm) status, stromal invasion, or tumor differentiation (p>0.05). Similarly, the expression of nf-b in the cervical cancer tissues was significantly correlated with tumor size (=7.421, p=0.006), figo histology grade (=10.182, p=0.001), and lymph node metastasis (lnm) status (=4.762, p=0.029). However, there was no statistical correlation was found between nf-b expression with patient age and stromal invasion and tumor differentiation (p>0.05). Therefore, the results demonstrate that higher sdf-1 and nf-b expression in cervical cancer tissues is positively correlated with tumor metastasis and cancer progression, suggesting that sdf-1and nf-b play important roles in the progression . To further evaluate the relationship between sdf-1and nf-b expression and prognosis of cervical cancer, we performed log - rank survival analysis according to the sdf-1 and nf-b expression level and patient survival data . The survival analysis demonstrates that the cervical survival rate of the patients with negative sdf-1 and nf-b expression is significantly better than that of the patients with positive ones (p<0.05, figures 3, 4). Furthermore, a multivariate cox regression analysis demonstrated that sdf-1 expression and lymph node metastasis are independent predictors of the os in cervical cancer patients . The expression of sdf-1 was positively correlated with the expression of nf-b (r=0.201, p=0.040; table 3). Typical immunohistochemistry images of sdf-1 and nf-b in cervical cancer tissues and paired adjacent non - tumor tissues are shown in figures 1 and 2 . The positive percentage of sdf-1 expression in cervical cancer and adjacent non - tumor tissues were 68.6% (72/105) and 8.6% (9/105), respectively . The positive percentage of nf-b expression in cervical cancer and adjacent non - tumor tissues were 60.0% (63/105) and 5.7% (6/105), respectively . The chi - square test was used to confirmed that the difference in the expression level of sdf-1and nf-b between cervical cancer tissues and paired adjacent non - tumor tissues was statistically significant (p<0.05). The relationship between patients clinical parameters and the expression of sdf-1and nf-b is shown in tables 1 and 2, respectively, showing that the expression of sdf-1 in the cervical cancer tissues is significantly correlated with tumor size (=10.794, p=0.001) and figo histology grade (=7.000, p=0.008). However, there was no statistical correlation between sdf-1 expression and patient age, lymph node metastasis (lnm) status, stromal invasion, or tumor differentiation (p>0.05). Similarly, the expression of nf-b in the cervical cancer tissues was significantly correlated with tumor size (=7.421, p=0.006), figo histology grade (=10.182, p=0.001), and lymph node metastasis (lnm) status (=4.762, p=0.029). However, there was no statistical correlation was found between nf-b expression with patient age and stromal invasion and tumor differentiation (p>0.05). Therefore, the results demonstrate that higher sdf-1 and nf-b expression in cervical cancer tissues is positively correlated with tumor metastasis and cancer progression, suggesting that sdf-1and nf-b play important roles in the progression . To further evaluate the relationship between sdf-1and nf-b expression and prognosis of cervical cancer, we performed log - rank survival analysis according to the sdf-1 and nf-b expression level and patient survival data . The survival analysis demonstrates that the cervical survival rate of the patients with negative sdf-1 and nf-b expression is significantly better than that of the patients with positive ones (p<0.05, figures 3, 4). Furthermore, a multivariate cox regression analysis demonstrated that sdf-1 expression and lymph node metastasis are independent predictors of the os in cervical cancer patients . The expression of sdf-1 was positively correlated with the expression of nf-b (r=0.201, p=0.040; table 3). It has 2 major isoforms: and . Both are derived from a single gene due to alternative splicing . It is well acknowledged that sdf-1 mediates a large number of crucial biological processes such as neuronal and cardiac development, angiogenesis, apoptosis, and stem cell motility . Substantial evidence indicates that sdf-1 can mediate the activation of sdf-1/crcr4-pi3-mark - nf-b pathway, pi3k / akt, wnt, and erk pathways, and further promotes the invasion and progression of cancers [1719]. Sdf-1 can also improve the expression of mmps and vegf, which are crucial for the aggressive behavior of cancers . Many studies have revealed that the over - expression of sdf-1 is found in various malignancies, including lung cancer, prostate cancer, breast cancer, and pancreatic cancer . The nf - kb transcription factor family includes 5 genes: nf-b1 (p50/p105), nf-b2 (p52/p100), rela (p65), c - rel, and relb . Normally, nf - kb dimmers are transcriptionally inactive due to their interaction with nf - kb inhibitors (i kbs). Activation of nf-b may result from the enhanced expression of epidermal growth factor receptor, insulin growth factor receptor, and tumor necrosis factor receptor families . Activation of some other pathways, like pi3k / akt and ras / mapk, also participate in the activation of nf-b . Some different kinds of molecular alterations in cancer cells may lead to impaired regulation of nf-b activation . In this situation, nf - kb becomes constantly activated, which leads to aberrant expression of downstream genes of nf-b, including those involved in regulation of cell cycle, proliferation, adhesion, and apoptosis . These pathophysiological changes may finally lead to the initiation, development, and metastasis of cancer . The results of this study indicate that the expression of sdf-1 is increased in cervical cancer tissues and is correrated with tumor size and figo histology grade . We found that the expression of nf-b is increased in cervical cancer tissues and is correrated with tumor size, figo histology grade, and lymph node metastasis . The potential mechanism may be that sdf-1 activates the nf-b pathway by the interaction with cxcr4 . The sdf-1/crcr4-nf-b pathway participates in the regulation of cell prliferation, apoptosis, and angiogenesis in cervical cancer . The increased expression of vegf and mmps caused by sdf-1-cxcr4 interaction and many nf-b - related biological macular - involved cell cycle, apoptosis, and chronic inflammation responses may mediate the initiation and progression of cervical cancer [3032]. It is well known that cancer pathogenesis is a multi - factor, multi - step, complicated process involved in gene - gene and gene - environment interactions . Large and well - designed studies are still needed to elucidate the pathogenesis of cervical cancer . Results of our study indicate that the expression of sdf-1 is significantly associated with tumor size and figo histology grade . The expression of nf-b is significantly associated with tumor size, figo histology grade, and lymph node metastasis . We also found that positive sdf-1 or nf-b expression is significantly correlated with poor prognosis.
Pycnodysostosis was first reported in 1923 by montanari, and he called it as atypical achondroplasia . The main characteristics are short stature, cranial dysplasia, increased bone density and fragility . Other clinical features include open cranial sutures, hypoplastic paranasal sinuses, dysplastic lateral clavicle, shortened terminal phalanges, proptosis, blue sclera and frontal or occipital bossing . Oral manifestations include obtuse gonial angle, grooved palate, anterior cross - bite, malpositioned teeth associated with increased incidence of dental caries and periodontitis, hypoplastic maxilla, receded chin, delayed eruption of permanent teeth, delayed exfoliation of deciduous teeth and hypoplasia of root - obliterated pulp spaces . A 47-year - old man reported to the department with a complaint of deformed lower jaw for the past 10 years . History revealed that the patient had undergone extraction of his teeth that was uneventful, following which there was fracture of jaw at the extraction site . Subsequently, there was frequent exfoliation of teeth with fracture at different sites in the lower jaw . His medical history revealed that he had multiple fractures of the upper and lower limbs and a history of diabetes mellitus and hypertension . General examination revealed that the patient's height was 127 cm and weight was around 49 kg, with proportionate dwarfism [figure 1]. The hand and feet had short digits with overlying cutaneous wrinkles that tapered off with large overriding nails [figure 2]. A 47-year - old man, 127 cm in height shortened digits and cutaneous wrinkles on extraoral examination, there was facial dysmorphia with prominent forehead (frontal bossing), proptosis, beaked nose, deep nasolabial skin folds, micrognathia and obtuse mandibular angle on the right side [figure 3]. Mild proptosis and facial dysmorphia intraoral examination revealed multiple clinically missing teeth, chronic periodontitis, narrow and grooved palate, no features of enamel hypoplasia, malposed teeth [figure 4] and evidence of sequestrum in relation to the tooth 46 [figure 5]. Based on the history and clinical presentation, a provisional diagnosis of a bone dysplasia, probably pycnodysostosis, was made and differential diagnosis of cleidocranial dysplasia and osteopetrosis was included . Grooved palate and clinically missing maxillary anteriors sequestrum in relation to tooth 46 laboratory findings were within normal limits, including hemoglobin conc ., differential count calcium, phosphate, alkaline and acid phophatase level . Computed tomography of the bone window of the skull showed open sutures and fontenalles with nonaerated paranasal sinuses, flattening of the mandibular angle on the right side with evidence of fracture and loss of bone architecture on the left side involving the ramus of the body of the mandible and hypoplastic maxilla [figures 68]. Orthopantomograph revealed generalized bone loss, multiple missing teeth and obtuse gonial angle with loss of bone structure on the left side of the mandible involving the body and ramus [figure 9]. 3d - reconstructed computed tomography showing open sutures and fontanelles 3d - reconstructed computed tomography showing fractured body of the mandible and obtuse gonial angle 3d - reconstructed computed tomography showing fractured body of the mandible opg showing generalized bone loss, multiple missing teeth, and obtuse gonial angle lateral skull showing open fontanelles with nonaerated paranasal sinus and fractured body of the mandible the patient was surgically managed for osteomyelitis by removal of the sequestrum and curettage, and further mandibular reconstruction was performed . The patient presents with characteristic facies, dwarfism, beaked nose, prominent head and generalized increase in the density of bones not sufficient to obliterate medullary canals or cranial orifices . Frequent fractures due to trauma can aid in diagnosing this condition . In our case, the patient was negligent about the condition and reported to us with a fractured jaw . Intraoral clinical presentation included altered pattern of exfoliation of deciduous teeth and eruption of the permanent dentition . The disease is diagnosed at an early age, wherein the main reasons for consultation are generally short stature and open anterior fontanelles . In later stages, consultation is usually for fracture resulting from slight or moderate trauma, given the severe bone fragility . Symptoms include dental abnormalities, with hypoplasia of the enamel, obliterated pulp chambers and hypercementosis . Protrusion of the incisors with anterior open bite may be found, and dental crowding associated with extensive caries and periodontitis is frequent . In our case, multiple clinically missing teeth, chronic periodontitis, narrow and grooved palate, features of enamel hypoplasia and malposed teeth these conditions cause the premature loss of dentition that may already be complete by the fourth decade of life, similar to our patients . Our patient showed evidence of sequestrum in relation to the tooth 46 as a result of osteomyelitis . Orofacial infections are commonly encountered by the dentist and there are wide ranges of modalities that can be implemented in managing them . This is caused by the increased bone volume of the sella turcica that, on compressing the pituitary gland, causes its hypoplasia and a deficient production of the growth hormone . Our patient's height was 127 cm and weight was around 49 kg, with proportionate dwarfism . The hand and feet had short digits with overlying cutaneous wrinkles that tapered off with large overriding nails . Diagnosis of pycnodysostosis is based on the clinical presentation, and medical treatment for the condition is symptomatic . The differential diagnosis of pycnodysostosis includes osteopetrosis, acroosteolysis, mandibular acral dysplasia and cleidocranial dysplasia . Unlike osteoporosis, hepatosplenomegaly and difference between pycnodysostosis and cleidocranial dysplasia is that dense and brittle bones are found in pycnodysostosis but not in cleidocranial dysplasia . New treatment modalities like gene therapy and bone marrow transplant can be expected to be the mainstay in the future, now that the abnormal expression of cathepsin k and the gene defect has been located . Now - a - days, symptomatic treatment is provided for patients with pycnodysostosis, with the main intention of prevention of fractures . As pycnodysostosis is associated with inappropriate bone remodeling, it can pose a challenge for a dental health care professional to provide treatment as there can be serious complications, such as osteomyelitis arising as a result of dental infections . Tooth extractions in these patients demand certain special care, such as carrying out the surgery atraumatically with proper asepsis . Oral hygiene practices and frequent visits to
The accumulation of metals in the environment, stemming from their origination in the earth's crust, as well as from anthropogenic sources, creates the potential for significant human exposures and subsequent health hazards . Deleterious metal - induced health effects, including carcinogenesis and neurodegeneration, have been reported in all body systems, with exposure stemming from multiple sources, including contact with contaminated food, water, air, or soil . The particular metals considered in this review, antimony, arsenic, cadmium, manganese, mercury, silver, and uranium, are among the classes of essential nutrients, as is the case with copper and manganese, as well as the nonessential, naturally occurring metals, such as arsenic, cadmium, and mercury, all of which can induce toxicity depending on the concentration level and exposure duration . Over time, organisms have developed protective mechanisms to deal with metal exposure, most of which function in one of three ways: (1) decreasing the uptake of the metal, (2) stimulating the expulsion of the metal, or (3) activating the organism's general stress response mechanisms . Metals can disrupt homeostasis by generating oxidative stress, inhibiting enzyme activity, impairing dna repair, and disrupting protein binding and normal cellular function, including proliferation, cell cycle progression, and apoptosis [14]. Elucidating the mechanism(s) of metal detoxification has been difficult due to the complexity of mammalian systems and the reductionist approach inherent to cell culture systems . The model organism, caenorhabditis elegans (c. elegans), offers the advantage of an in vivo system that is less complex than the mammalian system while still sharing high homology . C. elegans possess ~60%80% of human genes and contain conserved regulatory proteins [68]. This soil nematode has been used in a number of toxicity studies due to its well - characterized genetic, physiological, molecular, and developmental stages . Some of the advantages afforded by the c. elegans model system are small size (~1.5 mm adult), short lifespan (~3 weeks), and rapid lifecycle (~3 days) [810]. At adulthood, a single c. elegans hermaphrodite is capable of producing ~300 progeny . C. elegans are hermaphrodites, but approximately 1% of c. elegans are male, allowing for genetic experimentation . The nematode's small genome and relative anatomical simplicity (less than 1000 cells) contribute to the appeal of this model system for genetic manipulation . In addition, the use of rna interference (rnai) and chromosomal deletion in worms has provided valuable information regarding the increased sensitivity of mutant strains to metal toxicity [1214]. Maintenance of nematode strains is relatively simple; they grow on bacteria - seeded plates and can be maintained at 20c . Strains can also be frozen indefinitely, easily allowing for the accumulation of large stocks of worms . C. elegans provide the researcher with a uniquely powerful model, as the worm's translucent body allows for the in vivo visualization of fluorescently labeled individual cells and proteins . The in vivo c. elegans model system is especially valuable for the investigation of metal detoxification and is particularly amenable for examining gene - environment interactions, albeit with a few considerations to take into account (table 1). Several toxicity endpoints are readily detected and well documented in the nematode, including mortality, lifespan, reproduction, and feeding [1719]. Acute toxicity can also be assessed in the nematode through behavioral endpoints, such as locomotive behavior, head thrashing, body bending, and other basic movements [2024]. Recently, the role of c. elegans as a biomonitor in environmental risk assessment has also been explored [2528]. Several cellular systems such as the glutathione (gsh), metallothioneins (mts), heat shock proteins (hsps), as well as various pumps and transporters work in concert to detoxify and excrete metals . It remains to be established whether the knockdown or overload of one detoxifying system upregulates other compensatory mechanisms . In this review, we offer a brief summary of the ways in which the c. elegans model has shed novel insights on the various mechanisms of metal detoxification . Metals considered herein are antimony (sb), arsenic (as), cadmium (cd), lead (pb), mercury (hg), silver (ag), and uranium (u). Glutathione (gsh) is a cysteine - containing tripeptide, consisting of glutamic acid, cysteine, and glycine and is found in most life forms . Gsh possesses antioxidant properties, since the thiol group of cysteine is a reducing agent and can be reversibly oxidized (gssg) and reduced (gsh). Gsh is maintained in the reduced form by the enzyme, glutathione reductase (gr), and functions by reducing other metabolites and enzyme systems . Glutathione peroxidase (gpx) catalyzes the oxidation of gsh to gssg in the presence of ros (figure 1). Gssg can be converted back into gsh via gr and the conversion of nadph to nadp+ . Proton - translocating mitochondrial nicotinamide nucleotide transhydrogenase (nnt) catalyzes the reduction of nadp+ by nadh and is an important source of nadph, as has been demonstrated using nnt-1 deletion mutants . Glutathione s - transferases (gsts) can catalyze the conversion of gsh to gs, which can then form complexes with various xenobiotics to facilitate excretion . These enzymes are found at particularly high levels in the liver, and gsh is typically the most abundant sulfhydryl - containing compound in cells . Additionally, gsh is known to offer protection against metal - generated ros by binding free radicals . Gshs have been reported to increase, decrease, or remain constant after exposure to metals [5557]. The gsh system is found in animals, plants, and microorganisms . C. elegans express approximately 50 gsts . Additionally, phytochelatins (pcs), a family of metal - inducible peptides synthesized enzymatically from gsh by pc synthase (pcs) in the presence of heavy metal ions, have been identified in c. elegans . Although pcs are synthesized from gsh, they are broadly classified as class iii metallothioneins and have been shown to be important in the detoxification of heavy metals . Liao and yu investigated the involvement of the gsh system in response to inorganic