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a tripartite fiv vector system was used to produce vesicular stomatitis g (vsv - g) pseudotyped lentiviral vectors consisting of envelope plasmid pmd.g, packaging plasmid pfp93, and fiv transfer vector ginsin - encoding enhanced green fluorescent protein (egfp) and neomycin resistance via an internal ribosomal entry site (ires). lentiviral particles were generated using a new, scaled up transient transfection system with polyethylenimine (pei) as a polycationic, polymeric transfection agent to provide a simplified and stable platform that allows three full harvests from a single transfection. prior to transfection, 2.5 10 293 t cells that were cycling at a high cell division rate were seeded in a large surface culture vessel (cell factory 10 ; nunc, naperville, il, usa) with 2 l of dulbecco 's modified eagle 's medium (dmem with 4.5% glucose, 4 mm l - glutamine, without phenol red, without sodium pyruvate ; # sh30284.02, hyclone ; ge healthcare life sciences, piscataway, nj, usa) at 37c and supplemented with 10% fetal calf serum and penicillin g sodium / streptomycin sulfate (100 units / ml and 100 g / ml, pen strep, life technologies, grand island, ny, usa). on the day of transfection (day 0) this transfection mix contained 116 g of vsv - g envelope plasmid pmd - g, 339 micrograms of packaging plasmid pfp93, and 339 micrograms of transfer plasmid ginsin which were suspended in 18 ml of 37c reduced, serum - free media (opti - mem ; gibco, thermo fisher scientific, waltham, ma, usa) that had been combined with 3.2 ml of pei (polyethylenimine, linear ; polysciences, inc., warrington, pa, usa), had been vortexed for 10 seconds, and then allowed to stand for 5 minutes at room temperature. this solution was then pipetted into 1.4 l of fresh media which replaced the one present in the cell factory by gently pouring it along the exchange port without disturbing the cell layers. the cell factory was balanced to distribute the media equally between the layers and placed in an incubator. following an 8-hour incubation at 37c, expression of egfp was imaged by fluorescence microscopy to confirm a transfection rate above 80%. on days 2, 4, and 6, vector particles were pelleted by ultracentrifugation in a large volume rotor (type 19 ; beckman coulter life sciences, brea, ca, usa), as previously described, and titrated 24 hours later by fluorescence - assisted flow cytometry (facs) analysis on crfk cells. porcine eyes were obtained from the local abattoir and processed within approximately 2 hours of death. the eyelids and adnexal structures were excised, eyes dipped into 5% povidone - iodine ophthalmic solution (betadine 5% ; alcon, fort worth, tx, usa) for 30 seconds and then transferred into sterile pbs (dulbecco 's pbs ; mp biomedicals, llc, santa ana, ca, usa). in a tissue culture hood, the eyes were hemisected along the equator followed by removal of the vitreous, lens, ciliary body, iris, retina, and choroid. pigment shedding was avoided by carefully dissecting the choroid off the sclera directly along the equatorial incision in one piece while leaving the vitreous and lens in place as a barrier toward the anterior chamber. the anterior segments were then immediately mounted in perfusion chambers connected to a microinfusion pump (phd 22/2000 ; harvard apparatus, holliston, ma, usa) and perfused with serum - free media without phenol red (dmem with penicillin g sodium / streptomycin sulfate [100 units / ml and 100 g / ml, respectively ]) at a constant flow rate of 3 l / min. the intraocular pressure was continuously monitored with pressure transducers (physiological pressure transducer, sp844 ; memscap, skoppum, norway) and recorded using a software system (labchart ; adinstruments, colorado springs, co, usa). the perfusion system was calibrated using a pressure transducer tester (veri - cal ; utah medical products, midvale, ut, usa). eyes that experienced a contamination, showed erroneous iop recordings in the negative pressure range, or readings above 30 mm hg during the first 24 hours were considered a failure. such negative iop recordings were observed for instance when debris blocked the transducer lumen while early high iop was interpreted as relative tm failure or blockage. gravity perfused anterior segments (cogr) were similarly processed and mounted in perfusion chambers connected to a gravity flow system and perfused with serum free clear dmem supplemented with penicillin g sodium and streptomycin sulfate (100 units / ml and 100 g / ml, respectively). the gravity flow system utilized a fluid column at a constant height of 20.4 cm above perfusion chambers to maintain pressures at 15 mm hg. porcine anterior segments were allowed to stabilize at a constant flow rate for 48 hours before any manipulation. on day 0, the central infusion and transducer lines were disconnected and a bolus of 1 ml of dmem with 1 10 transducing units (tu) ginsin was injected at a rate of 1ml / min, displacing the same amount of intracameral volume. we redesigned established anterior chamber perfusion dishes by reducing the thickness of the bottom to approximately one - third and widening the now flatter central mounting hub (fig. there was reduced image distortion compared with regular chambers with a thick bottom. on days 1, 3, 7, and 14, anterior segments mounted as described above were inverted and placed under a stereo dissecting microscope equipped for fluorescent imaging (olympus szx16 with gfp filter cube and dp80 monochrome / color camera ; olympus corp., center valley, pa, usa) with continued perfusion. the dish was stabilized at a 30 angle and each quadrant was imaged using an exposure of 100 ms, 1360 1024 resolution and 8-bit depth. (b) view through bottom of inverted dish allows the observation of the tm directly with central in - line and peripheral pressure transducer line. fluorescence of tm was graded on a scale of 0 to 4 as previously established : grade 0, no detectable fluorescence ; grade 1, single fluorescent spots in the tm ; grade 2, numerous, nonconfluent fluorescent spots with some confluent areas ; grade 3, extensive, mostly confluent, midlevel transduction ; grade 4, extensive, high - level, and confluent fluorescence. at the conclusion of the experiment, the anterior segments were removed from the perfusion dish, rinsed in pbs, cut into quadrants, and fixed with 4% paraformaldehyde in pbs for a period of 48 hours before being placed in 70% ethanol. paraffin - embedded specimens were cut at 6-m thickness and stained with hematoxylin eosin (h&e). the ratio of transduced to total tm cells was determined as described previously by counting all tm cells in each of four sections per eye (one from each quadrant). the trabecular meshwork was identified and cell counts of 6-m h&e - stained paraffin sections obtained for two adjacent quadrants in four transduced and nontransduced anterior segments. photographs of fluorescent tm were taken of each quadrant at each examination and grades determined in a masked manner using a previously established continuous grading scale. total tm cells present in random 6-m sections from above quadrants were determined by counting the labeled and unlabeled cells and calculating the mean value for each quadrant. an unpaired student 's t - test was used for statistical analysis to compare transduced and nontransduced anterior segments. because of the linear correlation between transduced cells and grades, expression grades were handled as continuous data during the statistical comparison of marker protein accumulation. a tripartite fiv vector system was used to produce vesicular stomatitis g (vsv - g) pseudotyped lentiviral vectors consisting of envelope plasmid pmd.g, packaging plasmid pfp93, and fiv transfer vector ginsin - encoding enhanced green fluorescent protein (egfp) and neomycin resistance via an internal ribosomal entry site (ires). lentiviral particles were generated using a new, scaled up transient transfection system with polyethylenimine (pei) as a polycationic, polymeric transfection agent to provide a simplified and stable platform that allows three full harvests from a single transfection. prior to transfection, 2.5 10 293 t cells that were cycling at a high cell division rate were seeded in a large surface culture vessel (cell factory 10 ; nunc, naperville, il, usa) with 2 l of dulbecco 's modified eagle 's medium (dmem with 4.5% glucose, 4 mm l - glutamine, without phenol red, without sodium pyruvate ; # sh30284.02, hyclone ; ge healthcare life sciences, piscataway, nj, usa) at 37c and supplemented with 10% fetal calf serum and penicillin g sodium / streptomycin sulfate (100 units / ml and 100 g / ml, pen strep, life technologies, grand island, ny, usa). on the day of transfection (day 0) this transfection mix contained 116 g of vsv - g envelope plasmid pmd - g, 339 micrograms of packaging plasmid pfp93, and 339 micrograms of transfer plasmid ginsin which were suspended in 18 ml of 37c reduced, serum - free media (opti - mem ; gibco, thermo fisher scientific, waltham, ma, usa) that had been combined with 3.2 ml of pei (polyethylenimine, linear ; polysciences, inc., warrington, pa, usa), had been vortexed for 10 seconds, and then allowed to stand for 5 minutes at room temperature. this solution was then pipetted into 1.4 l of fresh media which replaced the one present in the cell factory by gently pouring it along the exchange port without disturbing the cell layers. the cell factory was balanced to distribute the media equally between the layers and placed in an incubator. following an 8-hour incubation at 37c, expression of egfp was imaged by fluorescence microscopy to confirm a transfection rate above 80%. on days 2, 4, and 6, vector particles were pelleted by ultracentrifugation in a large volume rotor (type 19 ; beckman coulter life sciences, brea, ca, usa), as previously described, and titrated 24 hours later by fluorescence - assisted flow cytometry (facs) analysis on crfk cells. porcine eyes were obtained from the local abattoir and processed within approximately 2 hours of death. the eyelids and adnexal structures were excised, eyes dipped into 5% povidone - iodine ophthalmic solution (betadine 5% ; alcon, fort worth, tx, usa) for 30 seconds and then transferred into sterile pbs (dulbecco 's pbs ; mp biomedicals, llc, santa ana, ca, usa). in a tissue culture hood, the eyes were hemisected along the equator followed by removal of the vitreous, lens, ciliary body, iris, retina, and choroid. pigment shedding was avoided by carefully dissecting the choroid off the sclera directly along the equatorial incision in one piece while leaving the vitreous and lens in place as a barrier toward the anterior chamber. the anterior segments were then immediately mounted in perfusion chambers connected to a microinfusion pump (phd 22/2000 ; harvard apparatus, holliston, ma, usa) and perfused with serum - free media without phenol red (dmem with penicillin g sodium / streptomycin sulfate [100 units / ml and 100 g / ml, respectively ]) at a constant flow rate of 3 l / min. the intraocular pressure was continuously monitored with pressure transducers (physiological pressure transducer, sp844 ; memscap, skoppum, norway) and recorded using a software system (labchart ; adinstruments, colorado springs, co, usa). the perfusion system was calibrated using a pressure transducer tester (veri - cal ; utah medical products, midvale, ut, usa). eyes that experienced a contamination, showed erroneous iop recordings in the negative pressure range, or readings above 30 mm hg during the first 24 hours were considered a failure. such negative iop recordings were observed for instance when debris blocked the transducer lumen while early high iop was interpreted as relative tm failure or blockage. gravity perfused anterior segments (cogr) were similarly processed and mounted in perfusion chambers connected to a gravity flow system and perfused with serum free clear dmem supplemented with penicillin g sodium and streptomycin sulfate (100 units / ml and 100 g / ml, respectively). the gravity flow system utilized a fluid column at a constant height of 20.4 cm above perfusion chambers to maintain pressures at 15 mm hg. porcine anterior segments were allowed to stabilize at a constant flow rate for 48 hours before any manipulation. on day 0, the central infusion and transducer lines were disconnected and a bolus of 1 ml of dmem with 1 10 transducing units (tu) ginsin was injected at a rate of 1ml / min, displacing the same amount of intracameral volume. we redesigned established anterior chamber perfusion dishes by reducing the thickness of the bottom to approximately one - third and widening the now flatter central mounting hub (fig. there was reduced image distortion compared with regular chambers with a thick bottom. on days 1, 3, 7, and 14, anterior segments mounted as described above were inverted and placed under a stereo dissecting microscope equipped for fluorescent imaging (olympus szx16 with gfp filter cube and dp80 monochrome / color camera ; olympus corp., the dish was stabilized at a 30 angle and each quadrant was imaged using an exposure of 100 ms, 1360 1024 resolution and 8-bit depth. (b) view through bottom of inverted dish allows the observation of the tm directly with central in - line and peripheral pressure transducer line. fluorescence of tm was graded on a scale of 0 to 4 as previously established : grade 0, no detectable fluorescence ; grade 1, single fluorescent spots in the tm ; grade 2, numerous, nonconfluent fluorescent spots with some confluent areas ; grade 3, extensive, mostly confluent, midlevel transduction ; grade 4, extensive, high - level, and confluent fluorescence. at the conclusion of the experiment, the anterior segments were removed from the perfusion dish, rinsed in pbs, cut into quadrants, and fixed with 4% paraformaldehyde in pbs for a period of 48 hours before being placed in 70% ethanol. paraffin - embedded specimens were cut at 6-m thickness and stained with hematoxylin eosin (h&e). the ratio of transduced to total tm cells was determined as described previously by counting all tm cells in each of four sections per eye (one from each quadrant). the trabecular meshwork was identified and cell counts of 6-m h&e - stained paraffin sections obtained for two adjacent quadrants in four transduced and nontransduced anterior segments. photographs of fluorescent tm were taken of each quadrant at each examination and grades determined in a masked manner using a previously established continuous grading scale. total tm cells present in random 6-m sections from above quadrants were determined by counting the labeled and unlabeled cells and calculating the mean value for each quadrant. an unpaired student 's t - test was used for statistical analysis to compare transduced and nontransduced anterior segments. because of the linear correlation between transduced cells and grades, expression grades were handled as continuous data during the statistical comparison of marker protein accumulation. vector production using the scaled up pei transfection platform yielded 1.1 tu total on first, 7.2 tu on second and 8.0 tu on third harvest with a titer range of approximately 29% at each time point (n = 3). 