arsenic exposure . Results confirmed that oxidative stress plays a role in arsenic - induced toxicity by mutating glutamylcysteine synthetase (gcs) (gcs-1), the rate - limiting enzyme in gsh synthesis, in worms . The gcs-1 loss - of - function strain demonstrated hypersensitivity to arsenic exposure in lethality testing, as compared to wild - type animals, an effect that was rescued by the addition of gsh to the medium, indicating that these enzymes are crucial for mediating arsenic - induced toxicity . Furthermore, helmcke and aschner reported a significant increase in fluorescence in a gst-4::gfp strain following both acute and chronic exposure to mehg . It was also demonstrated that knockout gst-4 worms did not display greater sensitivity to mehg than did n2 wild - type worms . Gsh levels were found to be increased in worms subjected to acute exposure, whereas worms subjected to chronic exposure exhibited depleted levels of gsh . In their hormetic model, helmcke and aschner demonstrated an increase in gst-4::gfp expression after low concentration, acute exposure, a finding which indicates that gst-4 may be involved in the hormetic response to mehg . Taken together, the data from these studies suggest that gst-4 contributes to the response to mehg exposure, but that knockdown of this gene does not affect with overall lethality . In a study examining the role of pcs in the elimination of cadmium, vatamaniuk and colleagues reported that the c. elegans pcs-1 gene encodes a functional pc synthase critical for heavy metal tolerance . Using double stranded rnai against pcs-1, this group showed that, although the progeny of worms injected with the dsrnai and exposed to cadmium (525 m) managed to reach adulthood, these worms were small, necrotic, sterile, and had a much shorter lifespan than did the wild - type controls . After being exposed to 50 and 100 m concentrations of cadmium, the pcs-1 worms arrested at the l2l4 stage were necrotic and died by day 6 . The results were definitively dependent on the presence of cadmium, because pcs-1-deficient worms, in the absence of cadmium exposure, responded identically to the wild - type controls . Hughes and colleagues examined metabolic profiles following cadmium exposure in phytochelation synthase-1 (pcs-1) mutants . Results from these studies showed that the primary response to low levels of cadmium is the regulation of the transsulfuration pathway, due to the fact that cadmium exposure caused a decrease in cystathionine concentrations and an increase in phytochelation-2 and -3 . These results were corroborated by additional studies which demonstrated that pcs-1 mutants were an order of magnitude more sensitive to cadmium than were the metallothionein mutants . Furthermore, the mt - pcs-1 triple mutant was found to display an additive sensitivity toward cadmium . Metallothioneins (mts) belong to a family of cysteine - rich low - molecular - weight metal - binding proteins (mw 3,50014,000 da) involved in metal detoxification and homeostasis . Mts bind both metals of physiological importance such as copper and zinc, as well as xenobiotics including arsenic, cadmium, mercury, and silver . The binding of these metals occurs via the interaction of the cysteine residues with thiol groups . Binding of metals by mts may be transient, as mts are capable of rapidly releasing metal ions . The protective roles of mts can be ascribed to their three primary functions: (1) metal homeostasis, (2) heavy metal detoxification, and (3) protection from oxidative stress . Additionally, these proteins have been identified as contributors to the hormetic response [39, 61]. Mammals express four known metallothionein isoforms (mt - i, mt - ii, mt - iii, mt - iv). Mt - i and mt - ii are expressed in almost all tissues and have been best characterized with regard to their protection of the brain . Mt - iii is especially enriched in the central nervous system, although its role has not yet been clearly defined . Mt - iv is most abundantly expressed in the stratified squamous epithelia [6466]. Mt expression has been shown to be induced under stressful cellular conditions such as exposure to cytokines, glucocorticoids, reactive oxygen species (ros), and metal ions . Mts can bind directly and sequester the toxicant; they also can provide protection by acting as antioxidants (figure 1). Further, mts can limit apoptosis and promote the survival of mitochondrial dysfunctional cells by serving as highly efficient reducing elements against reactive oxygen species (ros). C. elegans contain two distinct isoforms of mts, known as mtl-1 and mtl-2, which can be induced in response to exposure to various metals . Jiang and colleagues examined the effects of mts on depleted uranium (du) in c. elegans . Results from their study showed that mtl-1 was an important factor in uranium accumulation in c. elegans as knockouts displayed increased cellular accumulation . In a study investigating lead and methylmercury toxicity, ye and colleagues demonstrated the involvement of mts in affording a protective cross - adaptation response to neurobehavioral toxicity . This endpoint was assessed by observing behavioral alterations (head thrashing and body bending) in worms that were exposed during the l2 phase to either pb or mehg . The study was conducted in conjunction with mild heat shock, wherein pretreatment of the larva with heat shock prevented the neurobehavioral deficits and the stress response at lower concentrations (50100 m) but not at higher concentrations (200 m). Additionally, mild heat shock coupled with exposure to a low concentration of either metal was found to induce mtl-1 and mtl-2 promoter activity and gfp gene expression, results that were not observed in either the metal - exposed or heat - shocked cohort alone . Finally, the overexpression of mtl-1 or mtl-2 at the l2 stage was shown to significantly repress neurobehavioral toxicity, suggesting that the accumulation of mt protein is necessary to confer the protective response to the toxicant . Similarly, helmcke and aschner reported that mtl knockouts displayed increased lethality upon exposure to mehg . This group also demonstrated increases in mtl-1::gfp fluorescence in response to acute mehg exposure at the l1 stage; however, chronic mehg exposure produced no change in florescence . Their results indicate that mtl-1 is important in mediating mehg toxicity and that the effects occur in a concentration- and time - dependent manner . Meyer and colleagues examined the aggregation of silver nanoparticles (agnps) in wild type and mtl-2 c. elegans . Results from these studies showed that the mtl-2 strain displayed greater agnp sensitivity than did the wild - type controls . These data indicate that there may be a differential preference for mtl-1 over mtl-2 depending on the particular metal to which an organism is exposed . In a 2004 study by swain and colleagues, using gfp - expressing transgenes, mt - null alleles, and the rnai knockdown of mts, this group demonstrated that cadmium but not copper or zinc was able to influence a concentration - dependent, temporal transcription response . Cadmium exposure caused a reduction in body size, generation time, brood size, and lifespan, effects that were magnified in the mt knockdown worms . Hughes and colleagues studied metabolic profiles using proton nmr spectroscopy and uplc - ms following cadmium exposure in single and double mtl knockouts . Results showed that the metallothionein status did not influence the metabolic profile in cadmium - exposed or unexposed worms . The primary response to low levels of cadmium was the regulation of the transsulfuration pathway, due to the fact that cadmium exposure resulted in a decrease in cystathionine concentrations and an increase in phytochelation-2 and -3 . These results were corroborated by data showing that pcs-1 mutants (phytochelation synthase-1) were an order of magnitude more sensitive to cadmium than were mt mutants . Further, an additive sensitivity toward cadmium was observed in the mt - pcs-1 triple mutant . A study by bofill and colleagues examined zinc and cadmium toxicity; results indicated differential metal binding behavior for mt-1 as compared to mt-2 . Specifically, the mt-1 isoform showed optimal behavior when binding zn, and mt-2 showed optimal behavior when binding cd . Accordingly, it was hypothesized that, due to its induction following cd exposure, mt2 is primarily responsible for detoxification, whereas mt1 possesses some degree of constitutive expression and is, therefore, primarily involved in physiological metal metabolism (e.g. Zinc). These findings were corroborated by additional studies which showed that mt - knockout worms exhibited significantly decreased levels of overall fitness after the knockout of mt1 than after mt2 knockout . Further, both mt isoforms displayed a clear preference for divalent metal ion binding as opposed to copper coordination, likely due to the presence of histidines in the mts . Using both in vitro and in vivo models, zeitoun - ghandour and colleagues examined zinc and cadmium exposures and showed different roles for mtl-1 and mtl-2 . Both isoforms their affinities and stoichiometries were measured, and both isoforms displayed equal zinc- binding ability; however, mtl-2 had a higher affinity for cd than did mtl-1 . These experiments were repeated in vivo in mtl-1, mtl-2, and double knockouts following exposure to 340 m zn or 25 m cd . Zinc levels were found to be significantly increased in all knockout strains, but mtl-1 knockout worms demonstrated the most acute level of sensitivity . However, cadmium accumulation was found to be the highest in the mtl-2 knockout and double mutant strains . Additional studies assessed metal speciation, and results indicated that o - donating ligands play an important role in maintaining zinc levels, independent of metallothioneins status . Further, cadmium was shown to interact with thiol groups, and cd speciation was significantly different in the mtl-1 strain when compared with both the mtl-2 strain and the double knockout strain, suggesting that the two mt isoforms have distinct in vivo roles . The authors suggested that mts are not functioning as metal storage proteins but, rather, are mediating the accumulation and excretion of metals . A follow - up study, showed in vitro evidence for the partitioning of zinc and cadmium with different metallothionein isoforms . Employing electrospray ionization mass spectrometry (esi - ms) to directly observe zinc and cadmium binding preferences, more cadmium ions were found to be preferentially bound to mt-2 than to mt-1; however, cd was shown to be capable of inducing both isoforms . Finally, partitioning was also demonstrated to be more effective at lower cd: zn ratios . Using daf-2 (insulin receptor - like protein) and age-1 (phosphatidylinositol-3-oh kinase catalytic subunit) mutants, barsyte et al . Examined the expression of mt genes under noninducing conditions and after exposure to cadmium and copper . They reported that mt-1 mrna levels were significantly higher in daf-2 mutants compared to both age-1 mutants and wild - type worms under basal conditions . Exposure to cadmium treatment resulted in a three - fold induction of mt-1 and a two - fold induction of mt-2 mrna in daf-2 mutants as compared to wild - type controls . Copper did not induce mt-1 or mt-2 mrna expression in any of the strains tested . Collectively, these studies show differential metal preferences for one mt isoform over another depending on the metal to which an organism is exposed . Most significantly, these studies indicate that the mts play crucial roles in metal detoxification (table 2). Indeed, mts have been associated with a protective effect in cells under numerous states of disease and stress . Interestingly, serum mt levels of cancer patients are three times higher than those of control patients . A study conducted in denmark revealed the increased expression of mt-1 and mt-2 mrna and protein in many human cancers such as breast, kidney, lung, nasopharynx, ovary, prostate, salivary gland, testes, urinary bladder, cervical endometrial skin carcinoma, melanoma, acute lymphoblastic leukemia, and pancreatic cancers . This information is of particular import given the use of metals for the treatment of certain cancers, for example, arsenic as treatment for promyelocytic leukemia . It is interesting to postulate that higher levels of mts may enhance the efficacy of metal therapeutic agents or, conversely, may lead to resistance to such therapies . Understanding the factors that modulate mt expression will allow for the improved understanding of metalloid toxicity and will provide more effective therapeutic approaches to metalloid - based chemotherapy . There are a number of pumps and transporters that have been implicated in metal detoxification . These include atp - binding cassette (abc) transporters, such as the multidrug resistance - associated protein (mrp) as well as two members of the p - glycoprotein subfamily (pgp-1 and pgp-3), which have been shown to contribute to heavy metal tolerance through the use of c. elegans deletion mutants . In c. elegans, there are approximately 60 genes encoding abc transporters, and these genes make up the largest family of transporters . C. elegans have four mrp homologues and fifteen pgp homologues . The pgps are ubiquitously expressed and are most abundantly found in the apical membranes of the gut and in the excretory organs of the worm . Pgp-2 is expressed in the intestine and is required for the acidification of lysosomes and lipid storage; pgp-1 and -3 contribute to heavy metal and drug resistance . Tseng and colleagues investigated the arsa protein - mediated detoxification of the metalloids, as(iii) and sb(iii). Bacterial arsa atpase is the catalytic component of an oxyanion pump that is responsible for resistance to arsenite and antimonite . In this study, wild - type and asna-1-mutant nematodes were evaluated for as and sb response and toxicity . The asna-1 gene of c. elegans was found to be stimulated by as(iii); further, sb(iii) was determined to be crucial for establishing tolerance . Although these results occurred in response to as and sb exposure, the ubiquity of the arsa atpase - dependent pathway has not been observed in other species or in response to other metals . The role of multidrug resistance - associated protein (mrp) in arsenite and cadmium toxicity was explored in a study by broeks and colleagues . The targeted inactivation of mrp-1 rendered the arsenite and cd exposed worms incapable of recovering from temporary exposure to high arsenic and cadmium, whereas the wild - type controls were able to recover . Additionally, worms were also shown to be hypersensitive to arsenite and cd exposures when both mrp-1 and pgp-1 (p - glycoprotein-1) were deleted . Lastly, no increased sensitivity in response to exposure to antimony was observed in mrp-1-deletion mutants as compared to wild - type controls . Vatamaniuk and colleagues characterized the half - molecule abc transporter of the heavy metal tolerance family-1 (hmt-1) subfamily in response to cadmium exposure . The suppression of hmt-1 expression by rnai was shown to produce punctuate refractive inclusions within the vicinity of the nucleus of the intestinal epithelial cells upon exposure to toxic levels of cadmium . Similarly, schwartz and colleagues described the c. elegans hmt-1 following exposure to arsenic, copper, and cadmium . Hmt-1 conferred tolerance in response to exposure to all three metals as shown by lethality testing following the knockdown of hmt-1 . Kurz and colleagues demonstrated the three - fold induction of pgp-5 following cadmium exposure . Results of this study showed that strong fluorescence was induced in the intestinal cells of pgp-5::gfp worms, where the gfp - encoding gene is under the control of the upstream pgp-5 promoter . Copper and zinc were also found to be capable of inducing pgp-5 expression in these worms . Accordingly, it was concluded that pgp-5 is required for establishing full resistance to cadmium and copper . In addition, the rnai knockdown of tir-1, an upstream component of the p38 mapk pathway in the pgp-5 transgenic reporter strain, was shown to significantly reduce pgp-5 induction following exposure to cadmium . However, the double - stranded rna knockdown of erk (mpk-1) and jnk (med-1 and kgb-1) did not affect the induction of pgp-5 in response to cadmium exposure . Au and colleagues studied the divalent - metal transporter (dmt1) following exposure to manganese . The dmt1-like family of proteins has been shown to regulate manganese and iron in the cell . The deletion of the three worm dmt1-like genes resulted in differential effects on manganese toxicity . The deletion of smf-1 and smf-3 increased mn tolerance, whereas the deletion of smf-2 increased mn sensitivity . Heat shock proteins (hsps) are cytosolic molecular chaperones . Hsps promote the refolding and repair of denatured proteins and facilitate protein synthesis upon activation by cellular stress [75, 76]. Hsps, particularly those in the hsp70 family, have also been shown to participate in the hormetic response . Hsp70s are atp - binding proteins that convert atp to adp and bind to peptides, thereby, inactivating them and preventing aggregation (figure 1). Oxidative stress can cause a reduction in cellular atp levels . Decreased levels of atp result in the continued prevention of the aggregation of damaged proteins . The functions of the hsp70 products are mediated by the conserved n - terminal atpase and the c - terminal peptide - binding region . The human and c. elegans hsp70 genes have a high degree of homology and share a conserved core the hsp16 family of stress proteins is produced in c. elegans only under stress conditions [8082]. In a study examining the effects of mehg exposure, hsp-4::gfp was measured immediately following the treatment of l1 worms for 30 minutes and l4 worms for 15 hours with this toxicant . After 30 minutes of acute exposure to mehg, however, in l4 worms chronically exposed to mehg for 15 hours, hsp-4::gfp was induced . At the same time point in the chronic treatment paradigm, a four - fold increase in gst-4 fluorescence was detected, but there were no changes in either mtl-1 or mtl-2::gfp expression . Jones and candido exposed nematodes to cadmium or mercury and measured feeding behavior . For these studies, transgenic lines containing the promoter sequence for hsp16 genes which regulate the production of e. coli-galactosidase were used . Accordingly, to measure stress, levels of this protein were assessed . Results showed that cadmium inhibited feeding behavior significantly but not completely, as a minimal rate of feeding continued at high cadmium concentrations . Further, exposure to cadmium (1 ppm) induced a detectable production of -galactosidase without inhibiting feeding behavior . The stress response was induced at a concentration of cadmium that was ten times lower than the lc50 . Mercury also was shown to inhibit feeding at concentrations similar to those necessary for the induction of a stress response; however, the difference in this instance was less than two fold . The use of c. elegans as an experimental model has produced considerable insight and valuable information regarding the multiple and varied processes of metal detoxification . Conclusive biochemical evidence has indicated that different metals are not handled in the same capacity . The selectivity and sensitivity of each of these proteins is highlighted in the large body of accumulated research on different metal toxicities as well as various systems of metal detoxification . However, the overall mechanisms, temporal activation, and interplay between different cell detoxification systems remain elusive . Future studies are necessary in order to enhance our understanding of the complex interplay of multiple - cell detoxification systems in response to exposure to different metals . The c. elegans model system will be critical for these investigations, as knockouts are easily generated and provide a wealth of information about metal detoxification in a genetically retractable, inexpensive, and in vivo model.
Obesity is a complex heterogeneous disease that is caused by genes, environmental factors, and the interaction between the two . Obesity is also a multifactorial condition, and many endocrine and inflammatory pathways are involved in its development and in obesity - related diseases . Excess weight in obesity may come from muscles, bone, fat, and/or body water, but obesity specifically refers to having an abnormally high proportion of total body fat . The world health organization defines overweight as a body mass index (bmi) of 25 or more and obesity as a bmi of 30 or more . The prevalence of obesity has been stated as being near epidemic size [13, 57], and obesity has been associated with type ii diabetes, hypertension, coronary artery disease, stroke, and many forms of cancer [8, 9]. Therefore, it is important that the underlying pathophysiology of obesity - related diseases is understood . Obesity results from the combined effects of genes, lifestyle, and the interactions of these factors, and both familial and nonfamilial factors play an important role in its development . A genetic predisposition to obesity has been reported as a major risk factor for individuals . With the increasing prevalence of obesity, studies on candidate genes for obesity most obesity - predisposing genes encode the molecular components of physiological systems related to energy balance . Leptin is a protein product of the ob gene and is expressed and secreted by adipose tissue in amounts proportional to the body weight content; studies on its receptor have greatly advanced the comprehension of the mechanism for regulating body weight and energy homeostasis . The lipostat system, mediated by leptin and its hypothalamic receptor, reduces food intake and increases thermogenesis [10, 12]. The leptin (lep) and leptin receptor (lepr) genes have been evaluated for polymorphisms that could potentially be related to the pathophysiology of obesity and its complications . Although the polymorphisms in these genes have been evaluated [1315], the association of these polymorphisms with obesity is still controversial . Therefore, we investigated whether the lep gene g2548a polymorphism and lepr gene 668a / g (q223r) polymorphism might be involved in the pathogenesis of obesity . This study included 127 obesity patients (93 women, 34 men) and 105 controls (62 women, 43 men) provided from the department of internal medicine, gazi osmanpaa university in tokat, turkey . Informed consent was in accordance with the study protocol, and all patients and controls signed a written consent form . All patients received a complete clinical evaluation, and all individuals in the control group were healthy and were selected by excluding the diagnosis of obesity . Genomic dna was isolated from white blood cells by a kit procedure (invitrogen life technologies, carlsbad, ca, usa) and stored at 20c . Lep g2548a and lepr 668a / g polymorphisms were analyzed by polymerase chain reaction based restriction fragment length polymorphism (pcr - based rflp) methods . The pcr protocol consisted of an initial melting step of 2 min at 94c, followed by 35 cycles of 30 s at 94c, 30 s at 55c (for lep), 30 s at 60c (for lepr), and 30 s at 72c, and a final elongation step of 5 min at 72c . Amplification was carried out using primers forward 5-ttt cct gta att ttc ccg tga g-3 and reverse 5-aaa gca aag aca ggc ata aaa a-3 for the lep gene and forward 5-tcc tct tta aag cct atc cag tat tt-3 and reverse 5-agc tag caa ata ttt ttg taa gca at-3 for the lepr gene . Pcr was performed with a 25 l reaction mixture containing 2550 ng/l dna, 1 l of 10 pmol/l of each primer, 1 l of dntp mixture (5 mm dntp, 1 l 2.5 mm mgcl2, 1 u taq dna polymerase), 2.5 l 10x pcr buffer (mg free, invitrogen life technologies, carlsbad, ca, usa), and dh2o . Amplified products were digested with hhai at 37c for lep and mspi at 37c for lepr, and the resulting fragments were separated by 2% agarose gel electrophoresis . The fragments were stained with ethidium bromide and visualized through a vilber - lourmat gel quantification and documentation system (quantum - st4; vilber lourmat bp 66, torcy, france). Analysis of the data was performed using spss 16.0 (spss, chicago, il, usa) and openepi info (http://www.openepi.com). The frequencies of the alleles and genotypes (hardy - weinberg equilibrium) in patients and controls were compared with analysis, and 95% confidence intervals were calculated . A p value less than 0.05 (two - tailed) was regarded as statistically significant . The mean age and bmi were 44.86 1.51, 35.45 4.56 and 34.25 15.43, 21.57 1.89 in patients and control groups, respectively . The mean age of obesity patients with bmi> 35 kg / m and bmi <35 kg / m was 45.01 1.38 and 44.7 1.65, respectively, while the mean bmi of these patients was 38.68 3.11 (bmi> 35 kg / m) and 32.06 1.56 (bmi <35 kg / m). Patients and controls were genotyped for both the g2548a polymorphism in the lep gene promoter and the 668a / g polymorphism in the lepr gene . The distribution of the lep g2548a and lepr 668a / g polymorphisms of the patients and control groups are presented in tables 2 and 3 . We found no statistically significant difference in the genotype frequencies of the lep gene polymorphism in patients and control groups (p> 0.05). The lepr genotypes differed between the obesity patients and controls, but this was not statistically significant after the bonferroni correction (p = 0.05). Allele frequencies in the lepr gene showed no statistically significant association (p> 0.05) (table 3). The lepr gene a allele was 61.4% in patients and 69.5% in the control group, while the g allele frequency was 38.5% in patients and 30.4% in the control group . In the combined analysis of the lep and lepr genes, the lep / lepr gg / gg combined genotype was found to increase the risk of obesity compared to the controls (p <0.05) (table 4). In the combined genotype analysis based on the mean bmi of obesity patients, there was no association of the lep / lepr combined genotype and obesity between patients with a bmi 35 kg / m and patients with a bmi near 30 kg / m (p> 0.05) (table 4). Human obesity is a complex trait determined by the interaction of multiple genes and environmental factors . Obesity may arise as a result of increased energy intake, decreased energy expenditure, or increased partitioning of nutrients into fat, either alone or in combination . The prevalence of obesity and being overweight continues to increase worldwide, not only causing serious personal health problems but also imposing a substantial economic burden on societies . Genetic influences are difficult to elucidate, and identification of the involved genes is not easily achieved . In the present study, we analyzed the frequencies of lep g2548a and lepr 668a / g polymorphisms in obesity patients in a turkish population . There was no statistically significant difference between the groups with respect to the lep genotype distribution (p> 0.05) and allele frequencies (p> 0.05). Suggested that the lep g2548a variant may influence gene expression of leptin and leptin secretion by adipose tissue . Noted that the lep g2548a polymorphism may influence a bmi increase by means of its effects on leptin secretion; however, they identified a significant and independent association between the lep 2548gg carrier status and higher leptin levels . An association of the lep g2548a polymorphism and increased bmi was reported in overweight europeans and in taiwanese subjects with obesity and the combined lep 759c / t and lep g2548a genotype may be a determinant of obesity . The results of our study do not support the results of these studies but do support those of other studies that showed no association between the lep g2548a polymorphism and obesity - related phenotypes [11, 14, 15, 21]. We found that lepr genotypes show a difference, but not statistically significant, between obesity patients and controls . We attribute this lack of significance to the low number of patients included in our study, but finding obese patients that have no other disease is difficult . Some researchers have proposed that the polymorphisms of the leptin receptor gene (especially lepr 668a / g polymorphism) may contribute to common forms of human obesity [11, 14, 2224]. Our results with respect to the lepr polymorphism are in agreement with the results of these studies . Our results showed a statistically significant difference between groups with respect to the distribution of the lep / lepr gg / gg combined genotype . Obesity results from both gene - gene and gene - environment interactions, and in our study we examined the gene - gene interactions of the lep / lepr genes and their link to obesity . Demonstrated that the haplotype association of the lep g2548a and lepr q223r variants was related to a 58% increase in obesity risk, and they considered the interactions between lep and lepr gene polymorphisms to intensively influence modulation of energy homeostasis . In agreement with the findings of our study, boumaiza et al . Reported that the lep g2548a and lepr q223r polymorphisms and haplotype combination were associated with a metabolic syndrome and obesity risk in tunisian subjects . The g2548a and 3hvr variants of the lep gene have been noted as being in linkage disequilibrium, and i / g combined genotypes are associated with obesity . In addition, the interactions between the polymorphisms of the lep and lepr genes have been shown to increase the risk of non - hodgkin's lymphoma and influence insulin plasma concentrations and blood pressure levels . Our findings indicate that the lep g2548a polymorphism is not a relevant obesity marker and that the lepr 668a / g polymorphism may be related to obesity in a turkish population . Additionally, the lep / lepr gg / gg combined genotype was found to increase the risk of obesity in patients compared to controls . However, the association of these polymorphisms with obesity is still controversial, and further research with larger patient populations is necessary.
The basic concept of this technique is maintaining phase information into the final image, discarding phase artefacts and keeping just the local phase of interest . Sensitivity to susceptibility effects increases, progressing from fast spin - echo (se) to conventional se to gradient - echo (ge) sequences, from t2-weighting to t2 * -weighting, from short to long echo times and from lower to higher field strengths . Before the clinical implementation of swi, susceptibility imaging relied only on ge sequences . Swi differs significantly from a t2 * -weighted ge sequence: it is based on a long echo time (te) high - resolution, flow - compensated, three - dimensional (3d) ge imaging technique with filtered phase information in each voxel . The combination of magnitude and phase data produces an enhanced contrast magnitude image that is particularly sensitive to haemorrhage, calcium, iron storage and slow venous blood, thus allowing a significant improvement compared with t2 * ge sequences . After imaging acquisition, the phase mask is then multiplied with the magnitude data to enhance the visualisation of vessels or foci with susceptibility effects . Swi is therefore especially helpful in the detection of calcifications and microhaemorrhages, which are both characterised by low signal . The evaluation of the corrected phase images allows the differentiation between the two substances, as calcifications appear bright because of a positive phase shift and haemorrhages appear dark because of a negative phase shift . A supplementary source of information in swi swi represents a technical improvement in high - resolution blood oxygenation level - dependent venography (hrbv), originally developed by reichenbach et al ., which was based on 3d long te, flow - independent ge sequences and manipulation of the images with the phase data . The paramagnetic properties of deoxyhaemoglobin [bold (blood oxygen level dependent) effect] and the prolonged t2 * of venous blood were used as an intrinsic contrast agent, leading to a phase difference between vessels containing deoxygenated blood and surrounding brain tissue, resulting in signal intensity cancellation . Thus, deoxyhaemoglobin can behave like a contrast agent with long tes for differentiating arteries from small veins, which can be as small as 100200 m and therefore difficult to detect with conventional mr angiography techniques, such as time of flight (tof) or phase contrast (pc). For this reason, the phase - added information that is usually not available in the conventional magnitude image makes swi well suited for the visualisation of very small vessels such as the caput medusae of venous angiomas and telangiectasias as a result of a combination of slow flow with changes in deoxyhaemoglobin concentration . Latest advances have allowed the technique to be refined, thereby expanding its clinical applicability to brain imaging as a complementary source of information to conventional t1-weighted and t2-weighted imaging sequences . At our institution, mr imaging (mri) is performed by using a 1.5-t system (magnetom avanto; siemens, erlangen, germany) with a 12-channel head coil . Swi is obtained with a long - te, fully flow - compensated 3d ge sequence with the following parameters: repetition time (tr)/te, 49/40 ms; flip angle, 15; rectangular field of view (fov), 7/8; matrix size, 280 320; slice thickness, 1.6 mm (80 slices in a single slab matrix size); ipat factor, 2; acquisition time, 5 min . The swi sequences are reconstructed with the minimum intensity projection algorithm (minip) and multiplanar reformation (mpr) techniques to obtain images with thickness (310 mm) and position comparable to those of conventional sequences . The minip algorithm has the characteristic to enhance the visualisation of veins while attenuating the signal from the brain tissue (fig . 1). B swi, minimal intensity projection (minip; 10 mm) image: cortical and subependymal veins are well displayed . C swi, phase map: the distinction between the pars reticulata (arrow) and compacta (asterisk) of the substantia nigra is enhanced compared with conventional fast se and ge sequences . The outer margins of the red nucleus are also better displayed (see d for comparison). B swi, minimal intensity projection (minip; 10 mm) image: cortical and subependymal veins are well displayed . C swi, phase map: the distinction between the pars reticulata (arrow) and compacta (asterisk) of the substantia nigra is enhanced compared with conventional fast se and ge sequences . The outer margins of the red nucleus are also better displayed (see d for comparison). D t2-weighted axial image swi sequences have some intrinsic disadvantages, which are mainly represented by artefacts caused by undesirable sources of magnetic susceptibility that occur at air tissue interfaces, therefore limiting the investigation of areas next to paranasal sinuses and temporal bone . The blooming artefact, useful in most cases, might also not be needed in some situations, producing normal tissue signal cancellation and loss of anatomical borders . The sequence acquisition time on a 1.5-t system ranges from 5 to 8 min, depending on the spatial resolution and the coverage of the brain needed, leading to an increased incidence of movement artefacts . Imaging at a high field strength has some advantages over 1.5 t in the delineation of even smaller vessels belonging to the venous network, with shorter imaging times because of the higher signal - to - noise ratio, higher spatial resolution and increased susceptibility effects . Tissue interfaces or other sources of local field heterogeneity are much more severe at higher fields, thus reducing swi s usefulness in the evaluation of the posterior fossa and skull base . Swi has been investigated at 3 t with excellent results, especially concerning brain tumours and detection of cerebral microbleeds [6, 7]. Sporadic cerebral amyloid angiopathy (caa) is a common small - vessel disease associated with ageing, dementia and alzheimer s disease, that can only be diagnosed histopathologically following biopsy or at post - mortem examination . It consists of deposition of amyloid protein within the small and medium - sized cerebral arteries, which is likely responsible for increased vessel fragility and it is one of the major causes of lobar intraparenchymal haemorrhages in the elderly . Computed tomography (ct) and conventional mr techniques are usually unable to show cerebral microbleeds (cmbs), which can be frequently observed on t2 * -weighted gradient - echo mri and have a typical lobar distribution . Recent findings indicate that cmbs in the general elderly population are relatively common and are even more frequently observed in patients with ad . Swi is much more sensitive than ge sequences in demonstrating cmbs in and around the arteriole vessel wall, which appear as black dots better displayed by minip images (fig . 2). Histologically, they represent perivascular clusters of haemosiderin - laden macrophages resulting from leakage from cerebral small vessels . Higher field strengths, as expected, can improve cmb detectability, increasing the lesion s contrast enhancement . Fig . 2recent onset of altered mental status and cognitive impairment in a 67-year - old man . A fluid attenuated inversion recovery (flair) mri: diffuse hyperintensity of the white matter, more pronounced in the right parietal region . B diffusion - weighted imaging (dwi), apparent diffusion coefficient (adc) map: the white - matter hyperintensity represents vasogenic oedema . C ge t2 * -weighted image: small punctate hypointense foci in the right parietal cortex (open arrow). D swi minip: increased visualisation of markedly hypointense foci surrounding the white matter abnormalities which correspond to multiple cerebral microbleeds . On the basis of swi imaging findings, caa with diffuse inflammatory changes was suspected and steroid therapy was administered . E follow - up mri at 3 months showed significant reduction of the vasogenic oedema recent onset of altered mental status and cognitive impairment in a 67-year - old man . A fluid attenuated inversion recovery (flair) mri: diffuse hyperintensity of the white matter, more pronounced in the right parietal region . B diffusion - weighted imaging (dwi), apparent diffusion coefficient (adc) map: the white - matter hyperintensity represents vasogenic oedema . C ge t2 * -weighted image: small punctate hypointense foci in the right parietal cortex (open arrow). D swi minip: increased visualisation of markedly hypointense foci surrounding the white matter abnormalities which correspond to multiple cerebral microbleeds . On the basis of swi imaging findings, caa with diffuse inflammatory changes was suspected and steroid therapy was administered . E follow - up mri at 3 months showed significant reduction of the vasogenic oedema caa is also characterised by white matter hyperintensities on conventional mri sequences, which have been associated with cognitive impairment . Vascular amyloid deposition is believed to be involved in the pathophysiological mechanisms that determine white matter hypoperfusion through either vessel stenosis or vascular dysfunction . Caa should also be suspected in elderly patients with clinical signs of a progressive encephalopathy syndrome with seizures, in which extensive white matter abnormalities are discovered in brain mri together with multiple cmbs with lobar and subcortical distribution (fig . 