2) had an iop of 15.8 1.9 mm hg during constant perfusion at a rate of 3 l / min compared with gravity - perfused control anterior segments with an imputed flow rate of 3.7 1.6 l / min at constant gravity perfusion with a pressure of 15 mm hg. control anterior segments were cultured in the redesigned culture system (fig. 1) and pilot anterior segments kept for up to 21 days during initial viability testing (data not shown). trabecular meshwork could be observed directly through the bottom of the inverted dishes (fig. 1) with best detection of egfp fluorescence when held at a 30 angle (fig. view was relatively free of distortion for whole eye and quadrant view at up to 10 magnification but became vertically compressed when viewed at higher magnification or when held at a steeper angle (fig. could be seen over 360 but with segmental focus and short areas of nontransduction (fig. the most notable iop changes occurred when the perfusion system was disconnected for media change (fig. 4). intraocular pressure graph of perfused anterior segment controls (n = 22, left). direct, repeated visualization of whole eye with view of transduced tm through the bottom of the redesigned perfusion system. left : macroscopic view without magnification. right : fifty - fold magnified view of tm with only minor image distortion (tm is 700 m wide). intraocular pressure and directly observed, same eye egfp expression in tm of eyes transduced with 10 tu of egfp - expressing fiv vector at time points indicated., porcine tm showed low level egfp marker gene expression after transduction at the highest titer tested, 10 tu (fig. expression peaked within 48 hours. at this titer, tm transduction was extensive and could be observed throughout the entire periphery. the culture system enabled us to find precise landmarks during serial observation as in the example of the less transduced, more pigmented tm in the center of the fluorescent band of tm in figure 5 (fig. intraocular pressure and directly observed, same eye egfp expression in tm of eyes transduced with 10 tu of egfp - expressing fiv vector at time points indicated. anterior segments of ginsin transduced with 10 tu experienced a significant (p = 0.03) but transient posttransduction iop increase of 44% at 24 hours that resolved at 48 hours (fig. histology obtained at the conclusion of the experiment indicated a well - preserved tm architecture (fig., these anterior segments experienced a small but significant reduction of iop (p = 0.03) before returning to 14.1 2.4 mm hg, which was not significantly different from control anterior segments (p = 0.2). expression was initially faint and could only be observed in some anterior segments (figs. 4, 5) before increasing. at 24 hours, relative ginsin expression grading in anterior segments transduced with 10 tu had an average of 0.5 0.7 (p < 0.01) at 24 hours on a scale from 0 to 4 that increased to 2.6 1.1 at 168 hours (p < 0.01). anterior segments transduced with 10 tu had a significantly lower expression than those transduced with 10 tu. it was 0.2 0.4 at 24 hours which increased to 0.7 1.0 (fig. peaks for both titers were achieved at the same observation time point. despite weak or absent fluorescence, the new culture chambers allowed to identify anatomic landmarks in serial observation (fig. expression grades of eyes transduced with 10 and 10 tu (left, n = 8 for each group ; error bars = sd) and corresponding tm cellularity (right, n = 8 eyes for each group with two sections per eye counted from adjacent quadrants ; error bars sd). there are significant supply challenges for human donor eyes in research which is partially due to the increased rate of corneal endothelium and limbal stem cells transplantation. however, a local abattoir can serve as a source of porcine eyes to produce reliable, high quality anterior segment cultures. compared with human donor eyes that have a wide age range, disease, and variable time from explantation to culture, the consistency of pig eyes resulted in a considerably lower failure rate in our hands. low costs and close correlation with human anatomy make pig eyes an attractive model for research and training in ophthalmology. the pig 's similarity in size and anatomy also led to research into xenotransplantation with a focus on kidney, liver, and cornea and, more recently, the outflow tract. here, we describe a method to track transgene expression in an ex vivo pig eye culture system by direct observation that is made possible by redesigned, thinner culture dishes. the ability to directly observe the tm from the inside will provide a valuable tool to correlate expression of transgenes in the tm with the resulting segmental outflow changes as measured in quantitative canalograms obtained from the outside (loewen r,. the intraocular pressure of 15.8 1.9 mm hg we observed at the normal aqueous flow rate of 3 to 4 l / min is close to the physiologic iop of 15.2 1.8 mm hg of pigs and has been used in other porcine ex vivo studies. this intraocular pressure, obtained with a perfusion rate of 3 l / min, matches our imputed outflow of 3.7 1.6 l / min using the reverse approach of infusing these eyes at 15 mm hg in our constant pressure experiments. in contrast to these studies, we did not observe any prominent corneal swelling that can occur without a physiological transcorneal pressure gradient that allows proper nutrient transport into the stroma and preservation of normal corneal thickness and contour. the short time from enucleation to culture and limited exposure to povidone - iodine may have further reduced corneal compromise. problems encountered in prior use of pig eyes include pigment release resulting in trabecular meshwork obstruction, the larger diameter that not all human eye culture systems can accommodate, and the more plexus - like, discontinuous segments of schlemm 's canal. the discontinuity of schlemm 's canal may be an advantage for new outflow research strategies concerned with focal tm modification by limiting effects to the immediate downstream drainage system and providing the opportunity for localized flow measurements. segmental flow has recently been observed in other species as well. by dissecting the choroid off the sclera directly along the equatorial incision and using the lens as a barrier toward the anterior chamber copious irrigation alone was not sufficient to dislodge pigment that had already made it into the tm. we developed a protocol for scaled - up fiv vector production with pei transfection that provided reliable high titers eliminating the ph and titer variability of traditional production systems using calcium chloride. because transfected cell layers remained adherent, the vector could be harvested three times for an entire week with only moderate decline of titers. feline immunodeficiency virus, a non - primate lentiviral vector that has the safety advantage of not being based on a human pathogen, can transduce primate, feline, and rodent trabecular meshwork at a high level. this observation was principally the same in pig eyes, including a limited and temporary effect on outflow function at the highest titers. different from prior species, the corneal endothelium and sclera that were in proximity to the tm in pig eyes appeared to be transduced more often. this could be caused by different flow kinetics due to a relatively large anterior chamber with a less selective contact and perfusion of the target tissue. this vector does not currently deploy receptor or transcriptional targeting strategies that may become necessary in cytoablative models. the somewhat lower expression levels compared with human and feline eyes may indicate that lentiviral restriction is present but can be overcome at higher titers. defense mechanisms to curb lateral intrusion of foreign code from retro - elements are known in many species and the most important restriction factors found in human cells are also present in the pig as a genome search for apobec-3f, trim5, bst2, samhd1, and zap demonstrates. restriction has not presented an obstacle to lentiviral germline transgenesis with hiv and eiav vectors and should not impede future use of these vectors in anterior segments. relevant to biosafety in tm engineering studies involving xenotransplantation, the pig genome harbors porcine endogenous retroviruses (perv - a, -b, and -c), but these encounter trim and apobec3-mediated restriction in human cells. in conclusion, we found that pig eyes can be used as a reliable and high quality source for anterior segment perfusion cultures. the ability to manipulate and directly observe the tm in this culture system, combined with the relative similarity to human eyes, will provide useful tools for new approaches of bioengineering in glaucoma research. | purposeto establish a consistent and affordable, high quality porcine anterior segment perfusion and transduction model that allows direct visualization of the trabecular meshwork.methodsporcine anterior segments were cultured within 2 hours of death by removing lens and uvea and securing in a specially designed petri dish with a thin bottom to allow direct visualization of the trabecular meshwork with minimal distortion. twenty - two control eyes (co) with a constant flow rate were compared to eight gravity perfused eyes (cogr, 15 mm hg). we established gene delivery to the tm using egfp expressing feline immunodeficiency virus (fiv) vector ginsin at 108 transducing units (tu) per eye (ginsin_8, n = 8) and 107 tu (ginsin_7, n = 8). expression was assessed for 14 days before histology was obtained.resultspig eyes were a reliable source for consistent and high quality anterior segment cultures with a low failure rate of 12%. control eyes had an intraocular pressure (iop) of 15.8 1.9 mm hg at fixed pump perfusion with 3 l / min compared to gravity perfused cogr with imputed 3.7 1.6 l / min. vector ginsin_8 eyes experienced a transient posttransduction iop increase of 44% that resolved at 48 hours ; this was not observed in ginsin_7 eyes. expression was higher in ginsin_8 than in ginsin_7 eyes. trabecular meshwork architecture was well preserved.conclusionscompared with previously used human donor eyes, this inexpensive porcine anterior segment perfusion model is of sufficient, repeatable high quality to develop strategies of tm bioengineering. trabecular meshwork could be observed directly. despite significant anatomic differences, effects of transduction replicate the main aspects of previously explored human, feline and rodent models. |