2). It has been recently reported that this combination of mr findings should be interpreted as caa - related inflammation, which can be treated with steroid therapy with a prompt resolution of the symptoms . In those cases, the demonstration of an apoe 44 genotype can definitely support the neuroradiological diagnosis . Hypertensive encephalopathy and especially posterior reversible encephalopathy syndrome (pres), when the signal abnormalities of the white matter are more symmetrical and located in parieto - occipital regions, should be taken into consideration for differential diagnosis . Hypertensive encephalopathy is characterised by multiple cmbs which are usually silent and can be discovered when the patient is investigated with mri in order to understand the cause of an intraparenchymal haemorrhage located outside the basal ganglia . Swi is more sensitive to cmbs than t2 * -weighted ge sequences in blood pressure (bp)-related small vessels disease . Cmbs are usually discovered both in deep basal ganglia and subcortical white matter (fig . Histologically they represent focal accumulations of haemosiderin - containing macrophages in the perivascular space of small brain vessels, indicating previous extravasation of blood, and are often associated with the presence of a symptomatic haemorrhage in the corresponding area . The number of cmbs, which remain detectable for years, is significantly associated with bp levels . Moreover, the presence of deep cmbs can be a useful marker of bp - related small vessels disease, helping in the differential diagnosis with caa . A t2-weighted image shows a thin hypointense band in the right thalamus, which represents haemosiderin deposition (arrow). B ge t2 * -weighted image shows multiple microbleeds, the largest one in the right thalamus (arrow). C swi minip: the number of identifiable microbleeds is increased compared with ge images . They can be recognised bilaterally in the basal ganglia and in the subcortical temporal white matter (arrows) patient with long - standing hypertension . A t2-weighted image shows a thin hypointense band in the right thalamus, which represents haemosiderin deposition (arrow). B ge t2 * -weighted image shows multiple microbleeds, the largest one in the right thalamus (arrow). C swi minip: the number of identifiable microbleeds is increased compared with ge images . They can be recognised bilaterally in the basal ganglia and in the subcortical temporal white matter (arrows) iron deposition increases in the brain as a function of age, primarily in the form of ferritin and particularly in oligodendrocytes, but also in neurons and microglia . Typical sites of iron deposition include the globus pallidum, substantia nigra, and red and dentate nuclei . Swi filtered - phase images are particularly suitable for showing increased iron content in the brain (. Abnormally elevated iron levels are evident in many neurodegenerative disorders, including parkinson s disease, alzheimer s disease, huntington s disease and amyotrophic lateral sclerosis . The ability to measure the amount of ferritin in the brain can be used for a better understanding of the progression of the disease and is also helpful in predicting the treatment outcome . Phase images allow a better distinction between the pars compacta and the pars reticulata of the substantia nigra, which contains iron (fig . 1). As in patients with idiopathic parkinson s disease, there is evidence of increased iron in the substantia nigra, swi sequences can be proposed as a useful imaging tool to identify iron deposition as a biomarker for disease progression, although longitudinal studies are required to support the usefulness of this specific application . Accurate localisation of the subthalamic nucleus can also be achieved in the swi phase maps at 3 t, allowing safe direct targeting for placement of electrodes in the treatment of parkinson s disease . In 7098% of patients with alzheimer s disease recent evidence has shown that patients with multiple cmbs have more white matter hyperintensities and perform worse on mini mental state examination compared with patients with no cmbs, despite similar disease duration . A significant correlation has also been reported recently between patients with at least one hypointensity in gre - t2 * imaging and those homozygous for the apolipoprotein e 44 gene, a well - known risk factor for alzheimer s disease (fig 4). B coronal reformatted swi section shows a cerebral microbleed in the right frontal white matter (arrow) alzheimer s disease . B coronal reformatted swi section shows a cerebral microbleed in the right frontal white matter (arrow) swi has been demonstrated to be very useful in the acute phase of stroke for several reasons . First of all, it is very sensitive to the presence of cmbs, whose early identification is believed to predict the probability of potential haemorrhagic transformation after thrombolytic treatment . It has also been hypothesised that cmbs can represent a link between cerebral haemorrhage and ischaemia . Swi is also capable of identifying the acute intravascular clot in the main and distal branches of the cerebral arteries . The evaluation of the intravascular content of deoxyhaemoglobin in hypoperfused brain is one of the most interesting applications of swi . This technique has been proved to be useful for the assessment of tissue viability in patients with hyperacute cerebral ischaemia, as it provides functional information about the ischaemic penumbra . In acute arterial stroke, flow reduction or absence causes an increase in oxygen extraction, leading to a growth in the amount of deoxyhaemoglobin in the hypoperfused brain tissue . Deoxyhaemoglobin represents a supplementary source of contrast enhancement in swi because of its paramagnetic properties, which determine a reduction in t2 * as well as a phase difference between the vessel and its surrounding parenchyma . As the t1 and t2 properties of blood depend on the oxygen saturation of the blood, there are differences between arterial and venous vessels that cannot be discovered with conventional sequences . The visualisation of draining veins within areas of impaired perfusion allows the identification of penumbral brain tissue in a different way from the current perfusion - weighted imaging (pwi) techniques (fig . 5left internal carotid artery (ica) dissection with acute watershed infarct in a 42-year - old man with sudden onset of speech disturbance . A mr angiography, single partition at the level of the skull base showing dissection of the left ica, which is characteristically enlarged with high - signal - intensity methaemoglobin (arrowheads) representing the intramural thrombus and a small eccentric lumen (arrow). B mr angiography, coronal mip: narrowing of the ica in its vertical segment just below the skull base (arrow). D swi minip: improved visualisation of the veins of the left cerebral hemisphere, which is related to increased oxygen extraction in the ischaemic penumbra and corresponds to the hypoperfusion deficit shown by the mean transit time (mtt) map . E perfusion mri, mtt map, showing delayed transit time in the left middle cerebral artery (mca) territory . F intra - arterial digital subtraction angiography (dsa), left carotid angiogram, confirming the ica stenosis (arrow). G intra - arterial dsa: the patient was treated using stent placement, which resulted in partial restoration of the vessel lumen . H perfusion mri, mtt map 24 h after stent placement: complete resolution of the perfusion deficit . I swi minip, normalisation of the venous drainage of the left hemisphere left internal carotid artery (ica) dissection with acute watershed infarct in a 42-year - old man with sudden onset of speech disturbance . A mr angiography, single partition at the level of the skull base showing dissection of the left ica, which is characteristically enlarged with high - signal - intensity methaemoglobin (arrowheads) representing the intramural thrombus and a small eccentric lumen (arrow). B mr angiography, coronal mip: narrowing of the ica in its vertical segment just below the skull base (arrow). C dwi, axial image: acute watershed infarct in the left centrum semiovale . D swi minip: improved visualisation of the veins of the left cerebral hemisphere, which is related to increased oxygen extraction in the ischaemic penumbra and corresponds to the hypoperfusion deficit shown by the mean transit time (mtt) map . E perfusion mri, mtt map, showing delayed transit time in the left middle cerebral artery (mca) territory . F intra - arterial digital subtraction angiography (dsa), left carotid angiogram, confirming the ica stenosis (arrow). G intra - arterial dsa: the patient was treated using stent placement, which resulted in partial restoration of the vessel lumen . H perfusion mri, mtt map 24 h after stent placement: complete resolution of the perfusion deficit . I swi minip, normalisation of the venous drainage of the left hemisphere swi has become a functional method for evaluating cerebral venous sinus thrombosis (cvst) by demonstrating engorgement of the venous system as a result of venous hypertension and collateral slow flow . Dural sinus thrombosis causes an increase in deoxyhaemoglobin concentration in the veins involved that appears as a prominent area of hypointense signal intensity on swi; if cvst is treated successfully, this effect will disappear . Swi is also capable of identifying the thrombosed pial vein, which is responsible for the development of oedema into the white matter, due to its high sensitivity in the demonstration of an intravascular clot . Swi can depict at the same time the features of parenchymal or extra - axial haemorrhages that can occur in the case of infarction, with higher sensitivity compared with conventional ge sequences (fig . A sagittal t1-weighted image demonstrates hyperintensity of the posterior third of the sagittal sinus (open arrow). B mr venography confirms the thrombosis of the posterior third of the sagittal sinus ending at the torcular (open arrow). C dwi, b = 1,000 image demonstrates a right occipital acute ischaemic lesion . D swi minip shows a right thalamic haemorrhagic lesion in the vascular territory of the right deep internal cerebral vein (arrow) epileptic seizures and sagittal sinus thrombosis in a 3-year - old child . A sagittal t1-weighted image demonstrates hyperintensity of the posterior third of the sagittal sinus (open arrow). B mr venography confirms the thrombosis of the posterior third of the sagittal sinus ending at the torcular (open arrow). C dwi, b = 1,000 image demonstrates a right occipital acute ischaemic lesion . D swi minip shows a right thalamic haemorrhagic lesion in the vascular territory of the right deep internal cerebral vein (arrow) cerebral arteriovenous malformations (avms) are easily displayed by conventional mri and mr angiography because of their characteristic high flow, whereas venous malformations such as cavernomas, developmental venous anomalies and capillary telangiectasias cannot be adequately visualised without contrast medium administration and ge sequences, as they mainly consist of slow - flow small vessels . Mr angiography techniques are often inadequate for visualising small vessels with slow flow . On the contrary, swi sequences are well suited for the visualisation of very small vessels, such as the caput medusae of venous angiomas and telangiectasias as a result of a combination of slow flow with changes in deoxyhaemoglobin concentration . The combined information derived from both phase and magnitude images is responsible for enhanced visualisation of such lesions . Swi plays a substantial role in the identification and characterisation of cerebral vascular malformations, first of all improving their detection rate . On the other hand, allowing simultaneous visualisation of the different compartments of cerebral avms and of the relationship with one another and with the brain parenchyma, it is particularly valuable for therapeutic planning . The clinical usefulness of a similar technique, bold mr venography, was first demonstrated by essig et al . . Phase changes caused by the susceptibility differences between oxygenated and deoxygenated red blood cells and the different relaxation rates between venous and arterial blood provide a natural separation of arteries and veins . The 3d dataset of swi can therefore be used as an alternative to mr angiography in order to better differentiate afferent arteries from draining veins . This advantage becomes relevant with small avms with recent bleeding, in which it is mandatory to identify the nidus in relation to eloquent areas in order to plan the best therapeutic approach, endovascular or surgical or both (fig . Veins appear dark due to t2 * loss and processing of the phase image, while arteries show bright signal from time - of - flight (tof) inflow enhancement . The use of higher flip angles and shorter tr make it possible to increase the contrast enhancement in the arteries without overly degrading the venography . For the evaluation of high - flow vascular malformations (i.e. Avms and dural arteriovenous fistulas) the use of both minip and mip post - processing techniques can improve the image quality . The identification of small niduses, the exact location of the fistulous point and the depiction of the venous drainage patterns are the most relevant information that should be obtained from this technique . The elevated diagnostic accuracy of swi compared with digital subtraction angiography (dsa) has been recently reported for the detection of arteriovenous shunting (avs) in brain avms . B the swi minip image clearly depicts the brainstem haemorrhage, which is markedly hypointense . C, d swi, axial and reformatted sagittal images, processed with a maximum intensity projection algorithm (mip), show flow - related enhancement within the haemorrhagic lesion (arrow) and a hypertrophied right posterior inferior cerebellar artery (pica; open arrow) which are consistent with a small ruptured brainstem avm . E cerebral angiography, selective injection of the right vertebral artery: brainstem avm (arrow) fed by branches of the right pica and early venous drainage into a cerebellar vein (arrowheads) brainstem haemorrhage in a 43-year - old patient . B the swi minip image clearly depicts the brainstem haemorrhage, which is markedly hypointense . C, d swi, axial and reformatted sagittal images, processed with a maximum intensity projection algorithm (mip), show flow - related enhancement within the haemorrhagic lesion (arrow) and a hypertrophied right posterior inferior cerebellar artery (pica; open arrow) which are consistent with a small ruptured brainstem avm . E cerebral angiography, selective injection of the right vertebral artery: brainstem avm (arrow) fed by branches of the right pica and early venous drainage into a cerebellar vein (arrowheads) dural av fistulas (davfs) are usually more difficult to identify than avms on conventional mri, especially when venous ectasia is absent, as in type i and ii davfs; the patients have vague symptoms (i.e. Headache or tinnitus) and there are no localising signs . Swi is capable of better identifying the extent of the abnormal venous drainage compared with mr angiography . The conspicuity of the venous structures on swi can be explained by the combination of a prolonged cerebral circulation time that favours increased oxygen extraction resulting in increased concentration of deoxyhaemoglobin, with the typical venous engorgement (fig . B, c swi axial and coronal reformatted minip images show dilated right temporal veins (arrows), which arouse suspicion of a tentorial dural av fistula . D cerebral angiography, selective injection of the right external carotid artery: dural av fistula of the right tentorium, fed by the right middle meningeal artery (open arrow), with early venous drainage into the tentorial and temporal veins (arrows) patient with headache and pulsatile tinnitus of the right ear . A t2-weighted image shows a dilated right temporal vein (arrows). B, c swi axial and coronal reformatted minip images show dilated right temporal veins (arrows), which arouse suspicion of a tentorial dural av fistula . D cerebral angiography, selective injection of the right external carotid artery: dural av fistula of the right tentorium, fed by the right middle meningeal artery (open arrow), with early venous drainage into the tentorial and temporal veins (arrows) concerning slow - flow vascular malformations (i.e. Cavernomas, developmental venous anomalies and capillary telangiectasias) the phase information contained in swi is responsible for an artefactual enhancement that dramatically improves mri sensitivity to these pathological conditions . Without swi individuals with cavernous malformations can present with epilepsy and focal neurological deficits or acute intracranial haemorrhage, although these vascular malformations are often discovered as incidental findings . Cavernomas that have previously bled are usually detectable on routine mri because of the prominent signal intensity of haemorrhagic products . However, these lesions tend to be barely visible when they have not bled, except for faint enhancement after contrast medium administration that is often non - specific . Patients with multiple lesions can be diagnosed with familial cavernous angiomatosis and should be referred for genetic evaluation and counselling (fig . Individuals with symptomatic, growing, or haemorrhagic malformations should be considered for surgical resection . Close follow - up after diagnosis and treatment is helpful to identify lesion progression or recurrence . A t2-weighted image demonstrates a left frontal cavernous malformation surrounded by a thick ring of haemosiderin . B ge t2 * -weighted image shows multiple punctate hypointense foci located in both hemispheres, corresponding to small cavernous malformations (arrows). C swi minip: there are supplemental cavernous malformations that are not detected by ge sequences (arrows). D swi minip, reformatted sagittal section: multiple small cavernous malformations located in the brainstem (arrows) multiple cavernous malformations in a 32-year - old man investigated for intraparenchymal haemorrhage . A t2-weighted image demonstrates a left frontal cavernous malformation surrounded by a thick ring of haemosiderin . B ge t2 * -weighted image shows multiple punctate hypointense foci located in both hemispheres, corresponding to small cavernous malformations (arrows). C swi minip: there are supplemental cavernous malformations that are not detected by ge sequences (arrows). D swi minip, reformatted sagittal section: multiple small cavernous malformations located in the brainstem (arrows) developmental venous anomalies (dva) consist of a radially arranged venous complex converging on a centrally located venous trunk, which drains the normal brain parenchyma . They are better displayed with swi than with t1-weighted contrast - enhanced images (fig . 