skin - bleaching practices, such as using skin - lightening creams and soaps to achieve a lighter skin tone or to whiten skin, are common among non - white populations throughout the world, triggered by deep historical, economic, sociocultural, and psychosocial roots [15 ]. the exact prevalence of this practice among different population groups across different geographic areas is not known, and the existing estimates (e.g., 2567%) have most likely been underestimated, since some women may be reluctant to admit the practice because of the stigma surrounding these complexion - altering behaviors [6, 7 ]. in addition, skin bleaching is becoming more common among men and young adults, including teenagers [3, 5, 8 ]. prenatal exposure is also likely since pregnant women have been reportedly using skin - bleaching products [9, 10 ]. while skin - bleaching research has been heavily focused on darker - skinned african populations, it has become an increasing concern in jamaica and other afro - caribbean countries [6, 8, 11 ]. more specifically, hope has posited that for jamaicans, skin - bleaching practices have transitioned from historically being a response to economic and complexion - related oppression of the darker - skinned working class and poor populations to a current expression of fashion and ungendered rites of beauty. skin bleaching has been associated with a variety of known adverse health effects ranging from dermatitis to exogenous ochronosis (fig. 1), steroid acne (fig. 2), mercury (hg) poisoning, and nephrotic syndrome, which are linked to ingredients such as hydroquinone, corticosteroids, and hg [1, 4, 6, 1218 ]. due to health concerns, however, studies have found products containing the aforementioned ingredients with above legal limits, including products from the usa and european union, where regulations are stricter and better implemented. in addition, access to a variety of legal and illegal skin - bleaching products including prescription strength hydroquinone and topical corticosteroids is made easy through online purchases, global travel, and immigration [17, 18, 2426].fig. 1exogenous ochronosis on the face of a woman with a history of hydroquinone use fig. 2steroid acne on the chest following use of a corticosteroid containing bleaching cream for over 1 year exogenous ochronosis on the face of a woman with a history of hydroquinone use steroid acne on the chest following use of a corticosteroid containing bleaching cream for over 1 year skin bleaching is not uncommon in the us, and the health burden of skin - bleaching practices is not evenly distributed among the population. the 2004 new york city (nyc) local health and nutrition examination survey identified skin care product use as an important route of hg exposure, especially among caribbean - born blacks and dominicans in nyc, with mean urine hg concentrations 1.32.1 times higher than that of whites and other racial / ethnic groups. despite these reports, surprisingly sparse epidemiological studies are available regarding the health impacts of skin - bleaching practices on afro - caribbean immigrant groups in the us. in fact, this topic has gained very little attention among clinical and translational researchers in the us, with existing studies primarily limited to investigations of outbreaks of hg poisoning related to the use of skin - whitening products [2729 ]. first, we must comprehensively estimate the prevalence of skin bleaching among both previously studied populations and populations, like those from afro - caribbean countries such as jamaica, for which this practice is newly emerging at an alarming rate. while it is easier to focus just on newly emerging populations by geography, we should also examine the prevalence of this practice among other under - studied risk groups such as men, younger populations, and pregnant mothers. second, besides the known dermatologic effects of skin bleach, such as dermatitis, exogenous ochronosis, and acne, we should also expand our focus to long - term health consequences such as cancer as well as interactions between physical and psychosocial health outcomes. the potential carcinogenic effects of this practice may not be solely limited to exposure to hydroquinone, a primary metabolite of benzene, which is not currently classified as carcinogenic to humans because of conflicting results, though recent evidence suggests that hydroquinone can generate dna damages and immunosuppressive responses [18, 25, 26, 3034 ]. attention should also be given to potential increased susceptibility to skin cancer resulting from alterations of melanin production among those who practice skin bleaching, especially among populations living in tropical regions with strong uv radiation. while their darker skin is considered to be protective of sun - induced skin cancer, the carcinogenic effect of melanogenesis inhibition resulting from skin bleaching third, we should identify all skin - bleaching products used and scientifically evaluate ingredients such as metals, hydroquinone, and the steroid composition of these exposures using specialized laboratory methods [37, 38 ]. most existing studies focus on only one of the ingredients in these beauty products, which has hindered investigations of the health effects of exposure to mixtures from multiple ingredients. in addition, because self - mixing of a variety of chemicals (e.g., combining multiple commercial skin - bleaching products with hydrogen peroxide) is not uncommon, investigations of harmful chemical compositions in these home - made products should also be considered. fourth, we should empirically compute the dose - response relationships between the aforementioned exposures and associated dermatologic outcomes across various usage patterns and durations, which also require using innovative biomarkers to assess short- and long - term exposures to skin - bleaching products. last, but not the least, we posit that an integrative approach that brings together a multidisciplinary team of experts in dermatology, environmental epidemiology, biostatistics, pharmacology, behavioral psychology, and community health will be best suited to tackle this complex public health problem. this would allow for a large - scale, methodologically rigorous approach that can simultaneously explore the psychosocial motivations underlying this practice, the toxicological risks, and causal relationships between skin - bleaching product - related exposures and health outcomes. existing studies often employ a single - aspect design highlighting only one of three topic areas : (1) psychosocial implications, (2) dermatological health effects, or (3) an exposure assessment of the skin - bleaching practice. a well - designed, comprehensive integrative study would give researchers the latitude to investigate the underlying roots and epidemiologic status of skin bleaching while simultaneously providing evidence of exposure - outcome associations, which would serve as a necessary strategy if our ultimate goal is to develop efficacious interventions that can effectively reduce the health burdens of this practice. however, we understand that this interdisciplinary undertaking is challenging, given many in these communities, as we have observed at our institution, may not associate skin - related changes to their skin - bleaching practices. limited access to dermatologists among the population at risk may also contribute to under - detection of complications of skin bleaching. moreover, the underlying stigma surrounding this behavior might prevent these individuals from communicating their concerns to clinicians whom they perceive to have inadequate understanding of the cultural context. while these are all challenges that we must work on with these communities to overcome, from a research perspective, we still have a major difficulty in identifying patients with specific diagnoses resulting from skin bleaching because identifying cases requires an index of suspicion on the part of the clinician and specific inquiry into skin care practices, which may not be performed consistently or documented in the medical record. in summary, if we are to adequately care for clinically vulnerable, skin - bleaching subpopulations of the african and afro - caribbean immigrant communities, and other under - studied populations in the us, we must first understand the magnitude of the problem, draw valid causal inference about the harmful exposures from the skin - bleaching products and their resulting health problems, and subsequently provide culturally sensitive solutions that do not further stigmatize these populations. this is especially important since skin bleaching may be a rational response to being born in racially and economically segregated societies, even if this practice may be viewed less favorably in the clinical and translational sector. | skin - bleaching practices, such as using skin creams and soaps to achieve a lighter skin tone, are common throughout the world and are triggered by cosmetic reasons that oftentimes have deep historical, economic, sociocultural, and psychosocial roots. exposure to chemicals in the bleaching products, notably, mercury (hg), hydroquinone, and steroids, has been associated with a variety of adverse health effects, such as hg poisoning and exogenous ochronosis. in new york city (nyc), skin care product use has been identified as an important route of hg exposure, especially among caribbean - born blacks and dominicans. however, surprisingly sparse information is available on the epidemiology of the health impacts of skin - bleaching practices among these populations. we highlight the dearth of large - scale, comprehensive, community - based, clinical, and translational research in this area, especially the limited skin - bleaching - related research among non - white populations in the us. we offer five new research directions, including investigating the known and under - studied health consequences among populations for which the skin bleach practice is newly emerging at an alarming rate using innovative laboratory and statistical methods. we call for conducting methodologically rigorous, multidisciplinary, and culturally sensitive research in order to provide insights into the root and the epidemiological status of the practice and provide evidence of exposure - outcome associations, with an ultimate goal of developing potential intervention strategies to reduce the health burdens of skin - bleaching practice. |
distinguishing males from females and the differences in ethnic groups by analyzing the morphological characteristics of bone is important in the fields of physical and forensic anthropology. the mandible is the strongest bone in the human body and persists in a well - preserved state longer than any other bone. therefore, the use of morphological features of the mandible is a common approach used by anthropologists and forensic dentists in the determination of sex. skeletal characteristics vary by population ; therefore, there is a need for population - specific standards. among many anatomical landmarks in human skull, it is a funnel - like opening in the lateral surface of the mandible at the terminus of the mental canal. it provides the ability to view the entire body of the mandible and allow a more accurate location of the mental foramen in both horizontal as well as in vertical dimensions. the aim of the present study was to signify the average measurements from the superior and the inferior borders of the mental foramen to the lower border of the mandible on panoramic radiographs in determining the gender in a north indian population. a retrospective study was performed on the panoramic radiographs of patients aged between 18 and 62 years, which were obtained for orthodontic, periodontic and endodontic purposes. the radiographs were taken between january 2011 and june 2011 within a duration of 6 months. all the radiographs were taken using the planmeca proline 2006 ec machine with tube potential 60 - 80 kv, tube current 6 - 8 ma, total filtration 2.5 mm al, focal spot 0.3 and time 18 s. only high - quality radiographs with correct positioning were included in the study. the exclusion criteria for the radiographs were : distortion of images, presence of artefacts, surgical interventions, presence of any pathology, patient under 18 years and non - visualization of mental foramen. of the total 220 screened radiographs, 100 radiographs were selected for the analysis in which the mental foramen was identified as a separate type. the tangents were drawn to the superior and inferior borders of the foramen and perpendiculars were drawn from tangents to the lower border of the mandible bilaterally. an indelible pencil was used for marking the tangents and perpendiculars and a vernier calliper was used for the measurements. the distances were measured from the superior border of the mental foramen to the lower border of the mandible (s - l) and the inferior border of the mental foramen to the lower border of the mandible (i - l). statistical analysis was performed that included mean values in males and females on both the right and the left sides, confidence interval test at 95% and t - test and p values. the mean distance from the upper border of the mental foramen to the lower border of the mandible (s - l) on the right side in males was 17.650 mm, whereas it was 16.150 mm in females. on the left side, it was 17.475 mm in males and 15.787 mm in females [tables 1 and 2 ]. the mean distance from the lower border of the mental foramen to the lower border of the mandible (i - l) on the right side in males was 12.670 mm, whereas it was 11.462 mm in females. on the left side, it was 12.583 mm in males and 11.250 mm in females [tables 3 and 4 ]. the comparison of s - l between males and females showed a very high significant difference (p < 0.001) on both the right and the left sides [table 5 ]. similarly, the comparison of i - l between males and females suggested a highly significant difference (p = 0.0022) on both sides [table 6 ]. comparison of s - l between males and females comparison of i - l between males and females the comparison of s - l and i - l between the right and the left sides in males described a non - significant difference (p = 0.67 and p = 0.84) [table 7 ]. in the same way, the comparison of s - l and i - l between the right and left sides in females also showed a non - significant difference (p = 0.57 and p = 0.76) [table 8 ]. comparison of s - l and i - l between the right and left sides in males comparison of s - l and i - l between the right and left sides in females the mandible is the strongest bone in the human body and persists in a well - preserved state longer than any other bone. therefore, mandibular characteristics are extremely useful for determining sex. in 1974, wical and swoope described that despite the alveolar bone resorption above the mental foramen, the distance from the foramen to the inferior border of the mandible remains relatively constant throughout life. guler. in 2005 also suggested that the stability of this region does not depend on resorption of alveolar process above the foramen. therefore, the vertical measurements in panoramic radiography are clinically applicable for the quantification of height of alveolar bone in this region. because of the stability of the basal bone and mental foramen, these landmarks were selected as a point of reference for the present study. panoramic radiography is a curved plane tomographic radiographic technique used to depict the body of the mandible, maxilla and the lower one - half of the maxillary sinuses on a single image. the ability to view the entire body of the mandible allows a more accurate location of the mental foramen in both a horizontal and a vertical dimension on panoramic radiographs. yosue and brooks in 1989 described that the radiographic appearance of mental foramen can be classified into four types. in the continuous type,, the foramen is distinctly separated from the mandibular canal and appears as a well - defined radiolucency with a distinct border of condensing bone. in the diffuse type, the foramen has an indistinct border while in the unidentified type, the foramen can not be seen. the separate type is easy to identify on panoramic radiograph and so only this type was selected for the present study. it was suggested by akgul and toygar in 2002 that in comparative analyses between genders, the morphometric study by means of panoramic radiography reveals differences and inherent alterations in the evaluated groups. in the present study, the mean values of s - l and i - l were significantly high in males as compared with females, and the results were in accordance with those of thomas., mahima. and catovie. on the contrary, vodanovic. found that the mean value of i - l does not exhibit sexual dimorphism. the difference may be due to racial diversity of the study population. in our study, this value was also significantly high in males, which also corresponds to the studies of enlow. and amorim. the distances (s - l and i - l) for the right and left sides of an individual showed that the values were almost similar, with a non - significant difference, and this applies for both the male and the female groups. therefore, the distances from any of the sides can be used as a representative for gender discrimination. in the present study, the 95% confidence interval range analysis described that the s - l in males comes within the range of 16.921 - 18.160 mm, and in females it fall within the range of 15.041 - 17.032 mm. the i - l in males ranges between 11.944 and 13.281 mm and in females it comes within the range of 10.424 and 12.384 mm. these results suggest that if a distance above 16.921 mm for s - l and 11.944 mm for i - l is obtained on the panoramic radiograph ; the gender will be male in 95% of the cases. similarly, if a distance less than 17.032 mm for s - l and 12.384 mm for i - l is obtained, the gender will be female in 95% of the cases. the results were similar to the study conducted on a south indian population by mahima. in 2009, who described that if a distance above 1.7 cm is obtained for s - l and 1.48 cm for i - l, the gender is male in 99% of the cases. in the same way, if a distance is less than 1.69 cm for s - l and 1.3 cm for i - l, the gender is female. based on the results of this study, it is possible to conclude that the distances from the mental foramen to the lower border of the mandible exhibit sexual dimorphism in the north indian population. panoramic radiography is efficient for making the proposed measurements and can be considered as an additional radiographic method to determine gender from the skeletal remains. the technique is particularly important in mass disaster events, in which the jaws are available in fragments. larger study groups and comprehensive assessment of various other parameters related to the mental foramen are required to further confirm the results. | aim : identification and determination of sex of unknown human skeletal remains has been one of the most challenging tasks for forensic dentistry. the purpose of this study was to determine the gender from the analysis of mental foramen on panoramic radiographs in a north indian population.materials and methods : one hundred radiographs were selected for the analysis of mental foramen. tangents were drawn to the superior and inferior borders of the foramen and perpendiculars were drawn from the tangents to the lower border of the mandible (s - l and i - l). the data obtained were tabulated and subjected to statistical analysis.results:the average values of s - l and i - l were significantly higher in males than in females, while the distances for the right and left sides of an individual were almost similar in both the male and the females group, and the results were non-significant.conclusion:the distances from the mental foramen to the lower border of the mandible exhibit sexual dimorphism in the north indian population. |
temporal lobe epilepsy (tle) has been associated with hippocampal sclerosis and pathological changes in the closed neighboring structures, including entorhinal cortex, amygdala and dentate gyrus [13 ]. scalp electroencephalogram (eeg) recordings from patients with tle usually demonstrate interictal and ictal epileptiform abnormalities over the mid / anterior temporal region. studies using depth electrodes further confirmed the electrographic origin of these seizures in the hippocampal formation. however, recent studies have shown that the pathological substrate of tle is not confined to the temporal lobe. structural and metabolic imaging studies, such as magnetic resonance imaging (mri), functional mri, diffusion tensor imaging, and positron emission tomography, demonstrated abnormalities extending from hippocampus to subcortical structures and bilateral cortical regions in the precentral gyrus in patients with tle [6, 7 ]. in clinical practice, resection of epileptic focus within the temporal lobe might still fail to control the seizures in some patients with tle. the main factor contributing to these failures is probably the incomplete resection of the epileptogenic zone or that the patients might suffer from a complex epileptogenic network, which may involve the subcortical structures [8, 9 ]. in brief, more and more studies have shown that the hippocampal formation, although very important, may be not the only epileptogenic zone of tle. so we should extend investigations outside the temporal lobe and focus on the neuronal circuits that may support the seizures [10, 11 ]. thalamus is one of the potential critical components in the neuronal circuits that may take part in the initiation and spread of seizures in tle. it has rich and reciprocal connections with cerebral cortex and limbic system [12, 13 ]. these connections participate in relaying sensory and motor signals, along with regulation of consciousness, sleep, and alertness in the normal physiological condition and also could be involved in the genesis or propagation or both of focal and generalized seizures in the pathological state. the role of thalamus has been well demonstrated in animal models of absence seizures which are the most pure form of generalized epilepsy. the epileptic network was defined as a reciprocal circuit involving the neocortex and thalamic relay nuclei, and the thalamic reticular nucleus served as a key modulator. in this network, the cortex provided the excitatory drive, and the thalamic relay nuclei organized the drive into the epileptic spike wave pattern. recently, some clinical trials targeted various thalamic nuclei as therapeutic electrical stimulation zones in attempt to control the intractable epilepsy, mostly tle, with varying degrees of seizure reduction in more than half of the patients. moreover, there were evidences of thalamic involvement in seizures of tle in patients, although the role of the thalamus was not well defined. in animal models of tle, studies have provided the histopathologic evidence for cell loss in the medial subdivisions of thalamus that coupled with synaptic alterations, which could enhance the excitability of thalamic seizure circuits. enhancement of gaba activity in the medial dorsal nucleus (md) of thalamus resulted in a significant reduction of seizure duration [18, 19 ]. further investigations proposed that thalamus could act as an excitatory amplifier through divergent - convergent circuits in the seizures [10, 20 ]. besides md, other nuclei of thalamus electrical stimulation of the reticular nucleus suppressed the limbic motor seizures in a hippocampal kindling model. electrical stimulation of the anterior nucleus (ant) showed an improvement in seizure control in the status epilepticus (se) model of tle, and the possible mechanism was that the stimulation of ant caused decreases in concentrations of glutamate and increases in gaba in hippocampus. in the kainic acid model of tle, high - frequency electrical stimulation of the parafascicular nucleus (pf) interrupted the ongoing hippocampal paroxysmal discharges, and the administration of nmda antagonist and gabaa agonist suppressed the hippocampal discharges, whereas nmda agonist and gabaa antagonist increased the hippocampal discharges. these findings indicated that several nuclei of thalamus had close relation with the hippocampus in the neuronal circuits of seizures. however, in the study of absence seizures, enhancement of glutamate activity of pf significantly suppressed the spike - and - wave discharges. it is interesting that activating pf had the opposite effects on epileptic discharges in the temporal lobe epileptic seizures and the absence seizures, calling for more investigations on the role of pf in seizures. studies mentioned above mainly investigated the role of thalamus by pharmaceutically modulating or electrical stimulating the thalamus nuclei. in the present study we investigated the role of thalamus in the development of seizures by computational methods. we used multichannel microelectrode techniques to obtain the local field potentials (lfps) and single - unit discharges from ca1 of hippocampus and pf of thalamus during the development of epileptic seizures induced by pilocarpine in mice. computational methods from symbolic dynamics and information theory were used to analyze the electrophysiological characters of neuronal activities and the information flow between thalamus and hippocampus. we found that lfps became more regular during the seizure in both hippocampus and thalamus, and in some cases lfps showed a transient disorder state at the seizure onset. the information tended to flow from thalamus to hippocampus during seizure initiation period, and inactivation of thalamus by tetrodotoxin (ttx) resulted in a suppression of seizures. these findings indicated that thalamus may play an important role in the initiation of epileptic seizures. the mice were housed in individual cages with food and water ad libitum, and kept in a 12 h light / dark cycle. all animal experimentations were approved by the ethic committee, school of biomedical engineering, shanghai jiao tong university. all efforts were made to minimize the animal suffering and reduce the number of animals used in the experiments. the 16-channel electrodes, consisting of two independently movable bundles of 2 tetrodes, were secured to a recording microdrive (figure 1(a)). each tetrode was formed of four twisted polyester insulated nickel - chrome alloy wires (diameter, 13 m ; stablohm 675, california fine wire co, usa) with an impedance of 0.51 m. mice were handled for about one week prior to surgery to minimize the potential stress of human interaction. during the surgery, mice were anesthetized by pentobarbital sodium (100 mg / kg) intraperitoneally and mounted in a stereotaxic frame (51600, stoelting co, usa). the skin covering the skull was opened and the skull was exposed. the skull was perforated using a high speed dental drill (k.1070 high speed rotary micromotor kit, foredom co, usa) with 1.2 mm diameter drill tips. seven small holes were drilled : five for the positioning of anchor screws and two for the placement of electrodes. two cortical screws placed in the bilateral frontal bone were used as the reference and ground. the recording microdrive was then fixed to the skull using zinc phosphate cement (hoffmann dental manufaktur gmbh, germany). throughout the experiments, body temperature was maintained at 37.5c using a closed - loop animal blanket system (ss20 - 2, huaibei zhenghua biologic apparatus facilities ltd co, china). mice were injected intraperitoneally with atropine sulfate (1 mg / kg) and 30 minutes later with a single dose of pilocarpine (300 mg / kg). their behavior was scored according to the racine scale, and status epilepticus (se) was defined as continuous stage 3 or more serious seizures. se was terminated after 1 h by injection of diazepam (10 mg / kg). a bundle of electrodes was inserted in ca1 of the left hippocampus (with bregma as the reference, anteroposterior (ap), 2.3 mm ; mediolateral (ml), 2.1 mm ; dorsoventral (dv), 1.0 to 1.4 mm). the other bundle of electrodes was implanted into the left pf (ap, 2.3 mm ; ml, 0.5 mm ; dv, 3.0 to 3.4 mm). when the electrode reached the pyramidal cell layer of the ca1 region, single - unit activities were recorded. similarly, the electrodes in the pf region were adjusted between the depth of 3.0 mm and 3.4 mm until single - unit activities were recorded. the electrodes were lowered to the target areas at least one day before the seizure induction. the data were amplified (500), filtered (0.56,000 hz), and stored in a computer (16 bits ad converter, 40 khz sampling rate) using omniplex d neural data acquisition system (plexon co, usa). at the end of the electrophysiological recordings, mice were deeply anesthetized with pentobarbital sodium (120 mg / kg) and perfused transcardially with 20 ml of 0.9% saline solution