10incidental discovery of a cerebellar developmental venous anomaly in a 48-year - old man investigated for right - sided sensorineural hearing loss . A t2-weighted image shows thin hypointense bands of flow void, in the right cerebellar hemisphere (open arrow). B swi minip demonstrates multiple thin medullary veins (arrows) that converge into a dilated collector vein (open arrow), consistent with a cerebellar venous angioma, better defined as developmental venous anomaly . C post - contrast t1-weighted axial image shows the enhancing dilated veins (open arrow) incidental discovery of a cerebellar developmental venous anomaly in a 48-year - old man investigated for right - sided sensorineural hearing loss . A t2-weighted image shows thin hypointense bands of flow void, in the right cerebellar hemisphere (open arrow). B swi minip demonstrates multiple thin medullary veins (arrows) that converge into a dilated collector vein (open arrow), consistent with a cerebellar venous angioma, better defined as developmental venous anomaly . C post - contrast t1-weighted axial image shows the enhancing dilated veins (open arrow) capillary telangiectasias are asymptomatic venous vascular malformations, which are smaller and less common than cavernomas and can sometimes occur in mixed cavernoma / telangiectasia lesions . These may occur sporadically or may be infrequently associated with syndromes, like hereditary haemorrhagic telangiectasia or ataxia telangiectasia . They may also manifest as a result of endothelial injury, such as radiation - induced vascular injury, particularly in children who have received cranial irradiation . They are characterised by poor contrast enhancement and can therefore be missed on conventional t1-weighted and t2-weighted sequences (fig . 11incidental discovery of a pontine telangiectasia in a 34-year - old woman investigated for migraine . Swi minip axial (b) and sagittal reformatted section (c) show a markedly hypointense lesion with regular margins, located in the pons . . The capillary telangiectasia is characterised by mild enhancement with an arbor - like pattern incidental discovery of a pontine telangiectasia in a 34-year - old woman investigated for migraine . Swi minip axial (b) and sagittal reformatted section (c) show a markedly hypointense lesion with regular margins, located in the pons . . The capillary telangiectasia is characterised by mild enhancement with an arbor - like pattern ataxia telangiectasia is a rare autosomal recessive disorder characterised by an early - onset of progressive cerebellar ataxia, immunodeficiency, ocular and cutaneous telangiectasia and is associated with an elevated risk of intracranial tumours . Swi can discover brain gliovascular nodules with perivascular haemorrhage and small haemosiderin deposits presumably only in older patients, during the later stages of the disease; however, in our experience this technique can also be a useful screening tool in young patients (fig . A 31-year - old woman with a diagnosis of ataxia telangiectasia by the age of 7, investigated for dizziness and headache . C swi minip: additional hypointense black spots can be identified, corresponding to haemosiderin deposits presumably due to gliovascular nodules with perivascular haemorrhage ataxia telangiectasia . A 31-year - old woman with a diagnosis of ataxia telangiectasia by the age of 7, investigated for dizziness and headache . C swi minip: additional hypointense black spots can be identified, corresponding to haemosiderin deposits presumably due to gliovascular nodules with perivascular haemorrhage mri with ge sequences has been extensively used in the past for investigating young patients with traumatic brain injuries (tbi), either in the acute phase, when the clinical picture of a severe coma is not explained by ct, or months after trauma, in order to understand the causes of an unsuccessful recovery . In both cases, swi has been widely used in the identification of smaller haemorrhages due to a shear - strain mechanism of injury, thus refining the prediction of outcome . Haemorrhagic dai lesions on swi can be six - times better detected than t2 * -weighted conventional ge sequences and the recognisable volume of haemorrhage is approximately twofold greater (fig . Reformatted swi images in the sagittal plane are particularly helpful in the assessment of corpus callosum haemorrhages . Fig . 13diffuse axonal injury in a 14-year - old boy who had severe tbi following a motorcycle crash . Brain mr study was performed three days after the trauma, when the patient was in a severely comatose state (gcs 5). A flair image identifies multiple hyperintense lesions in the splenium of the corpus callosum and in frontal subcortical and periventricular white matter . B ge t2 * -weighted image: haemorrhagic shearing injuries are barely visible in the right fronto - opercular and parieto - occipital regions (arrows). C swi minip: additional microhaemorrhages are recognisable at the grey matter - white matter junction of the frontal lobes and in the right parieto - occipital white matter (black arrows). D swi minip, reformatted sagittal section shows microhaemorrhages in the corpus callosum (white arrows) diffuse axonal injury in a 14-year - old boy who had severe tbi following a motorcycle crash . Brain mr study was performed three days after the trauma, when the patient was in a severely comatose state (gcs 5). A flair image identifies multiple hyperintense lesions in the splenium of the corpus callosum and in frontal subcortical and periventricular white matter . B ge t2 * -weighted image: haemorrhagic shearing injuries are barely visible in the right fronto - opercular and parieto - occipital regions (arrows). C swi minip: additional microhaemorrhages are recognisable at the grey matter - white matter junction of the frontal lobes and in the right parieto - occipital white matter (black arrows). D swi minip, reformatted sagittal section shows microhaemorrhages in the corpus callosum (white arrows) brain mri predictors of tumour grade include contrast enhancement, oedema, mass effect, cyst formation or necrosis, haemorrhage, metabolic activity and cerebral blood volume . It is well known that the growth of solid tumours, such as gliomas, is dependent on the angiogenesis of pathological vessels . Swi can provide a thorough assessment of the internal angioarchitecture of brain tumours (increased microvascularity inside and beyond the tumour margins), together with the identification of foci of haemorrhage and calcification, thus representing an additional tool in the neuroradiological grading of cerebral neoplasms . The administration of a contrast agent (ce - swi) allows discrimination among those three entities, as only blood vessels will change their signal intensity, while calcifications and regions of inactive haemorrhage (which can be differentiated from each other by the evaluation of phase images, as described above) will not . The clinical potential of contrast - enhanced bold mr venography at 3 t and 1.5 t for the study of brain tumours was first reported by barth et al ., who demonstrated variable venous patterns in various types of tumours and in different parts of the lesions (oedema, contrast - enhancing areas, necrosis), which might represent increased blood supply and particular vascular patterns around fast - growing malignant tumours . Kim et al . Recently assessed the added value provided by swi in the differential diagnosis of solitary enhancing brain lesions compared with the use of conventional mri alone . Ce - swi has been found to be equivalent or even superior to ce - t1 images in the evaluation of most tumours with necrotic areas (figs . 14 and 15): the particular contrast combination within swi images permits the simultaneous visualisation of the information otherwise obtained by a multimodal imaging approach including ct, ce - t1 se, flair and t2 * conventional ge sequences . 14characterisation and grading of a glial tumour . A 30-year - old man with dizziness, long - standing behavioural changes and subsequent diagnosis of intra - axial cerebral tumour . B swi, unprocessed image: absence of intratumoural susceptibility signals (due either to calcifications, haemorrhages or venous vasculature). C contrast - enhanced swi: detailed visualisation of the margins of a large anaplastic area (asterisk), which is in good correlation with enhancement characteristics (d), hypervolaemia on pwi relative cerebral blood volume (rcbv) map (e) and mr spectroscopy (f). E pwi, rcbv map: area of increased cerebral blood volume (open arrow). F single - voxel mr spectroscopy, showing inversion of the cho / naa ratio and the presence of a lactate peak . Targeted stereotactic biopsy in the supposed necrotic area confirmed the hypothesis of gliomatosis cerebri who grade iii with several necrotic focifig . B t2-weighted image: the mass is irregularly hyperintense with cystic changes and small hypointensities representing small vessels and calcifications . C post - contrast t1-weighted image shows patchy enhancement of the lateral part of the lesion and some vascular structures . D ce - swi minip: calcifications appear as punctate hypointensities, unchanged after gadolinium injection (white arrows). Post - processing with minip algorithm allows the simultaneous visualisation of arteries (hyperintense, black arrows) and veins (linear hypointensities, open arrow) around and inside the tumour . Ce - swi is superior to t1-weighted post - gadolinium sequences in demonstrating a diffuse bbb rupture in the lateral necrotic area (bright signal, asterisk). Histopathology revealed a primary neuroectodermal tumour (pnet) with extensive cystic and necrotic changes and increased vascularity characterisation and grading of a glial tumour . A 30-year - old man with dizziness, long - standing behavioural changes and subsequent diagnosis of intra - axial cerebral tumour . B swi, unprocessed image: absence of intratumoural susceptibility signals (due either to calcifications, haemorrhages or venous vasculature). C contrast - enhanced swi: detailed visualisation of the margins of a large anaplastic area (asterisk), which is in good correlation with enhancement characteristics (d), hypervolaemia on pwi relative cerebral blood volume (rcbv) map (e) and mr spectroscopy (f). E pwi, rcbv map: area of increased cerebral blood volume (open arrow). F single - voxel mr spectroscopy, showing inversion of the cho / naa ratio and the presence of a lactate peak . Targeted stereotactic biopsy in the supposed necrotic area confirmed the hypothesis of gliomatosis cerebri who grade iii with several necrotic foci internal architecture of a high - grade tumour . A 2-year - old child with headache, vomiting and right hemiparesis . B t2-weighted image: the mass is irregularly hyperintense with cystic changes and small hypointensities representing small vessels and calcifications . C post - contrast t1-weighted image shows patchy enhancement of the lateral part of the lesion and some vascular structures . D ce - swi minip: calcifications appear as punctate hypointensities, unchanged after gadolinium injection (white arrows). Post - processing with minip algorithm allows the simultaneous visualisation of arteries (hyperintense, black arrows) and veins (linear hypointensities, open arrow) around and inside the tumour . Ce - swi is superior to t1-weighted post - gadolinium sequences in demonstrating a diffuse bbb rupture in the lateral necrotic area (bright signal, asterisk). Histopathology revealed a primary neuroectodermal tumour (pnet) with extensive cystic and necrotic changes and increased vascularity pinker et al . Demonstrated a correlation between intratumoural susceptibility effects, positron emission tomography (pet) results and histopathological grading . Swi has been proposed in the evaluation of clinical response to anti - angiogenetic drugs and in the differential diagnosis with pseudo - progression after chemo- and radiotherapy . A correlation with mr pwi has also been attempted . However, larger comparative studies of pwi and swi are still needed to determine a more precise role of the new techniques in the grading of cerebral neoplasms . Sporadic cerebral amyloid angiopathy (caa) is a common small - vessel disease associated with ageing, dementia and alzheimer s disease, that can only be diagnosed histopathologically following biopsy or at post - mortem examination . It consists of deposition of amyloid protein within the small and medium - sized cerebral arteries, which is likely responsible for increased vessel fragility and it is one of the major causes of lobar intraparenchymal haemorrhages in the elderly . Computed tomography (ct) and conventional mr techniques are usually unable to show cerebral microbleeds (cmbs), which can be frequently observed on t2 * -weighted gradient - echo mri and have a typical lobar distribution . Recent findings indicate that cmbs in the general elderly population are relatively common and are even more frequently observed in patients with ad . Swi is much more sensitive than ge sequences in demonstrating cmbs in and around the arteriole vessel wall, which appear as black dots better displayed by minip images (fig . 2). Histologically, they represent perivascular clusters of haemosiderin - laden macrophages resulting from leakage from cerebral small vessels . Higher field strengths, as expected, can improve cmb detectability, increasing the lesion s contrast enhancement . Fig . 2recent onset of altered mental status and cognitive impairment in a 67-year - old man . A fluid attenuated inversion recovery (flair) mri: diffuse hyperintensity of the white matter, more pronounced in the right parietal region . B diffusion - weighted imaging (dwi), apparent diffusion coefficient (adc) map: the white - matter hyperintensity represents vasogenic oedema . C ge t2 * -weighted image: small punctate hypointense foci in the right parietal cortex (open arrow). D swi minip: increased visualisation of markedly hypointense foci surrounding the white matter abnormalities which correspond to multiple cerebral microbleeds . On the basis of swi imaging findings, caa with diffuse inflammatory changes was suspected and steroid therapy was administered . E follow - up mri at 3 months showed significant reduction of the vasogenic oedema recent onset of altered mental status and cognitive impairment in a 67-year - old man . A fluid attenuated inversion recovery (flair) mri: diffuse hyperintensity of the white matter, more pronounced in the right parietal region . B diffusion - weighted imaging (dwi), apparent diffusion coefficient (adc) map: the white - matter hyperintensity represents vasogenic oedema . C ge t2 * -weighted image: small punctate hypointense foci in the right parietal cortex (open arrow). D swi minip: increased visualisation of markedly hypointense foci surrounding the white matter abnormalities which correspond to multiple cerebral microbleeds . On the basis of swi imaging findings, caa with diffuse inflammatory changes was suspected and steroid therapy was administered . E follow - up mri at 3 months showed significant reduction of the vasogenic oedema caa is also characterised by white matter hyperintensities on conventional mri sequences, which have been associated with cognitive impairment . Vascular amyloid deposition is believed to be involved in the pathophysiological mechanisms that determine white matter hypoperfusion through either vessel stenosis or vascular dysfunction . Caa should also be suspected in elderly patients with clinical signs of a progressive encephalopathy syndrome with seizures, in which extensive white matter abnormalities are discovered in brain mri together with multiple cmbs with lobar and subcortical distribution (fig . 2). It has been recently reported that this combination of mr findings should be interpreted as caa - related inflammation, which can be treated with steroid therapy with a prompt resolution of the symptoms . In those cases, the demonstration of an apoe 44 genotype can definitely support the neuroradiological diagnosis . Hypertensive encephalopathy and especially posterior reversible encephalopathy syndrome (pres), when the signal abnormalities of the white matter are more symmetrical and located in parieto - occipital regions, should be taken into consideration for differential diagnosis . Hypertensive encephalopathy is characterised by multiple cmbs which are usually silent and can be discovered when the patient is investigated with mri in order to understand the cause of an intraparenchymal haemorrhage located outside the basal ganglia . Swi is more sensitive to cmbs than t2 * -weighted ge sequences in blood pressure (bp)-related small vessels disease . Cmbs are usually discovered both in deep basal ganglia and subcortical white matter (fig . Histologically they represent focal accumulations of haemosiderin - containing macrophages in the perivascular space of small brain vessels, indicating previous extravasation of blood, and are often associated with the presence of a symptomatic haemorrhage in the corresponding area . The number of cmbs, which remain detectable for years, is significantly associated with bp levels . Moreover, the presence of deep cmbs can be a useful marker of bp - related small vessels disease, helping in the differential diagnosis with caa . A t2-weighted image shows a thin hypointense band in the right thalamus, which represents haemosiderin deposition (arrow). B ge t2 * -weighted image shows multiple microbleeds, the largest one in the right thalamus (arrow). C swi minip: the number of identifiable microbleeds is increased compared with ge images . They can be recognised bilaterally in the basal ganglia and in the subcortical temporal white matter (arrows) patient with long - standing hypertension . A t2-weighted image shows a thin hypointense band in the right thalamus, which represents haemosiderin deposition (arrow). B ge t2 * -weighted image shows multiple microbleeds, the largest one in the right thalamus (arrow). C swi minip: the number of identifiable microbleeds is increased compared with ge images . They can be recognised bilaterally in the basal ganglia and in the subcortical temporal white matter (arrows) iron deposition increases in the brain as a function of age, primarily in the form of ferritin and particularly in oligodendrocytes, but also in neurons and microglia . Typical sites of iron deposition include the globus pallidum, substantia nigra, and red and dentate nuclei . Swi filtered - phase images are particularly suitable for showing increased iron content in the brain (. Abnormally elevated iron levels are evident in many neurodegenerative disorders, including parkinson s disease, alzheimer s disease, huntington s disease and amyotrophic lateral sclerosis . The ability to measure the amount of ferritin in the brain can be used for a better understanding of the progression of the disease and is also helpful in predicting the treatment outcome . Phase images allow a better distinction between the pars compacta and the pars reticulata of the substantia nigra, which contains iron (fig . 