followed by 50 ml of fixative (4% paraformaldehyde in 0.1 m sodium phosphate buffer (pbs), ph 7.4). the brains were then removed and postfixed in 4% paraformaldehyde 0.1 m pbs solution at 4c for 24 h, equilibrated in 30% sucrose in 0.1 m pbs at least overnight. the whole brain was frozen and sectioned coronally on a cryostat microtome set at 70 m. electrodes outside the target regions were excluded in the analysis. the data were replayed and filtered with two different band - pass filters : 0.5100 hz to obtain lfps and 3006,000 hz to obtain spikes as illustrated in figures 1(b) and 1(c), respectively. the spike data were later sorted into single - unit activities using the offline sorter software (plexon co, usa). single - unit activities were sorted according to a threshold and shape detector using principal component analysis method (figure 1(d)). lfp signals were resampled by the frequency of 500 hz. in addition, lfps recorded from 4 channels of a tetrode were highly similar since the distance between wires in a tetrode was only about 1525 m. therefore we used the mean of lfp signals recorded from the four channels in a tetrode to represent the lfp signal of the tetrode. we analyzed the synchronization of firing activities between the recorded neurons by calculating the cross - correlation function (ccf). ccf is one of the most commonly used methods to evaluate the synchronization of firing activities between neurons. in order to calculate ccf, each neuron 's spike train is symbolized into 0 and 1 within a time bin (time bin = 2 ms, to ensure there is only up to one spike in one time bin), where 1 means that neuron i fires in the tth time bin and 0 means no firing. ccf is calculated as (1)ccf(t)=nt=1+|t|n|t|r1(t)r2(t+t)(n2|t|)t=1nr1(t)2t=1nr2(t)2, where ri(t) for i = 1, 2, denotes the spike train generated by the ith neuron at the moment t and n indicates the length of spike train. the maximum of ccf reflects a maximal synchronization of two sequences when the r1(t) was postponed for lag t. in order to examine the dynamics of neuron population activities, we used a moving window with length of 5 seconds and shifted in 1-second steps to calculate ccf. in each window, t ranges from 200 ms to 200 ms. various methods have been used to analyze the temporal evolution of brain activities from eeg or lfp recordings, ranging from traditional linear methods to nonlinear methods. to some extent, the nonlinear methods are superior to the traditional linear methods in extracting information from eeg or lfp data. however, these nonlinear methods assume that the signal is stationary and originates from a low dimensional nonlinear system. without doubt, recently, bandt and pompe proposed an ordinal time series analysis method, that is, permutation entropy, which measures the irregularity of nonstationary time series. this method concentrates on the order relations between the values of a time series but not the values themselves. the advantages of this method are its simplicity and low complexity in computation without further model assumptions [32, 33 ]. furthermore, the bandt - pompe method is robust in the presence of observational and dynamical noise. over the last few years, the permutation entropy and related metrics have emerged as particularly appropriate complexity measures in the study of time series from biological systems, such as the brain or the heart. redundancy is equal to one minus normalized permutation entropy ; thus, it is also an index to measures the irregularity of nonstationary time series, with the opposite trend to the permutation entropy. here, the redundancy of lfps was analyzed to deduce the signals ' irregularity changing during the development of epileptic seizures. given a time series of length l, { x1, x2,, xl }, a vector is generated by an embedding procedure : st = [xt, xt+,, xt+(m1) ], where m and are the embedding dimension and the lag, respectively. xt+(jm1). we set jr1 0 means that the information flows from the process x to y. dxy 0 as the percentage of information flow from hippocampus to thalamus and the percentage of dxy < 0 as the percentage of information flow from thalamus to hippocampus, respectively. for each mouse, the summation of percentages of the two directions is 100% during each specific period. for the is period, the information flowed from thalamus to hippocampus in most cases (7 of 9 seizures, including mouse # 2, # 4, # 5, # 6, # 7, # 8, and # 9). in the other two seizures, the information flow was symmetrically bidirectional, but none was found in which the information flowed from hippocampus to thalamus during the is period (figure 6(c)). in the other two periods, these results suggest that the thalamus may play an important role in the initiation of seizure. to further confirm the role of thalamus, we used the pharmacological method to inactivate thalamus to examine its effect on the seizure. after the animals had a baseline seizure induced by pilocarpine, ttx was injected into the thalamus (n = 2). the inactivation of thalamus caused a reduction of seizure activities (figures 7(a) and 7(c)), and there was a power reduction as shown in figure 7(b), though the power was larger than the baseline. but the injections of normal saline (n = 1) had little effect on the development of seizure activities and the power spectral (figures 7(d)7(f)). in both cases, the directionality index (dxy) did not show obvious change after the injection of ttx or normal saline (figures 7(a) and 7(d)). these results indicated that pf did not play the leading role after seizure initiation, but it still had an effect on the seizure activities. in this study, we used 16-channel microelectrode to record the lfps and single - unit activities from hippocampus and thalamus of mice. the computational methods from symbolic dynamics and information theory were used to investigate the dynamic changes of neural activities from each brain area and the direction of information flow between the two brain areas. these methods mapped the raw lfp signals into ordinal time series, which reflected the relational aspects between consecutive values of the original lfp signals but not the values themselves. our findings, mainly based on analysis of lfps, reveal two important points about the epileptic seizures induced by pilocarpine. first, we found a global increase of redundancy of ictal lfps, either in hippocampus or in thalamus (figures 3 and 4). however, in some cases, the redundancy of lfp (r) shows a transient decrease shortly after the seizure onset, which may last 6 to 12 s (9.1 2.3 s, mean s.d. the increase of r suggests more rhythmic lfps occurred during the ictal state, while the transient decrease of r suggests that the lfps are transiently disordered at the seizure onset. in a traditional view, epileptic seizures are commonly considered to be the result of monolithic, hypersynchronous activities arising from an imbalance between the excitation and inhibition in neuronal network. however, our results indicate that the epileptic seizure is not a monolithic state. in recent years schindler and his colleagues find that the correlation of multichannel eeg either remains approximately unchanged or decreases during the first half of the seizures in patients with pharmacoresistant focal epilepsy. in a smaller spatial scale, studies indicate that the lfp synchrony between the seizure generating brain and the other brain regions is lower in epilepsy patients than in control patients. another study shows that fractured microdomains exist in the epileptic brain, which can be observed by isolated microelectrodes rather than clinical macroelectrodes. in a much smaller scale, it was shown that neuronal spiking activity during the initiation and spreading of seizures in humans was highly heterogeneous, suggesting complex and variable interactions between different neuronal groups. since lfps are a summation of the synaptic activities of many of neurons around the recording electrode, we then examined how the single neurons acted during the peri - ictal period. the variation tendency of the peak values of ccf between neurons matches the variation tendency of r of lfp recorded from the same electrode very well, even in the period of transient decrease of r. as shown in figures 3(b) and 3(c), when the ccf peak value gets larger, r gets larger as well. so we speculate that the lager r may reflect higher level of the synchronization of single neurons to some extent. the global increase of r during the seizures may reflect the global synchronization of neurons around the electrode to some extent, while the transient decrease of r may reflect the transient desynchronization of neurons around the electrode to some extent. interestingly, cymerblit - sabba and schiller find the similar biphasic network dynamics, which composed of an early desynchronization phase and a late resynchronization phase, in the pre - ictal state of pharmacologically induced seizures in rats. the biphasic network dynamics may reveal a specific network mechanism underlying the development of epileptic seizure. we can not record lager amount of single - unit activities during the development of seizure in the present experiment conditions. we will try to improve the recording techniques to collect much more single - unit firing activities to confirm our conclusion. second, we found that the information tended to flow from thalamus to hippocampus during the initiation period of seizure (figures 5 and 6). this indicates that the thalamus led the hippocampus during the initiation period to some extent. in clinical study, it shows the similar coupling direction between hippocampus and thalamus during the seizure initiation period in some of patients with tle. in animal models of tle, studies have shown that thalamus indeed plays an important role in the initiation and spread of seizures, though the exact role is less understood [18, 19, 2125 ]. bertram put forward a framework to explain the neuronal circuits that support the different stages of seizures. it contains four parts, seizure focus, initiating circuits, paths of spread and neuromodulatory centers. in this framework, thalamus is served as part of the initiating circuit, which is the separate neuronal populations that are necessary to support the start of a seizure. although the framework need to be confirmed using a larger amount of data in the future, it provides a new view point to consider the role of brain structures out of the temporal lobe in tle at the level of neuronal circuits. our results are consistent with this framework, suggesting thalamus may play an important role in the initiation of epileptic seizures induced by pilocarpine. on the contrary, studies by toyoda and colleagues have demonstrated that dorsomedial thalamus has consistently late seizure onsets and is unlikely involved in the seizure initiation in pilocarpine - treated rat model. the controversial conclusion may be due to the different recording sites or different animal species. in addition, they drew the conclusion by simply comparing the seizure onset time across different brain regions. without detectable epileptiform discharges in thalamus at the first moment of seizure onset does not mean that there is no information exchange between thalamus and the brain areas where the seizure starts, and thalamus may be involved in the seizure initiation circuits. applying computational methods, such as pcmi, to analysis lfps may reveal the characteristics of epileptic network which might not be accessible by the examination of the waveforms of neuronal signals. of course, we can not draw a strong conclusion only by the mathematical results. but we provide a new perspective to understand the seizure generation induced by pilocarpine in mice. the pharmacological experiments show a suppression effect on the seizure activities by injection of ttx into thalamus but not completely. the power of lfps in hippocampus after ttx injection is smaller than that before ttx injection but is higher than the baseline (figure 7). since the injection site was located at pf of thalamus, this result reminds us that the pf of thalamus may be not the only structure which has effect on the development of seizure, but it does have effect partially. the ttx delivery experiment was done at the anesthetic state because of the limitation of our experiment conditions. if ttx was injected before the seizure induction, we can not determine whether the ttx injection had effect on the seizure or the seizure was not induced at all. so we can not observe the effect of ttx on the initiation of epileptic seizures. in our experiments, dxy did not change after ttx injection, suggesting that pf did not play the leading role after seizure initiation. this result does not conflict with our results in freely moving state, nor can confirm the results before. so we will keep working on it in freely moving state in the future. from a therapeutic perspective, researches on the role of thalamus in tle may provide a potential target to the clinical resection and the deep brain stimulation. they may also further the understanding of the nature of seizure initiation and spread. our data analysis brought new insight into the dynamic changes of single brain area and the interaction between brain areas during the development of seizures, and we still have many jobs to improve and further confirm our results. | studies have suggested that thalamus is involved in temporal lobe epilepsy, but the role of thalamus is still unclear. we obtained local filed potentials (lfps) and single - unit activities from ca1 of hippocampus and parafascicular nucleus of thalamus during the development of epileptic seizures induced by pilocarpine in mice. two measures, redundancy and directionality index, were used to analyze the electrophysiological characters of neuronal activities and the information flow between thalamus and hippocampus. we found that lfps became more regular during the seizure in both hippocampus and thalamus, and in some cases lfps showed a transient disorder at seizure onset. the variation tendency of the peak values of cross - correlation function between neurons matched the variation tendency of the redundancy of lfps. the information tended to flow from thalamus to hippocampus during seizure initiation period no matter what the information flow direction was before the seizure. in some cases the information flow was symmetrically bidirectional, but none was found in which the information flowed from hippocampus to thalamus during the seizure initiation period. in addition, inactivation of thalamus by tetrodotoxin (ttx) resulted in a suppression of seizures. these results suggest that thalamus may play an important role in the initiation of epileptic seizures. |
osteoma is a benign osteogenic tumor arising from the proliferation of the cancellous or compact ivory osteoma bone. it can be central osteoma arising from the endosteum, peripheral osteoma (po) arising from the periosteum or extra - skeletal soft tissue osteoma that usually develops within the muscle. these occur frequently in the sinuses, most common in the frontal sinus, followed by the ethmoidal and maxillary sinus. most cases of po appear to have a very slow growth rate, are asymptomatic and produce swelling and asymmetry. its pathogenesis is unclear ; some believe it to be a true neoplasm while others classify it as a developmental anomaly. association between maxillofacial osteomas, cutaneous sebaceous cysts, multiple supernumerary teeth and colorectal polyposis has also been reported (gardner 's syndrome). a 50-year - old female patient reported with a chief complaint of swelling in the anterior region of the upper jaw, present since 10 years. history revealed that the swelling initially started as a peanut size and had gradually increased to the present size. the patient gave a past history of trauma to the upper jaw 15 years back during a road accident. she was given some drugs (patient was unaware of this) and, within 1 week, she was asymptomatic. after 4 years, she observed an asymptomatic peanut - sized swelling in the same region of the jaw, which has been slowly increasing in size. extraoral examination revealed a diffuse swelling measuring about 1 cm 1 cm in the midline below the nose, with slight obliteration of the left nasolabial fold [figure 1 ]. facial view showing the extraoral diffuse swelling intraoral examination revealed a well - circumscribed swelling of about 2 cm 2 cm on the alveolar process obliterating the labial vestibule in the 11 - 22 region. on palpation, the swelling was non - tender, bony hard (non - compressible), non - fluctuant and non - pulsatile. the pulp vitality test confirmed all the anterior teeth (13, 12, 11, 21, 22, 23) to be vital. the intra - oral periapical radiograph revealed a well - demarcated radio - opaque mass superimposing the roots of 21 and 22, clearly demarcated from the surrounding normal bone. based on the clinical and radiological findings, a provisional differential diagnosis of peripheral ossifying fibroma, osteoma and maxillary torus was made. intraoral photograph revealing the extension and appearance of the swelling the lesion was excised under local anesthesia. the gross specimen was bony hard, round in shape and measured about 2 cm 2 cm. the superficial surface appeared pale and smooth whereas the cut surface was rough [figure 3 ]. the tissue was fixed in 10% neutral buffered formalin, decalcified in 5% formic acid and was routinely processed. gross surgical specimen showing the superficial and cut surfaces the decalcified section was stained with hematoxylin and eosin, which revealed surface epithelium overlying the stromal tissue, consisting of lamellar bone and adipose tissue in the lamina propria and submucosa. the lamina propria consisted of multiple trabeculae of mature lamellar bone with a variable amount of fibro - fatty marrow tissue [figures 4 and 5 ]. large and small trabeculae of lamellar bone were seen extending into the submucosa, suggestive of cancellous bone. based on these histopathologic features, a diagnosis of peripheral cancellous osteoma was made. photomicrograph showing stratified squamous epithelium overlying the stroma consisting of trabeculae of mature lamellar bone (hematoxylin and eosin, original magnification 4) photomicrograph showing trabeculae of mature lamellar bone with a variable amount of fibro - fatty marrow tissue (hematoxylin and eosin, original magnification 40) osteoma is a rare benign tumor characterized by excessive and persistent proliferation of the bone. it may arise from proliferation of the medullary bone (endosteal) or on the bone surface as a polypoid or sessile growth (periosteal). depending on the location the size of the tumor may cause facial deformity. in the maxillofacial region, these tumors are mainly located on the skull. po occurs most frequently in the sinuses, of which the frontal sinus is the most common site, followed by ethmoidal and maxillary sinuses. po has also been described in the external auditory canal and, rarely, in the temporal bone and pteygoid plate. the most common affected sites are angle and lower border of the body of the mandible, the sites most susceptible to trauma. there is a 3:1 female predilection, but different authors have reported both female and male predilection in case series of osteomas of the maxillofacial region. to best of our knowledge, and apart from osteomas located in the maxillary sinuses, there are only 10 cases of maxillary osteomas previously described in the english language literature [table 1],[11114 ] of which complete clinical data of five cases is not available. their age ranged from 16 to 76 years, with a mean age of 45.5 years. the mean age range of the patients with osteoma in the maxillofacial region has been reported to be 29.4 and 40.5 years. various hypotheses have been put forward, which include congenital and hereditary disorder, a developmental origin, neoplastic or a reactive mechanism to trauma or infection. location of po of the jaws is usually in close proximity to the area of muscle attachment, suggesting that muscle traction may also play an important role in its development. the combination of trauma and muscle traction has also been suggested in the pathogenesis of po. differential diagnosis includes exostosis, but these tend to stop growing after puberty ; periosteal osteoblastoma, osteoid osteoma and paraosteal osteosarcoma usually present as a rapidly growing painful swelling. summary of clinical data of maxillary osteomas previously published in the english language literature, including the present case surgical removal of po is not generally necessary unless and until there are symptoms like facial asymmetry or other secondary problems. the present case of po was seen in a 50-year - old female patient with swelling in the anterior region of the upper jaw that was present since 10 years following a history of trauma. the swelling was solitary, sessile, slow growing, non - tender, non - compressible, non - fluctuant and non - pulsatile. the lesion was surgically excised, histopathological examination revealed the presence of a surface - stratified squamous non - keratinized epithelium overlying the stromal tissue, consisting of lamellar bone and adipose tissue. the stromal tissue consisted of multiple trabeculae of medullary spaces consist of fibro - fatty marrow tissue with endothelial - lined blood vessels. the patient has been asymptomatic, with no evidence of recurrence since 2 years after surgery. the possibility of po should be kept in mind as a differential diagnosis for any peripheral, solitary, bony hard, slowly growing painless swelling encountered in the oral and maxillofacial region. to best of our knowledge this is the eleventh case of maxillary osteoma reported in the english language literature. although the exact etiology and pathogenesis of this type of lesion is unclear, additional molecular and genetic research is required to improve the understanding. | osteoma is a benign osteogenic lesion with a very slow growth, characterized by proliferation of either cancellous or compact bone. depending on its location, osteoma may be classified as central, peripheral or extraskeletal type. although peripheral osteomas of the upper jaw are rare, diagnosticians can however encounter them in their clinical practice, which are probably misdiagnosed. these should be considered in the differential diagnosis of any slow - growing, non - tender, bony hard, non - compressible, non - fluctuant and non - pulsatile swelling present on the oral and maxillofacial region. we report a rare case of maxillary peripheral osteoma in a 50-year - old female patient. to best of our knowledge this is the eleventh case of maxillary osteoma reported in the english language literature. |
the current issam (international society for study of the aging male), eau (european association of urology), and bssm (british society for sexual medicine association) definition of late onset hypogonadism [1, 2 ] isa biochemical syndrome associated with advancing age and characterised by a deficiency in serum androgen levels with or without a decreased genomic sensitivity to androgens. it may result in significant alterations in the quality of life and adversely affect the function of multiple organ systems. a biochemical syndrome associated with advancing age and characterised by a deficiency in serum androgen levels with or without a decreased genomic sensitivity to androgens. it may result in significant alterations in the quality of life and adversely affect the function of multiple organ systems. this state of hypogonadism causes a global decrease in energy and a decrease in the feeling of well - being. it also causes a change in sexual function and has other endocrine and metabolic repercussions. these can affect bones, muscles, and lipids, as well as cognitive function testosterone deficiency syndrome (tds) or late onset hypogonadism which is defined on the basis of clinical symptoms associated with abnormal testosterone levels the european male aging study (emas) studied 3369 men aged 4079 at 8 european centres and concluded that the 3 following cardinal symptoms were most likely to be related to low levels of testosterone. other symptoms such as hot flushes, sweats and tiredness, loss of vitality, reduced shaving frequency, gynaecomastia, depressed mood, poor concentration, and sleep disturbance were regarded as less specific. recent guidelines suggest that a level of total testosterone of 12 nmol / l or free testosterone of > 225 pmol / l does not. between these levels a trial of therapy for a minimum of 6 months should be considered based on symptoms [1, 2 ] total testosterone should be measured between the hours of 7 and 11 am on 2 occasions at least 1 month apart and ideally be assessed by mass spectrometry (id - gcms). equilibrium dialysis is currently the gold standard for free testosterone as immunoassays based on analogue displacement are currently inaccurate [1, 2, 4 ]. the endocrine society recommends that men with the following conditions should be screened for low testosterone routinely : type 2 diabetes, metabolic syndrome, moderate to severe chronic lung disease, osteoporosis, hiv, history of infertility, treatment with steroids, opiates (even medically prescribed), and anticonvulsants, alcohol abuse. moderate to severe chronic lung disease, history of infertility, treatment with steroids, opiates (even medically prescribed), and anticonvulsants, there is increasing evidence from multiple long - term studies that tds is associated with increased cardiovascular and all - cause mortality [513 ]. two recent meta - analyses have looked at a large number of long - term studies linking low testosterone to increased cardiovascular and all - cause mortality [14, 15 ]. araujo. concluded that the evidence for a link between low testosterone and increased mortality was strong but concluded that most studies involved issues in cohort selection and choice. they concluded that a decrease of 2.1 standard deviations in total testosterone was associated with a 25% increase in mortality.. looked at the data in terms of several models and found that even after strict adjustment for comorbidities there was a consistent link between mortality risk and testosterone level throughout the studies but that this did not prove causation (table 1). the emas group recently reported 4.3 year follow - up data on 2599 men aged 4079 and concluded that men with a baseline tt of 8 nmol / l or less and sexual symptoms had a 3-fold increased mortality and a 5-fold increased risk of cancer death. there authors concluded that there is a small number of men with low testosterone at considerable risk of early death. a recent 10 year study from western australia involving 3690 men followed up from 20012010 concluded that tt and ft levels in the normal range were associated with decreased all - cause and cardiovascular mortality, for the first time suggesting that both low and dht are associated with all - cause mortality and higher levels of dht reduced cardiovascular risk. six published studies usually involving small samples have shown that low tt and ft are associated with cad and 4 have shown no association. four studies have shown inverse associations between low tt or ft (table 2) and the severity of