1). As in patients with idiopathic parkinson s disease, there is evidence of increased iron in the substantia nigra, swi sequences can be proposed as a useful imaging tool to identify iron deposition as a biomarker for disease progression, although longitudinal studies are required to support the usefulness of this specific application . Accurate localisation of the subthalamic nucleus can also be achieved in the swi phase maps at 3 t, allowing safe direct targeting for placement of electrodes in the treatment of parkinson s disease . In 7098% of patients with alzheimer s disease, recent evidence has shown that patients with multiple cmbs have more white matter hyperintensities and perform worse on mini mental state examination compared with patients with no cmbs, despite similar disease duration . A significant correlation has also been reported recently between patients with at least one hypointensity in gre - t2 * imaging and those homozygous for the apolipoprotein e 44 gene, a well - known risk factor for alzheimer s disease (fig 4). B coronal reformatted swi section shows a cerebral microbleed in the right frontal white matter (arrow) alzheimer s disease . B coronal reformatted swi section shows a cerebral microbleed in the right frontal white matter (arrow) swi has been demonstrated to be very useful in the acute phase of stroke for several reasons . First of all, it is very sensitive to the presence of cmbs, whose early identification is believed to predict the probability of potential haemorrhagic transformation after thrombolytic treatment . It has also been hypothesised that cmbs can represent a link between cerebral haemorrhage and ischaemia . Swi is also capable of identifying the acute intravascular clot in the main and distal branches of the cerebral arteries . The evaluation of the intravascular content of deoxyhaemoglobin in hypoperfused brain is one of the most interesting applications of swi . This technique has been proved to be useful for the assessment of tissue viability in patients with hyperacute cerebral ischaemia, as it provides functional information about the ischaemic penumbra . In acute arterial stroke, flow reduction or absence causes an increase in oxygen extraction, leading to a growth in the amount of deoxyhaemoglobin in the hypoperfused brain tissue . Deoxyhaemoglobin represents a supplementary source of contrast enhancement in swi because of its paramagnetic properties, which determine a reduction in t2 * as well as a phase difference between the vessel and its surrounding parenchyma . As the t1 and t2 properties of blood depend on the oxygen saturation of the blood, there are differences between arterial and venous vessels that cannot be discovered with conventional sequences . The visualisation of draining veins within areas of impaired perfusion allows the identification of penumbral brain tissue in a different way from the current perfusion - weighted imaging (pwi) techniques (fig . 5). 5left internal carotid artery (ica) dissection with acute watershed infarct in a 42-year - old man with sudden onset of speech disturbance . A mr angiography, single partition at the level of the skull base showing dissection of the left ica, which is characteristically enlarged with high - signal - intensity methaemoglobin (arrowheads) representing the intramural thrombus and a small eccentric lumen (arrow). B mr angiography, coronal mip: narrowing of the ica in its vertical segment just below the skull base (arrow). D swi minip: improved visualisation of the veins of the left cerebral hemisphere, which is related to increased oxygen extraction in the ischaemic penumbra and corresponds to the hypoperfusion deficit shown by the mean transit time (mtt) map . E perfusion mri, mtt map, showing delayed transit time in the left middle cerebral artery (mca) territory . F intra - arterial digital subtraction angiography (dsa), left carotid angiogram, confirming the ica stenosis (arrow). G intra - arterial dsa: the patient was treated using stent placement, which resulted in partial restoration of the vessel lumen . H perfusion mri, mtt map 24 h after stent placement: complete resolution of the perfusion deficit . I swi minip, normalisation of the venous drainage of the left hemisphere left internal carotid artery (ica) dissection with acute watershed infarct in a 42-year - old man with sudden onset of speech disturbance . A mr angiography, single partition at the level of the skull base showing dissection of the left ica, which is characteristically enlarged with high - signal - intensity methaemoglobin (arrowheads) representing the intramural thrombus and a small eccentric lumen (arrow). B mr angiography, coronal mip: narrowing of the ica in its vertical segment just below the skull base (arrow). D swi minip: improved visualisation of the veins of the left cerebral hemisphere, which is related to increased oxygen extraction in the ischaemic penumbra and corresponds to the hypoperfusion deficit shown by the mean transit time (mtt) map . E perfusion mri, mtt map, showing delayed transit time in the left middle cerebral artery (mca) territory . F intra - arterial digital subtraction angiography (dsa), left carotid angiogram, confirming the ica stenosis (arrow). G intra - arterial dsa: the patient was treated using stent placement, which resulted in partial restoration of the vessel lumen . H perfusion mri, mtt map 24 h after stent placement: complete resolution of the perfusion deficit . Swi has become a functional method for evaluating cerebral venous sinus thrombosis (cvst) by demonstrating engorgement of the venous system as a result of venous hypertension and collateral slow flow . Dural sinus thrombosis causes an increase in deoxyhaemoglobin concentration in the veins involved that appears as a prominent area of hypointense signal intensity on swi; if cvst is treated successfully, this effect will disappear . Swi is also capable of identifying the thrombosed pial vein, which is responsible for the development of oedema into the white matter, due to its high sensitivity in the demonstration of an intravascular clot . Swi can depict at the same time the features of parenchymal or extra - axial haemorrhages that can occur in the case of infarction, with higher sensitivity compared with conventional ge sequences (fig . A sagittal t1-weighted image demonstrates hyperintensity of the posterior third of the sagittal sinus (open arrow). B mr venography confirms the thrombosis of the posterior third of the sagittal sinus ending at the torcular (open arrow). C dwi, b = 1,000 image demonstrates a right occipital acute ischaemic lesion . D swi minip shows a right thalamic haemorrhagic lesion in the vascular territory of the right deep internal cerebral vein (arrow) epileptic seizures and sagittal sinus thrombosis in a 3-year - old child . A sagittal t1-weighted image demonstrates hyperintensity of the posterior third of the sagittal sinus (open arrow). B mr venography confirms the thrombosis of the posterior third of the sagittal sinus ending at the torcular (open arrow). C dwi, b = 1,000 image demonstrates a right occipital acute ischaemic lesion . D swi minip shows a right thalamic haemorrhagic lesion in the vascular territory of the right deep internal cerebral vein (arrow) cerebral arteriovenous malformations (avms) are easily displayed by conventional mri and mr angiography because of their characteristic high flow, whereas venous malformations such as cavernomas, developmental venous anomalies and capillary telangiectasias cannot be adequately visualised without contrast medium administration and ge sequences, as they mainly consist of slow - flow small vessels . Mr angiography techniques are often inadequate for visualising small vessels with slow flow . On the contrary, swi sequences are well suited for the visualisation of very small vessels, such as the caput medusae of venous angiomas and telangiectasias as a result of a combination of slow flow with changes in deoxyhaemoglobin concentration . The combined information derived from both phase and magnitude images is responsible for enhanced visualisation of such lesions . Swi plays a substantial role in the identification and characterisation of cerebral vascular malformations, first of all improving their detection rate . On the other hand, allowing simultaneous visualisation of the different compartments of cerebral avms and of the relationship with one another and with the brain parenchyma, it is particularly valuable for therapeutic planning . The clinical usefulness of a similar technique, bold mr venography, was first demonstrated by essig et al . . Phase changes caused by the susceptibility differences between oxygenated and deoxygenated red blood cells and the different relaxation rates between venous and arterial blood provide a natural separation of arteries and veins . The 3d dataset of swi can therefore be used as an alternative to mr angiography in order to better differentiate afferent arteries from draining veins . This advantage becomes relevant with small avms with recent bleeding, in which it is mandatory to identify the nidus in relation to eloquent areas in order to plan the best therapeutic approach, endovascular or surgical or both (fig . Veins appear dark due to t2 * loss and processing of the phase image, while arteries show bright signal from time - of - flight (tof) inflow enhancement . The use of higher flip angles and shorter tr make it possible to increase the contrast enhancement in the arteries without overly degrading the venography . For the evaluation of high - flow vascular malformations (i.e. Avms and dural arteriovenous fistulas) the use of both minip and mip post - processing techniques can improve the image quality . The identification of small niduses, the exact location of the fistulous point and the depiction of the venous drainage patterns are the most relevant information that should be obtained from this technique . The elevated diagnostic accuracy of swi compared with digital subtraction angiography (dsa) has been recently reported for the detection of arteriovenous shunting (avs) in brain avms . B the swi minip image clearly depicts the brainstem haemorrhage, which is markedly hypointense . C, d swi, axial and reformatted sagittal images, processed with a maximum intensity projection algorithm (mip), show flow - related enhancement within the haemorrhagic lesion (arrow) and a hypertrophied right posterior inferior cerebellar artery (pica; open arrow) which are consistent with a small ruptured brainstem avm . E cerebral angiography, selective injection of the right vertebral artery: brainstem avm (arrow) fed by branches of the right pica and early venous drainage into a cerebellar vein (arrowheads) brainstem haemorrhage in a 43-year - old patient . B the swi minip image clearly depicts the brainstem haemorrhage, which is markedly hypointense . C, d swi, axial and reformatted sagittal images, processed with a maximum intensity projection algorithm (mip), show flow - related enhancement within the haemorrhagic lesion (arrow) and a hypertrophied right posterior inferior cerebellar artery (pica; open arrow) which are consistent with a small ruptured brainstem avm . E cerebral angiography, selective injection of the right vertebral artery: brainstem avm (arrow) fed by branches of the right pica and early venous drainage into a cerebellar vein (arrowheads) dural av fistulas (davfs) are usually more difficult to identify than avms on conventional mri, especially when venous ectasia is absent, as in type i and ii davfs; the patients have vague symptoms (i.e. Headache or tinnitus) and there are no localising signs . Swi is capable of better identifying the extent of the abnormal venous drainage compared with mr angiography . The conspicuity of the venous structures on swi can be explained by the combination of a prolonged cerebral circulation time that favours increased oxygen extraction resulting in increased concentration of deoxyhaemoglobin, with the typical venous engorgement (fig . B, c swi axial and coronal reformatted minip images show dilated right temporal veins (arrows), which arouse suspicion of a tentorial dural av fistula . D cerebral angiography, selective injection of the right external carotid artery: dural av fistula of the right tentorium, fed by the right middle meningeal artery (open arrow), with early venous drainage into the tentorial and temporal veins (arrows) patient with headache and pulsatile tinnitus of the right ear . Swi axial and coronal reformatted minip images show dilated right temporal veins (arrows), which arouse suspicion of a tentorial dural av fistula . D cerebral angiography, selective injection of the right external carotid artery: dural av fistula of the right tentorium, fed by the right middle meningeal artery (open arrow), with early venous drainage into the tentorial and temporal veins (arrows) concerning slow - flow vascular malformations (i.e. Cavernomas, developmental venous anomalies and capillary telangiectasias) the phase information contained in swi is responsible for an artefactual enhancement that dramatically improves mri sensitivity to these pathological conditions . Without swi individuals with cavernous malformations can present with epilepsy and focal neurological deficits or acute intracranial haemorrhage, although these vascular malformations are often discovered as incidental findings . Cavernomas that have previously bled are usually detectable on routine mri because of the prominent signal intensity of haemorrhagic products . However, these lesions tend to be barely visible when they have not bled, except for faint enhancement after contrast medium administration that is often non - specific . Patients with multiple lesions can be diagnosed with familial cavernous angiomatosis and should be referred for genetic evaluation and counselling (fig . . Individuals with symptomatic, growing, or haemorrhagic malformations should be considered for surgical resection . Close follow - up after diagnosis and treatment is helpful to identify lesion progression or recurrence . A t2-weighted image demonstrates a left frontal cavernous malformation surrounded by a thick ring of haemosiderin . B ge t2 * -weighted image shows multiple punctate hypointense foci located in both hemispheres, corresponding to small cavernous malformations (arrows). C swi minip: there are supplemental cavernous malformations that are not detected by ge sequences (arrows). D swi minip, reformatted sagittal section: multiple small cavernous malformations located in the brainstem (arrows) multiple cavernous malformations in a 32-year - old man investigated for intraparenchymal haemorrhage . A t2-weighted image demonstrates a left frontal cavernous malformation surrounded by a thick ring of haemosiderin . B ge t2 * -weighted image shows multiple punctate hypointense foci located in both hemispheres, corresponding to small cavernous malformations (arrows). C swi minip: there are supplemental cavernous malformations that are not detected by ge sequences (arrows). D swi minip, reformatted sagittal section: multiple small cavernous malformations located in the brainstem (arrows) developmental venous anomalies (dva) consist of a radially arranged venous complex converging on a centrally located venous trunk, which drains the normal brain parenchyma . They are better displayed with swi than with t1-weighted contrast - enhanced images (fig . 10incidental discovery of a cerebellar developmental venous anomaly in a 48-year - old man investigated for right - sided sensorineural hearing loss . A t2-weighted image shows thin hypointense bands of flow void, in the right cerebellar hemisphere (open arrow). B swi minip demonstrates multiple thin medullary veins (arrows) that converge into a dilated collector vein (open arrow), consistent with a cerebellar venous angioma, better defined as developmental venous anomaly . C post - contrast t1-weighted axial image shows the enhancing dilated veins (open arrow) incidental discovery of a cerebellar developmental venous anomaly in a 48-year - old man investigated for right - sided sensorineural hearing loss . A t2-weighted image shows thin hypointense bands of flow void, in the right cerebellar hemisphere (open arrow). B swi minip demonstrates multiple thin medullary veins (arrows) that converge into a dilated collector vein (open arrow), consistent with a cerebellar venous angioma, better defined as developmental venous anomaly . C post - contrast t1-weighted axial image shows the enhancing dilated veins (open arrow) capillary telangiectasias are asymptomatic venous vascular malformations, which are smaller and less common than cavernomas and can sometimes occur in mixed cavernoma / telangiectasia lesions . These may occur sporadically or may be infrequently associated with syndromes, like hereditary haemorrhagic telangiectasia or ataxia telangiectasia . They may also manifest as a result of endothelial injury, such as radiation - induced vascular injury, particularly in children who have received cranial irradiation . They are characterised by poor contrast enhancement and can therefore be missed on conventional t1-weighted and t2-weighted sequences (fig . 11incidental discovery of a pontine telangiectasia in a 34-year - old woman investigated for migraine . Swi minip axial (b) and sagittal reformatted section (c) show a markedly hypointense lesion with regular margins, located in the pons . The capillary telangiectasia is characterised by mild enhancement with an arbor - like pattern incidental discovery of a pontine telangiectasia in a 34-year - old woman investigated for migraine . Swi minip axial (b) and sagittal reformatted section (c) show a markedly hypointense lesion with regular margins, located in the pons . . The capillary telangiectasia is characterised by mild enhancement with an arbor - like pattern ataxia telangiectasia is a rare autosomal recessive disorder characterised by an early - onset of progressive cerebellar ataxia, immunodeficiency, ocular and cutaneous telangiectasia and is associated with an elevated risk of intracranial tumours . Swi can discover brain gliovascular nodules with perivascular haemorrhage and small haemosiderin deposits presumably only in older patients, during the later stages of the disease; however, in our experience this technique can also be a useful screening tool in young patients (fig . A 31-year - old woman with a diagnosis of ataxia telangiectasia by the age of 7, investigated for dizziness and headache . C swi minip: additional hypointense black spots can be identified, corresponding to haemosiderin deposits presumably due to gliovascular nodules with perivascular haemorrhage ataxia telangiectasia . A 31-year - old woman with a diagnosis of ataxia telangiectasia by the age of 7, investigated for dizziness and headache . C swi minip: additional hypointense black spots can be identified, corresponding to haemosiderin deposits presumably due to gliovascular nodules with perivascular haemorrhage mri with ge sequences has been extensively used in the past for investigating young patients with traumatic brain injuries (tbi), either in the acute phase, when the clinical picture of a severe coma is not explained by ct, or months after trauma, in order to understand the causes of an unsuccessful recovery . In both cases, swi has been widely used in the identification of smaller haemorrhages due to a shear - strain mechanism of injury, thus refining the prediction of outcome . Haemorrhagic dai lesions on swi can be six - times better detected than t2 * -weighted conventional ge sequences and the recognisable volume of haemorrhage is approximately twofold greater (fig . Reformatted swi images in the sagittal plane are particularly helpful in the assessment of corpus callosum haemorrhages . Both number and volume of haemorrhagic lesions correlate with neuropsychological deficits . Fig . 