cad. one involved 803 men assessed by gensini score, based on the location and number of stenotic coronary artery segments and degree of luminal narrowing. such studies do not establish whether low tt or ft is a cause or a consequence of cad. the prevalence of hypogonadism was 24%, mortality rates were 21% versus 12% (p = 0.002) for hypogonadal men versus eugonadal, and the study was halted early at 6.9 years. only beta - blocker therapy and left ventricular failure were found to have a greater influence on survival. muraleedaran. screened a primary care diabetic population of 587 patients and followed them up for 5.8 years. they found that 475 of men had normal tt levels, 22% were overtly hypogonadal (50%) without significant consequences and no deaths in the active treatment group versus 5 in the placebo cohort. at least 7 observational studies have reported no association of luts with serum testosterone level and 5 have shown an inverse relationship. no studies to date show an increase in luts / bph symptoms with higher serum testosterone levels. a recent long - term registry of 5 years trt shows sustained reduction in ipss, postresidual volume, and bladder wall thickness, despite minor increase in prostate volume. another 5-year study involving a smaller cohort showed no impact on luts / bph parameters. as trt has been shown to upregulate pde5 and enhance the effect of pde5is (now an accepted therapy for both ed and luts), it no longer seems logical to advice avoidance of trt in men with mild to moderate bph.. also found that patients on testosterone were 12 times more likely to get a prostate biopsy but no more likely to have a positive finding. several meta - analyses have failed to show a link between trt and development of prostate cancer but some studies have shown a tendency for more aggressive prostate cancer in men with low testosterone. one recent study of 279 consecutive patients referred for biopsy on the basis of abnormal dre or raised psa found that low bioavailable testosterone and high shbg were associated with a 4.9- and 3.2-fold risk of positive biopsy. current eau, issam, and bssm guidance [1, 2 ] is that there is no evidence trt is associated with increased risk of prostate cancer or activation of subclinical cancer. despite these conclusions, many patients are deprived of clinical and metabolic benefit because of minor physiological increases in psa and concerns that no long - term study has conclusively proved absolute safety. men with prostate cancer, treated with androgen deprivation, develop an increase of fat mass with an altered lipid profile. the risks of diabetes mellitus increase by 44% and mortality of cardiovascular diseases by 16% during a follow - up of up to 10 years. the authors concluded that before commencing adt, the overall health, comorbidities, and life expectancy of the patient need to be fully assessed. several studies have clearly shown that trt can be effective as monotherapy for ed [26, 28 ], in men without other multiple comorbidities. a recent double blind placebo controlled study in men with diabetes showed prompt improvement in iief in men with severe hypogonadism and a secondary improvement with 12 to 18 months of therapy, dependent on obtaining prolonged sustained levels in the normal range. results from a 5-year registry showed 12.3-point improvements in iief in 260 men treated with long acting tu. a systematic review and meta - analysis of placebo - controlled studies published in the past 30 years aimed to study effects of testosterone on the different domains of sexual life. this latter study concluded that t treatment might be useful for improving vasculogenic ed in selected subjects with low or low - normal t levels. erectile dysfunction is an established marker for future cardiovascular risk and the major presenting symptom leading to a diagnosis of low testosterone. current guidelines suggest that all patients presenting with ed, irrespective of age, should be screened for low testosterone, as it is a potentially curable cause of ed, especially in men without othercomorbidities [1, 2 ]. nice guidance suggests that all men with type 2 diabetes be assessed for ed annually and the gp contract now includes routine assessment for ed, and increased demand for testosterone supplementation will be a natural consequence of this correction of previous underdiagnosis and undertreatment. men with low testosterone usually present with bothersome symptoms, particularly ed, and require treatment to address those problems, not simply for cardiovascular prevention purposes. the benefits of conventional cardiovascular risk reduction with exercise and weight reduction are fundamental to management but are frequently unsuccessful. there is a considerable body of evidence that low testosterone is associated with increased cardiovascular and cancer mortality. a policy of taking little or no action for these men based on concerns of increased cardiovascular and cancer risk associated with physiological replacement would seem illogical. there is considerable evidence of modest cardiac and metabolic benefits that are shown to reduce cardiovascular risk plus sexual, mood, and quality of life changes associated with restoring testosterone levels. these benefits may potentially denied to patients by fears over prostate and cardiac risk that is not currently supported by evidence. ideally, we need large long - term studies to resolve these issues with certainty but such studies are unlikely to be done for logistic and financial reasons. | low levels of testosterone are manifested by erectile dysfunction, reduced sexual desire, and loss of morning erections with increasing numbers of men are being diagnosed and require treatment. the prevalence rates of testosterone deficiency vary according to different studies but may be as high as 40% in populations of patients with type 2 diabetes. there is increasing evidence that testosterone deficiency is associated with increased cardiovascular and all - cause mortality. screening for low testosterone is recommended in a number of high risk groups including those with type 2 diabetes and metabolic syndrome. there are recent data to suggest that testosterone replacement therapy may reduce cardiovascular mortality as well as improving multiple surrogate markers for cardiovascular events. specific clinical trials of testosterone replacement therapy are needed in selected populations but in the meantime we must treat patients based on the best current evidence. |
focal atrial tachycardia (fat) is a relatively uncommon entity causing supraventricular tachycardia. in many cases, it is well established that foci of atrial tachycardia tend to cluster at specific atrial structures. for the right atrium (ra), these foci are frequently localized along the crista terminalis. other common locations are the coronary sinus (cs) ostium, the tricuspid annulus, the perinodal region [4, 5 ] or rarely the cs body. in the left atrium (la), the structures most commonly giving rise to atrial tachycardia foci are the pulmonary veins (pv) ostia and the mitral annulus. less commonly foci may arise from the left aspect of the interatrial septum (ias). left and right atrial appendages could also be the sources of foci of atrial tachycardias [10, 11 ]. an increasingly recognized entity includes fats that are successfully ablated from the non - coronary aortic sinus [12, 13 ]. planning mapping and ablation strategy requires accurate determination of the atrium from which the focus originates. this is relatively straightforward based on p wave morphology and mapping data when the focus originates from the lateral walls of ra or la. however, when the focus is located at or close to the ias differentiating between ra and la origin often requires mapping both sides of the septum. this review will focus on the mapping techniques used to overcome this obstacle and to provide reliable information to guide ablation prior to acquiring access to the la. ias is a complex anatomical structure comprising the oval fossa with its rim and the adjacent ra and la walls. numerous anatomical studies demonstrate that the only structure that is truly interatrial in this regard is the oval fossa with its adjacent rim [15, 16 ]. the remaining parts represent infoldings of the ra wall separated by fibrofatty tissue posteriorly and superiorly and the two atrial vestibules and the interposed aortic root anteriorly. electrical activation of the ias also follows complex patterns reported in several studies. in an animal study using simultaneous biatrial mapping using two multipolar basket catheters deployed in the ra and la sun similar findings have also been reported by human studies as well [18 - 20 ]. an elegant study by lemery. used simultaneous noncontact biatrial mapping in 20 patients. their results demonstrate that in the majority of the studied patients endocardial activation of ra and la septal aspects was discordant, independent and asynchronous during sinus rhythm and during pacing from multiple sites in the ra and la. in all of these reports such routes have been described in detail in anatomical studies and include mainly the bachmann s bundle traversing the ias anterosuperiorly, oval fossa, cs and subepicardial muscular fibers located in the posterior interatrial groove [15, 16, 21 - 23 ]. the atrial electrograms (egms) recorded at the his bundle region were analyzed during paraseptal pacing from the tricuspid and the mitral annulus. during pacing from the tricuspid annulus the atrial egm at the his bundle region was found to be fragmented or double and consisted of two distinct components. in all patients, the first component was found to have high - frequency, and large - amplitude signal and the second component was of lower amplitude and lower - frequency. the timing of the second component was found to coincide with the local atrial egm at the mitral annulus in the vicinity of the ias. during mitral annular pacing this sequence was reversed in 7 of 8 patients with the lower - amplitude, lower - frequency signal preceding the higher - frequency, higher - amplitude component (fig. we have concluded that atrial egms recorded from the his bundle region consist of two components a high - frequency, higher - amplitude component representing a near - field potential from activation of the ra septal aspect and a lower - frequency, lower - amplitude component representing a far - field potential from activation of the la septal aspect. its proximal and middle part is covered by a cuff of striated muscle that extends from the ostium to the valve of vieussens and even more distally in the great cardiac vein as reported in a detailed anatomical study of 240 human hearts. according to another anatomical study the myocardial cuff is continuous with the ra myocardium at the ostium and extends to an average of 40 mm from there. it is connected to the la myocardium by discrete bundles of striated myocardium that represent the substrate for la to cs conduction and is continuous with the ra myocardium at the ostium. this explains the role of the cs as one of the routes of preferential interatrial electrical conduction as described above. in an experiment on 8 excised canine beating hearts these authors showed that cs egms are fragmented or double with two distinct components. one of them is a high - amplitude, high - frequency (near - field) component arising from the myocardial cuff of the cs and the other is low - amplitude, low - frequency (far - field) component arising from the activation of the adjacent la myocardium. the origin of the two components was confirmed by microelectrode recordings from the la myocardium and cs myocardial cuff. activation of the la and adjacent cs myocardial cuff was demonstrated to be discordant as well. during lateral la pacing activation of the atrial myocardium adjacent to the mitral annulus was shown to be activated from lateral to septal while the cs myocardial cuff was activated in a centrifugal pattern with a breakthrough at the middle cs where the presumed la - to - cs connection is located. this study also demonstrated the presence of ra - to - cs electrical connection located at the ostium of the cs. isolation of the cs ostium from the remaining part of the ra myocardium by a set of incisions resulted in complete reversal of atrial activation during ra pacing : la was found to be activated via the septum and bachmann s bundle. the near - field component in the cs electrograms followed the far - field component indicating activation of the cs myocardial cuff in a reversed fashion via an la - cs connection. with all these complex electrical activation patterns the cs seems to play an important role in interatrial conduction ideally, the p wave morphology should be assessed during episodes of spontaneous or induced av block or during ventricular pacing. many fats demonstrate 1:1 atrioventricular conduction necessitating carotid sinus massage or administration of av nodal blocking drugs (e.g. adenosine) to disclose p wave morphology. these maneouvers not infrequently result in tachycardia termination making analysis of p wave morphology impossible or inaccurate. despite these limitations p a couple of algorithms have been created for this purpose. in an early study tang they have found that p wave polarity in leads avl and v1 was most helpful to distinguish between la and ra foci. positive or biphasic p wave in lead avl was able to predict ra focus with a sensitivity of 88% and a specificity of 79%. positive p wave in the surface ecg lead v1 during fat predicted la origin of the focus with a sensitivity of 93% and a specificity of 88%. however, their series did not include