13diffuse axonal injury in a 14-year - old boy who had severe tbi following a motorcycle crash . Brain mr study was performed three days after the trauma, when the patient was in a severely comatose state (gcs 5). A flair image identifies multiple hyperintense lesions in the splenium of the corpus callosum and in frontal subcortical and periventricular white matter . B ge t2 * -weighted image: haemorrhagic shearing injuries are barely visible in the right fronto - opercular and parieto - occipital regions (arrows). C swi minip: additional microhaemorrhages are recognisable at the grey matter - white matter junction of the frontal lobes and in the right parieto - occipital white matter (black arrows). D swi minip, reformatted sagittal section shows microhaemorrhages in the corpus callosum (white arrows) diffuse axonal injury in a 14-year - old boy who had severe tbi following a motorcycle crash . Brain mr study was performed three days after the trauma, when the patient was in a severely comatose state (gcs 5). A flair image identifies multiple hyperintense lesions in the splenium of the corpus callosum and in frontal subcortical and periventricular white matter . B ge t2 * -weighted image: haemorrhagic shearing injuries are barely visible in the right fronto - opercular and parieto - occipital regions (arrows). C swi minip: additional microhaemorrhages are recognisable at the grey matter - white matter junction of the frontal lobes and in the right parieto - occipital white matter (black arrows). D swi minip, reformatted sagittal section shows microhaemorrhages in the corpus callosum (white arrows) brain mri predictors of tumour grade include contrast enhancement, oedema, mass effect, cyst formation or necrosis, haemorrhage, metabolic activity and cerebral blood volume . It is well known that the growth of solid tumours, such as gliomas, is dependent on the angiogenesis of pathological vessels . Swi can provide a thorough assessment of the internal angioarchitecture of brain tumours (increased microvascularity inside and beyond the tumour margins), together with the identification of foci of haemorrhage and calcification, thus representing an additional tool in the neuroradiological grading of cerebral neoplasms . The administration of a contrast agent (ce - swi) allows discrimination among those three entities, as only blood vessels will change their signal intensity, while calcifications and regions of inactive haemorrhage (which can be differentiated from each other by the evaluation of phase images, as described above) will not . The clinical potential of contrast - enhanced bold mr venography at 3 t and 1.5 t for the study of brain tumours was first reported by barth et al ., who demonstrated variable venous patterns in various types of tumours and in different parts of the lesions (oedema, contrast - enhancing areas, necrosis), which might represent increased blood supply and particular vascular patterns around fast - growing malignant tumours . Kim et al . Recently assessed the added value provided by swi in the differential diagnosis of solitary enhancing brain lesions compared with the use of conventional mri alone . Ce - swi has been found to be equivalent or even superior to ce - t1 images in the evaluation of most tumours with necrotic areas (figs . 14 and 15): the particular contrast combination within swi images permits the simultaneous visualisation of the information otherwise obtained by a multimodal imaging approach including ct, ce - t1 se, flair and t2 * conventional ge sequences . 14characterisation and grading of a glial tumour . A 30-year - old man with dizziness, long - standing behavioural changes and subsequent diagnosis of intra - axial cerebral tumour . B swi, unprocessed image: absence of intratumoural susceptibility signals (due either to calcifications, haemorrhages or venous vasculature). C contrast - enhanced swi: detailed visualisation of the margins of a large anaplastic area (asterisk), which is in good correlation with enhancement characteristics (d), hypervolaemia on pwi relative cerebral blood volume (rcbv) map (e) and mr spectroscopy (f). E pwi, rcbv map: area of increased cerebral blood volume (open arrow). F single - voxel mr spectroscopy, showing inversion of the cho / naa ratio and the presence of a lactate peak . Targeted stereotactic biopsy in the supposed necrotic area confirmed the hypothesis of gliomatosis cerebri who grade iii with several necrotic focifig . B t2-weighted image: the mass is irregularly hyperintense with cystic changes and small hypointensities representing small vessels and calcifications . C post - contrast t1-weighted image shows patchy enhancement of the lateral part of the lesion and some vascular structures . D ce - swi minip: calcifications appear as punctate hypointensities, unchanged after gadolinium injection (white arrows). Post - processing with minip algorithm allows the simultaneous visualisation of arteries (hyperintense, black arrows) and veins (linear hypointensities, open arrow) around and inside the tumour . Ce - swi is superior to t1-weighted post - gadolinium sequences in demonstrating a diffuse bbb rupture in the lateral necrotic area (bright signal, asterisk). Histopathology revealed a primary neuroectodermal tumour (pnet) with extensive cystic and necrotic changes and increased vascularity characterisation and grading of a glial tumour . A 30-year - old man with dizziness, long - standing behavioural changes and subsequent diagnosis of intra - axial cerebral tumour . B swi, unprocessed image: absence of intratumoural susceptibility signals (due either to calcifications, haemorrhages or venous vasculature). C contrast - enhanced swi: detailed visualisation of the margins of a large anaplastic area (asterisk), which is in good correlation with enhancement characteristics (d), hypervolaemia on pwi relative cerebral blood volume (rcbv) map (e) and mr spectroscopy (f). E pwi, rcbv map: area of increased cerebral blood volume (open arrow). F single - voxel mr spectroscopy, showing inversion of the cho / naa ratio and the presence of a lactate peak . Targeted stereotactic biopsy in the supposed necrotic area confirmed the hypothesis of gliomatosis cerebri who grade iii with several necrotic foci internal architecture of a high - grade tumour . B t2-weighted image: the mass is irregularly hyperintense with cystic changes and small hypointensities representing small vessels and calcifications . C post - contrast t1-weighted image shows patchy enhancement of the lateral part of the lesion and some vascular structures . D ce - swi minip: calcifications appear as punctate hypointensities, unchanged after gadolinium injection (white arrows). Post - processing with minip algorithm allows the simultaneous visualisation of arteries (hyperintense, black arrows) and veins (linear hypointensities, open arrow) around and inside the tumour . Ce - swi is superior to t1-weighted post - gadolinium sequences in demonstrating a diffuse bbb rupture in the lateral necrotic area (bright signal, asterisk). Histopathology revealed a primary neuroectodermal tumour (pnet) with extensive cystic and necrotic changes and increased vascularity pinker et al . Demonstrated a correlation between intratumoural susceptibility effects, positron emission tomography (pet) results and histopathological grading . Swi has been proposed in the evaluation of clinical response to anti - angiogenetic drugs and in the differential diagnosis with pseudo - progression after chemo- and radiotherapy . A correlation with mr pwi has also been attempted . However, larger comparative studies of pwi and swi are still needed to determine a more precise role of the new techniques in the grading of cerebral neoplasms . Swi, which is a combination of ge techniques with phase information, represents a useful tool for the identification and characterisation of vascular malformations and for a better understanding of cerebrovascular diseases . Despite some inherent limitations, swi has increasing indications for neuroradiology and should be included in the routine imaging protocols of trauma and vascular abnormalities . Further investigation is still needed into its extensive clinical application in neurodegenerative diseases and tumoural pathological conditions.
Shallots (wild garlic / osghordion) with the scientific name of allium hertifolium, is one of the most famous plants from the alliaceae family . For a long time, shallots have been used as a source of food and medicine in iran . The active ingredients of the plant could be referred to agapentagenin, allicin, omega-3, omega-6, and minerals such as potassium, sodium, magnesium, iron, copper, zinc, and manganese . This study was conducted to compare shallots in the traditional and modern medicine in order to make a better use of this precious plant . To collect appropriate data, resources and articles in trustworthy databases (e.g. Cochrane library, pubmed, google scholar) and traditional literature (e.g. Makhzan - ul - adwiah, canon, zakhireh - ye khwarazmshahi) were studied . Subsequently, the findings were reviewed, classified, and reported in a tabular format . Shallots are rich in fatty acids and minerals with many pharmacological effects such as its effect on the respiratory and nervous system and blood dilution, as reflected in the modern medicine . However, certain effects as mentioned in traditional medicine (e.g. Anti - warts, anti - lipoma, anti - kidney stone, and its diuretic effects) are not covered in research studies of the modern medicine . Depending on its natural habitats, shallots have different pharmacological effects for which many usages are mentioned in traditional medicine . Some of these effects have been investigated in modern medicine; however, further evaluation of its safety and dosages for clinical use is necessary . Furthermore, some cases have not been studied in modern medicine, which could be the basis for future research.
Multiple sclerosis (ms) is a chronic idiopathic disorder of the central nervous system (cns) sustained by a multifocal inflammatory process predominantly affecting myelin - sheathed axons . Although traditionally viewed as a white matter (wm) demyelinating disorder, ms is characterized by acute and chronic axonal and neuronal loss, as shown for long by pathological and neuroimaging studies [1, 2]. Acute inflammation causes the development of plaques, characterized by blood - brain barrier (bbb) breakdown, perivascular cellular infiltration, demyelination, and axonal degeneration . Notably, axonal damage occurs not only in the acute phase but also in inactive ms lesions [3, 4]. Plaques represent the underlying pathological substrate of clinical events, with occurrence of focal / multifocal neurological symptoms and signs that eventually subside in many cases as inflammation ceases . Lesions may also involve the cortical gray matter (gm) in which case they are characterized by myelin / axonal injury and microglial activation but not bbb disruption and less cellular infiltration compared to wm lesions [6, 7]. It is increasingly perceived that the severity of ms clinical outcome does not simply result from the extent of wm damage, but it rather represents a complex balance among wm and gm tissue damage, tissue repair, and cortical reorganisation [810]. The evidence that axonal loss highly correlates with neurological disability and disease progression has spurred the search for reliable markers of axonal degeneration . Although ms aetiology still remains undetermined, genetic and environmental risk factors have been identified or are suspected (i.e., female gender, hla - drb1 allele, genome - wide association studies candidate genes, epstein - barr virus infection, low vitamin d levels, cigarette smoking, etc .) Mainly influencing immune system modulation and although much less evidently myelin and axonal repair mechanisms [1115]. The complex and unique interplay between genetic background and environmental exposure in each case likely determines the clinical heterogeneity of ms both between and within subjects varying from benign or even subclinical types to highly disabling forms and making it a challenge to predict the clinical course at the individual level . Given that ms is mostly diagnosed in subjects in the third and fourth decade of life, the availability of reliable predictors of long - term prognosis is extremely important . The objective of this paper is to review the current literature and to discuss evidence on clinical, paraclinical, magnetic resonance imaging (mri), and cerebrospinal fluid (csf) markers as predictors of disability progression in ms . The typical clinical course of ms is relapsing - remitting (rr), characterized by an initial event of acute or subacute neurological disturbance, generally indicated as clinically isolated syndrome (cis) followed by recurrence of symptoms over time . Cis is the type of onset in around 85% of ms cases, while the remaining 15% of patients have a progressive disease from onset (primary progressive (pp) ms). Progressive onset is an unfavourable prognostic predictor per se, since motor, sphincter control, and cognitive impairment are prominent features of the clinical picture, neurological disability continues to worsen over time, and no effective treatment exists . Conversely, ciss generally recover well and may remain monophasic for a long time interval before conversion to clinically definite ms occurs . Since cis represents the earlier clinical manifestation of rr ms, this patient population is of great value for identification of predictive and prognostic disease markers compared to definite ms cases who are necessarily in a more advanced stage . Typical cis presentation includes acute partial myelitis (3050% of cases), brainstem / cerebellum syndromes (2530%) unilateral optic neuritis (2025%), and cerebral hemisphere syndromes (5%); more than 20% of ciss present with symptoms and/or signs of more than one anatomical location (multifocal presentation) [1820]. The percentage of cis patients who develop clinically definite ms in prospective observational studies ranges from 16% at 1 year to 80% at 25 years [21, 22]. However, these figures date back to studies conducted before the introduction of the most recent revision of ms diagnostic criteria according to which patients previously classified as cis already have ms at the time of initial symptoms if mri demonstrates space and time dissemination of demyelinating lesions . After ms develops, irreversible disability may be the result of accumulation of fixed sequelae after each attack or may be due to transition to a secondary progressive (sp) phase, in which insidious neurological deterioration substitutes the preceding rr stage of the disease (3258% of cases in major prospective studies). Clinical predictors of long - term disability in ms include male gender, older age, multifocal symptoms, efferent systems involvement, incomplete remission of the initial event, a short interval to the second event, and high relapse rate in the first 25 years after onset, although not all studies replicated the same findings (table 1) [2427, 30, 31, 48, 73]. One single study reported a shorter time to secondary progressive ms in patients with family history of ms . The relevance of age as a prognostic factor is subject to interpretation depending on the temporal frame in which disability levels are captured . Indeed, while older age at onset is associated with a more rapid disability progression likely due to prevalence of the primary progressive disease course, age - dependent degenerative processes, and dysfunction of repair mechanisms in older subjects early onset ms patients reach disability milestones at a younger age compared to late onset ms cases, even though in a longer time interval [26, 75]. In addition, it has been shown that the progressive phase of ms is an age- rather than a disease duration - dependent process, since age at pp and sp ms onset overlaps significantly in large observational studies and subsequent disability progresses along a common age - driven trajectory independent of onset epoch and previous clinical course . In this perspective, older age at onset may be viewed as a favourable prognostic factor, meaning a longer disease - free interval before ms symptoms occurrence in life and an older age at which significant disability milestones are reached, compared to early onset . Also the influence of relapses on later disability progression is debated . According to the authors who found a positive correlation between relapses and long - term disability, the association is stronger in younger patients (<25 years old at ms onset), it diminishes significantly after the first 25 years of disease, and it is minimal after the progressive phase has begun [32, 78]. A sizable proportion of ms patients does not accumulate clinically relevant disability during the entire natural history of the disease . This type of course is known as benign ms, although there is no general agreement on its definition and consequently, on its prevalence in the ms population . While initial definitions predominantly stressed the absence of significant ambulatory disability (expanded disability status scale (edss) score <3.5 or 2.5) after a reasonable time interval from initial symptoms (10 or 15 years) [81, 82], more recent studies highlighted the importance of carefully considering cognitive status and quality of life when defining benign ms . Indeed, edss, which is the most largely used disability scale in ms clinical practice, is clearly unbalanced towards ambulation impairment . Scores range from 0 meaning no disability to 10 meaning death due to ms: from score 1 to 3.5, there can be a wide range of neurological deficit but ambulation is unrestricted; from 4 to 5.5, independent ambulation is below 500 meters; from 6 to 7.5, ambulation is only possible with support; and from 8 on, the patient is wheelchair - bound or bedridden . Whatever the definition, it has been shown that benign status at 10 years after ms onset persist at 20 or more years in 5269% of patients, leading to the conclusion that benign ms is a transient condition for a considerable proportion of cases [82, 8486]. However, studies addressing this topic are generally limited by the clinic - based design in which ms patients with mild disease who are not seen on a regular basis in the neurology practices are not included in the analysis falsely reducing the proportion of benign cases . There are no diagnostic tools or validated markers to identify ms patients who will have a favourable clinical course; however, female gender, younger age, and absence of motor symptoms at onset have been associated with a benign disease form . Neurophysiological assessment with visual, somatosensory, motor, and brainstem auditory evoked potentials is traditionally used as a paraclinical tool for ms evaluation, although its diagnostic relevance has progressively decreased after mri became largely available as a more sensitive technique . However, evoked potentials still maintain a prognostic significance likely because they reflect the functional integrity of specific anatomical pathways and consequently tend to better correlate with neurological disability than conventional mri, which provide purely morphological information . Several cross - sectional and longitudinal studies established that the degree of evoked potentials abnormalities is significantly associated with the edss score at the time of neurophysiological evaluation and up to 14 years later in patients with ms [8893]. A recent study found that cis patients with at least three abnormal evoked potentials at baseline have an increased risk of reaching moderate disability over a mean follow - up period of six years, independent of initial mri features . In recent years optical coherence tomography (oct) has emerged as a powerful tool to detect retinal nerve fiber layer (rnfl) thinning in ms patients with and without optic neuritis history . Rnfl thickness decrease results from axonal loss in optic nerve, possibly reflecting diffuse neuroaxonal injury in the cns, and correlates with markers of ms activity such as relapses, new / gadolinium - enhancing lesions, and parenchymal atrophy on brain mri [96, 97]. The extent of rnfl thinning in optic neuritis patients predicts visual recovery and exhibits a modest correlation with overall neurological disability in ms patients . It has been recently suggested that thinning of inner and outer nuclear layers of the retina identifies a subset of ms patients with primary retinal neuronal pathology and more aggressive disease course [98, 99]. Given its increased availability and its sensitivity in detecting ms lesions, conventional mri has become the main imaging tool in the ms diagnostic work up as well as in monitoring treatment response to disease - modifying drugs . Its diagnostic sensitivity reflects the ability to identify clinically silent lesions, thus, favouring the early demonstration of dissemination in space and time of the lesions (figure 1) according to the recent revision of diagnostic criteria . However, while it may seem obvious that patients who develop new wm lesions are worse off than those without new lesions, conventional mri has been shown to have a prognostic value only in patients at disease onset: high t2-weighted lesion load in patients with a cis has been associated with an increased risk of subsequent conversion to clinically definite ms and long - term disability accumulation [101, 102]. By contrast, in a more advanced phase of the disease, the strength of relationship between conventional mri measures and subsequent disability progression is rather weak [33, 34]. In a recent study including 548 placebo - treated rr ms patients, the multivariate analysis indicated just edss score and t2 lesion load as factors independently predicting the clinical progression . Nevertheless, these two variables taken together were able to account for only 3% of the probability to have an edss increase over follow - up time, thus, confirming the limited value of these metrics in predicting short - term disability changes in rr ms . Such result is in line with those of several previous cross - sectional and longitudinal studies conducted on smaller groups of patients with different clinical characteristics, which have shown only modest correlation between t2- and t1-weighted brain mri activity and subsequent changes in disability [3638]. Although edss is not without limitations in terms of reliability and responsiveness to disease changes, the lack of a strong correlation between wm lesion load and clinical disability had prompted investigations of the so - called normal - appearing brain tissue . For this purpose, unconventional and quantitative mri techniques, having increased sensitivity and specificity to irreversible tissue damage, have been consistently applied to monitor and predict ms evolution . Given these premises, several studies have been focused on brain atrophy showing its relevant clinical impact not only in the diagnostic phase but also in predicting subsequent disability progression both in rr ms and in pp ms (figure 2). A recent study published by the magnims group, included 261 ms patients who had mr imaging at baseline and after 1 - 2 years and edss scoring at baseline and after 10 years; in the whole patient group, after correction for imaging protocol, whole brain and central atrophy were good predictors of edss at 10 years (r = 0.74). Despite the good sensitivity of brain atrophy, even better results in predicting disability progression have been further achieved by the regional analysis of brain atrophy . Jasperse and colleagues, for example, suggested that atrophy of central brain regions was related to decline in ambulatory function, whereas atrophy of both central and peripheral brain regions was associated to decline in neurologically more complex tasks for coordinated hand function . The best results, however, have been obtained by the study of gm and wm atrophy separately . Indeed several voxel - based and surface - based studies, both in rrms and in ppms, revealed strong relationship between gm, but not wm, atrophy and disability progression [4345]. Even when a very long followup, a very large sample size, or more sophisticated disability scales (i.e., ms functional composite) were considered, gm atrophy reflected disease subtype and disability progression to a greater extent than wm atrophy or lesions [4547]. A further step forward in the comprehension of the pathological mechanisms underlying the accumulation of irreversible disability in ms was obtained by the regional analysis of gm atrophy; since the first studies, indeed, it was clear that some cortical and deep gm structures were more prone to inflammatory and degenerative damage [49, 50] than others and that, when damaged, some cortical areas had a greater impact on the accumulation of physical [8, 51] and cognitive disability [52, 53] than others . In particular thalamus and cerebellum were consistently related to clinical disability and its progression over time . Thalamus was found to be one of the earliest structures involved by the neuropathological process taking place in the gm and the rate of thalamic atrophy in ms subjects was correlated with changes in edss . Moreover, in a longitudinal study, baseline thalamic fraction (odds ratio = 0.62) was identified as independent predictor of worsening disability at 8 years . Cerebellum has been indicated as a preferred site of demyelination, especially in patients with progressive ms, whose cerebellar cortex was found to be affected by ms - related pathology in up to 92% of its extension [105, 106]. In a recent 5-year longitudinal study cerebellar cortical atrophy, together with age and cortical lesion load, was indicated among the predictive parameters of progression in those rr ms patients who convert to the sp phase . Beyond diffuse gm damage, the relevance of cortical damage in determining disability has been pointed out by the strong correlation observed between focal gm damage as visible by double inversion recovery (dir) sequence (i.e., cortical lesions; figure 3) and clinical progression . Indeed, high number of cortical lesions has been demonstrated to characterize patients with the poorest prognosis and having early and severe cortical atrophy and cognitive impairment . In a 5-year longitudinal study on more than 300 ms patients with different clinical phenotypes, cortical lesion volume and gm atrophy were found to be associated to each other and to physical and cognitive disability progressions . Patients having high cortical lesion load at baseline showed the worse clinical evolution and a significant progression of cortical atrophy after 5 years . Of course a complete and accurate evaluation of the risk of clinical progression should not disregard the evaluation of spinal cord damage that has suggested as a major determinant of disability in patients with ms . In line with what has been happened for the brain damage, the application of quantitative mri techniques to the spinal cord damage has convincingly demonstrated that cord area, rather than t2 lesion load, might have a role in predicting the accumulation of disability [56, 57, 108]. In the last 10 years, finally, other non - conventional sequences have received considerable attention since their high sensitivity for the most disabling pathological features of ms (i.e., irreversible demyelination and neuroaxonal injury) and their ability to detect occult changes occurring in the normal - appearing brain tissue . Among these unconventional techniques magnetization transfer and diffusion tensor imaging gave the most interesting results . In 73 patients, who were followed prospectively with clinical visits for a median period of 8 years, a multivariable model identified baseline gm magnetization transfer ratio histogram peak height and average lesion magnetization transfer ratio percentage change after 12 months as independent predictors of disability worsening at 8 years (r = 0.28). In a longitudinal study on 54 primary progressive ms patients, lower level of disability and gm damage evaluated at study entry on the base of average gm mean diffusivity identified patients with high risk of disease progression over the following 5 years . In a more recent prospective study fractional anisotropy of normal appearing gm and t2 lesion load were independent predictors of edss score, while change in fractional anisotropy of normal appearing gm (b = 0.523) and disease duration (b = 0.342) were independent predictors of edss change . Finally, the application of diffusion tensor imaging to the spinal cord damage revealed that baseline cord cross - sectional area and its fractional anisotropy correlated with increase in disability at follow - up . All together these studies confirmed that neurological and neuropsychological disability in ms are likely the consequence of both visible and invisible wm and gm damage . The strength of correlation between gm tissue loss and progression of disability exceeds that related to wm lesions or atrophy (table 2). Unfortunately, gm damage is poorly evaluated by conventional mri and to achieve more accurate estimates of such a damage it requires multiparametric mri approach including unconventional and quantitative mri techniques, many of which are not yet available or practicable in routine diagnostics . The examination of csf represents a valuable procedure in investigating a number of inflammatory and degenerative neurological disorders . In addition to the classical biochemical and electrophoretic approaches, the proteome complexity of csf can be tackled today by a number of methods, hence, indicating that scientists involved in this frontier are fishing in the right pond . However, in a disorder with a complex pathogenesis, such as ms, individual biomarkers, taken singly, are likely to reflect only isolated components of ongoing neuroinflammation and neurodegeneration, hence, lacking prognostic significance . Moreover, most of the investigated ms biomarkers, while of invaluable diagnostic help, are currently unsuitable for predicting disease progression . According to their biological role, molecules of potential prognostic significance for ms may be classified as follows: (i) markers of immune activation (e.g., cytokines, chemokines, antibodies, complement factors, adhesion molecules, etc . ); (ii) markers of blood - brain barrier disruption (e.g., matrix metalloproteinases); (iii) markers of demyelination (e.g., myelin basic protein, myelin oligodendrocyte glycoprotein, proteolytic enzymes, proteases inhibitors, etc . ); (iv) markers of oxidative stress and cytotoxicity (e.g., advanced oxidation protein products, total thiol, hydroxyl radicals, divalent iron, etc . ); (v) markers of axonal / neuronal damage and gliosis (e.g., neurofilaments, tau, 14 - 3 - 3 protein, glial fibrillary acidic protein, etc . ); and (vi) markers of remyelination / neural repair (e.g., nerve growth factor, brain - derived growth factor, nogo - a, etc . ). A correlation with ms disability progression over time has been suggested for several csf markers, including but not limited to 14 - 3 - 3 protein; tau; neurofilament heavy chain [66, 67]; chitinase 3-like 1; and cystatin c . Csf igg oligoclonal bands, which have a recognized relevance for the diagnosis of ms and predict conversion from cis to ms, do not influence the long - term risk of disability, although a contrasting observation has been described . Conversely, csf oligoclonal igm, particularly if directed against myelin lipids, have been associated with a poor ms outcome in terms of frequency of relapses and disability progression [68, 69]. Several csf markers of inflammation have been investigated for potential prognostic value in cis and early ms patients . Some studies have identified novel biological predictors of conversion from cis to ms, for instance measles - rubella - varicella zoster virus igg antibody reaction (mrzr) and high levels of c - x - c motif ligand 13 (cxcl13) and in the csf . However, no predictive value for progression of disability has been shown for such molecular candidates [111, 112]. Since neurodegeneration is regarded as the biological determinant of irreversible neurological disability in demyelinating disorders, csf markers of neuroaxonal injury (e.g., tau, 14 - 3 - 3 protein, and neurofilaments) are the most promising candidates for predicting disease progression [113, 114]. Csf tau concentration in ms patients with both relapsing and progressive forms of the disease has been reported to be higher compared to controls in several studies [110, 115119], although other researchers did not replicate this finding [120122]. A correlation between csf tau and progression of disability in ms patients has been shown only in one 3-year follow - up study . In a small group of patients with cis and clinically definite ms, investigated either at the acute attack (i.e., within 30 days) or several weeks or months later, our group found values of tau within normal limits, a finding that we later confirmed in a larger mostly independent cohort of cis patients . Interestingly, it has been shown that csf tau levels decrease during the course of ms, as a likely effect of progressive parenchymal brain loss, hence, showing a negative correlation with clinical severity . The latter findings are consistent with studies showing progressive brain atrophy in ms patients, regardless of disease subtype . Taken together, while the determination of csf tau concentration in ms deserves further scrutiny, it is possible that in a subset of ms patients, this protein represent a reliable marker of axonal injury . Conversely, available evidence shows that determination of p - tau has no value as a biomarker . 14 - 3 - 3 protein has also been detected in the csf of subjects with cis / ms by several [61, 62, 116, 126, 127] but not all research groups . . Showed that a positive csf 14 - 3 - 3 assay at the first neurological event suggestive of ms predicted the development of significant neurological disability over a median follow - up period of 32 months, while colucci et al . Found that 14 - 3 - 3 positive ms patients had a higher rate of edss progression over 10 months compared to 14 - 3 - 3 negative cases . Fiorini et al . Found variable upregulation of csf 14 - 3 - 3 / in cis / ms patients investigated at active or inactive disease stages (as observed in other inflammatory / demyelinating conditions) but not overexpression of 14 - 3 - 3 and, the isoforms typical of disorders characterized by ongoing axonal and neuronal degeneration, such as sporadic creutzfeldt - jakob disease (scjd) and motor neuron disease . These findings encourage an in - dept - analysis in larger cohorts of patients, before ruling out the usefulness of this biomarker . Among csf biomarkers of neurodegeneration that have been tested in ms, concentrations are increased in the csf of ms patients compared to age - matched normal controls . Furthermore, csf nfl levels seem to better correlate with ms acute inflammatory activity (higher levels in cis patients who convert to ms and during relapse compared to remission phase), while csf nfh concentrations appear to be related to irreversible neuroaxonal injury as indicated by the correlation with confirmed edss score progression and brain atrophy both in cross - sectional and longitudinal studies [64, 65, 122, 128130]. Table 3 shows csf markers for which a correlation with ms disability progression has been reported in longitudinal studies . Which is not straightforward in all cases, clinicians have to be prepared for the challenge of prognostic predictions in order to give adequate responses to patients concern about their future life with ms . Among the determinants of ms burden, development of irreversible neurological disability, particularly when affecting motor and cognitive functions, has the highest impact on patients quality of life and health system costs . Therefore, prognostic markers of long - term disability progression are strongly needed in ms . A prognostic marker is a specific parameter or a combination of parameters that can be measured in a subject with a given condition and that is significantly correlated with a relevant clinical outcome of that condition . Ideally, reliable prognostic marker studies should fulfil the following methodological requirements: (1) prospective or longitudinal design; (2) long - term followup; (3) adequate marker and outcome measurement; (4) clinical significance of the marker (i.e., good correlation and consistency with relevant clinical outcomes); and (5) reproducibility . Clinical prognostic markers that are associated with an increased risk of disability progression in the longterm (e.g., male gender, older age, progressive onset, etc .) Have been identified in several ms natural history studies . However, besides identifying subjects who are more likely to experience a severe disease course, such markers do not offer real advantages in terms of prediction potential, since they are not modifiable risk factors, do not directly reflect biological processes, and do not generally distinguish between responders and nonresponders to available ms treatments . Conversely, mri and csf parameters, which can be classified as biomarkers as they express more closely biological mechanisms underlying the disease pathophysiology, have a good potential of quantitative assessment as well as variation according to disease stage . Considering the complex pathogenesis of ms however, families of biomarkers representative of specific pathogenetic pathways particularly those related to axonal / neuronal damage may correlate with irreversible neurological dysfunction and be used as prognostic indicators to identify patients at risk of a more aggressive disease course . Furthermore, such a biomarker might be helpful for identifying patients who could benefit from therapy in case it showed a reliable correlation with the response to a given treatment . Unfortunately, no conventional mri measure has shown strong correlation with long - term disability progression in ms, while unconventional mri techniques particularly those assessing gm damage are currently being investigated with promising results, although they are still difficult to apply in clinical settings . On the other hand, research on csf biomarkers has gathered convincing preliminary evidence only for nfh and nfl as predictors of disability progression . So far, biomarkers studies have mainly focused on selected candidates and have generally recruited relatively small sample of cases with a cross - sectional design, often showing conflicting results . It is likely that discrepancies across studies are at least in part explained by differences in selection of patients, marker measurement, and outcome assessment . Although a considerable level of international agreement has been reached on methodological requirements of mri studies in ms, an effort is being made by the csf markers research community in order to standardize collection and biobanking of samples from well clinically characterized ms patients to develop reproducible laboratory assays for csf analysis and to find common definitions of healthy and diseased controls [132, 133]. To identify reliable prognostic markers, future ms research will need to focus on large longitudinal observational studies and clinical trials exploring the correlation of unconventional mri measures and selected csf proteins with the development of irreversible neurological disability.

Subsets and Splits

No community queries yet

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