patients with septal foci. in an elegant study, kistler. they have analyzed p wave morphology during tachycardia in 126 patients with 130 fats undergoing successful catheter ablation. this data was used to create an algorithm that was further tested prospectively in 30 consecutive patients. the proposed algorithm could successfully predict the focus location in 93% of the studied patients. however, as stated by the authors diagnostic accuracy was much lower in foci located close to the ias. other groups have reported conflicting results in terms of p wave morphology in fats arising at or close to the ias. frey studied 16 patients with perinodal foci undergoing endocardial mapping of both sides of the septum and have reported that foci arising from the perinodal region on the la aspect of the septum demonstrated a monophasic positive p wave in lead v1 while foci from the ra aspect of the septum had an isoelectric or biphasic p wave in the same lead. a recent study of patients with left septal tachycardias has reported that all the 9 studied subjects demonstrated biphasic p waves with an initial negative deflection during fat in lead v1 and negative or biphasic p waves with an initial negative component in the inferior leads. with this p wave morphology differentiating left septal foci from foci from the cs ostium or superior mitral annulus might be difficult or impossible [2, 8 ]. according to published series fats successfully ablated from the non - coronary aortic in addition to that in a series of 5 patients marrouche. did not find a consistent p wave morphology during fats arising from the left aspect of the ias as confirmed by biatrial mapping and successful catheter ablation. reported the largest series of patients with para - hisian fat undergoing successful catheter ablation. in this study p wave morphology was also variable among study subjects with 20 of 35 patients demonstrating negative (or biphasic with terminal negative deflection) p waves in the inferior leads. p waves in lead v1 were biphasic with the predominant component being opposite to p wave polarity recorded in the inferior leads. p wave polarity in lead avr was also found to be opposite to that in the inferior leads. based on all this data it can be concluded that p wave morphology, although a classical tool to define the chamber of origin of fats, can not be used as a highly reliable method to differentiate ra from la origin and plan the ablation strategy in patients with fats arising at or close to the septum. this overlap in p wave morphology might be partially explained by the obvious spatial limitations of surface ecg. assessing the local endocardial activation timing during fat is the routine approach for localizing the focus. the first step to mapping would be to look into the timing of endocardial activation on the catheters located in the cs, his bundle region, and at the ra lateral wall. in fats that demonstrate early activation at the ra lateral wall or show distal to proximal cs activation predicting the chamber of origin of the focus is relatively straightforward even before any mapping with a roving catheter is carried out in the ra. however, when earliest endocardial activation at this first step is found at the his bundle region or at the proximal or middle cs poles, differentiating between ra and la foci frequently requires further detailed mapping with a roving catheter and often necessitates access to the la and biatrial septal mapping. right superior pulmonary vein) might demonstrate early endocardial activation at the ra posterior wall due to the proximity of these two structures. many groups have worked out different diagnostic tools based on earliest egm timing, sequence of endocardial activation or egm morphology in an attempt to define the chamber of origin of fats demonstrating early activation at or close to the septum. local egm prematurity in relation to surface p wave onset during fat has been studied as a parameter to differentiate ra from la origin when earliest activity after detailed ra mapping is found at the ias. following biatrial mapping, marouche. have identified 5 patients with left septal fat in a series of 120 consecutive patients with fat. the authors used an arbitrary value of 15 ms for the earliest ra septal activation to p wave onset as a cut - off to suggest la septal origin of the focus. however, in two of the patients in their series rf ablation was not successful at the ra septum and necessitated la access despite the egm prematurity exceeding 15 ms. this suggests the absence of a dichotomous cut - off limit for electrogram prematurity that can reliably differentiate la from ra septal origin. another very recent study reports early atrial endocardial activation (-155 msec ahead of surface p wave) at the his bundle region in patients with fats arising from the la septal aspect. values in this range can be recorded during fats coming from other la structures such as the mitral annulus or in those fats successfully ablated from the non - coronary aortic sinus [8, 12, 13 ]. in a study of 16 patients frey. have not found a significant difference in the timing of the earliest electrogram (recorded at the his bundle region in all patients) in perinodal fats ablated from the ra septal aspect, compared to those arising from la septum. in contrast fats arising from the left septum demonstrated significantly earlier egms on the left septal aspect compared to those originating from the ra septal aspect. therefore, the authors have concluded that biatrial septal mapping is necessary for reliable determination of the chamber of origin in fats arising in the vicinity of the av node. this is of extreme importance given the fact that applying ablation lesions in the region of the av node is associated with increased risk of iatrogenic av block. endocardial activation sequence can also be used to differentiate ra from la origin of fat. a focus at a specific region would generate a wavefront that would follow a specific pattern of endocardial activation. this pattern, compared to the endocardial activation pattern during sinus rhythm might serve to differentiate ra from la foci. in a series of patients with paroxysmal atrial fibrillation undergoing catheter studied the endocardial activation timing of the high ra, his bundle region and cs during sinus rhythm and during ectopic foci initiating atrial fibrillation. in 37 patients the sequence of endocardial activation was retrospectively correlated to foci location as confirmed by successful ablation. then, the difference in timing of high ra egm and the atrial egm at the his bundle region during atrial ectopy compared to that obtained during sinus rhythm was further prospectively studied in other 38 patients. when the difference in egm timing at the high ra and the his - bundle region during atrial ectopy subtracted from the same difference during sinus rhythm was 0 msec a right pv focus could be differentiated from an ra focus with a diagnostic accuracy of 100% before la access was obtained. chang. studied inter- and intraatrial activation sequence during fat and ectopic activity in comparison to sinus rhythm in 8 patients with foci originating from superior vena cava and 8 patients with ectopic activity from right superior pulmonary vein. they examined the difference in activation timing of high ra and distal cs as a reflection of interatrial activation and of high ra and his - bundle region as a measure of intra - ra activation. in an attempt to provide a simple measure of the difference in endocardial activation sequence during sinus rhythm and during ectopic activity they subtracted the values of interatrial and intra - ra activation during ectopic activity or fat from those during sinus rhythm. their results demonstrate that the difference in interatrial activation was significantly larger for ectopic activity arising from right superior pv in comparison to that originating from the superior vena cava. with a cut - off of 20 msec for this difference these authors could predict the focus location in all of the studied patients. from an electroanatomical standpoint these results could be explained by the fact that an ectopic beat from the superior vena cava and a sinus beat will follow the same or similar pattern of propagation across the atria. despite close anatomical proximity an ectopic beat from the right superior pulmonary vein would demonstrate a different pattern of electrical activation of the atria hence a different endocardial activation sequence that could explain the observed difference. analysis of egm morphology is another tool that might aid distinguishing between ra and la foci. unipolar electrogram showing qs morphology at the earliest site on the ra septal aspect might also signify ra origin while the presence of an r wave might support la septal origin as suggested by wetzel.. potentials consisting of components reflecting ra activity and right superior pulmonary vein activity may be recorded at the posterior ra due to the close anatomical proximity of these two structures. following studied 16 patients with la fat. in their series 14 patients had earliest ra activation recorded at the high posteromedial ra where double potentials were identified. during fat these double potentials consisted of a smaller first component and a larger second component. the sequence of these two components was reversed during sinus rhythm. the timing of the smaller component during fat coincided with the timing of the local la electrogram recorded at the same area on the la septum. ablation lesions at the sites of earliest electrograms during fat on the ra septum resulted in reduction of the amplitude of the larger component without any impact on the amplitude of the smaller component. based on these findings the authors concluded that the smaller component is a far - field signal from the activation of the left aspect of the posterior septum while the larger component is a near - field activation of the posteromedial ra myocardium. however, their series did not include patients with ra septal foci and pacing was used to simulate ra fat. in a prospective study yamada. similar dual - component egms were recorded at the posterior ra with the larger - amplitude, near - field component preceding the smaller - amplitude, far - field component during sinus rhythm and during fat in all cases of posterior ra foci. the sequence was reversed during fat in all patients with foci from the right pvs providing an excellent diagnostic accuracy in differentiating ra from la origin in these cases. despite its very high diagnostic accuracy the technique proposed by the two investigator groups is limited to fats originating from the right pulmonary veins and the posterior ra. it is not applicable to cases when earliest ra activation is found at other septal sites or proximal to middle cs. our group has recently reported a novel technique, based only on egms from his bundle region and cs, that can aid in differentiating ra from la origin of fat prior to any detailed mapping. we studied a series of 27 patients with fat demonstrating early activation of the his bundle region or proximal or middle cs assessed using a decapolar catheter in the cs and a quadripolar catheter at the his bundle region. fats demonstrating early activation at the ra lateral wall or at the distal cs clearly suggesting the chamber of origin were excluded. prior to any detailed conventional or three - dimensional mapping, we analyzed the morphology of the earliest egm recorded with the above mentioned catheter setting with regard to the sequence of near - field / far - field components. as already shown, atrial egms recorded from the cs and his bundle region are fragmented or double due to the recording of a near - field component from ra septal aspect or cs myocardial cuff and a far - field component from the la. we hypothesized that radially spreading wavefronts from a la tachycardia focus would likely activate the la septal aspect or the la myocardium adjacent to the cs earlier than the ra septal aspect or cs myocardial cuff, respectively. the activation of the latter two structures would occur via one or several interatrial connections. in this case the far - field component at the earliest bipole at the hb or proximal or mid - cs would precede the near - field one. the opposite sequence would occur in fats arising from the ra or from cs myocardial cuff. in both cases, due to the different timing of the two components the earliest electrogram would be double or fragmented, especially if the focus is in its immediate proximity (fig. electrogram recordings of the 27 cases were assessed by two independent observers with an interobserver agreement of 93% (kappa=0.83). in this series, a far - field / near - field sequence of the earliest egm could predict the need for left - sided ablation with a sensitivity of 78 and 89%, specificity of 94 and 89%, positive predictive value of 88 and 80%, and negative predictive value of 89 and 94%, respectively, for the two observers. this high diagnostic accuracy was achieved for a wide range of foci locations in comparison to other reports studying only fats originating from circumscribed areas as the la septal aspect, perinodal region or posterior septum [36, 37 ] (fig. determining the atrium of origin of foci located at or in the vicinity of the interatrial septum might be challenging and frequently necessitates biatrial septal mapping with all the risks associated with transseptal or transaortic access. while surface p wave morphology might be useful in this regard when the focus arises from ra or la structures located laterally, its utility remains limited for atrial tachycardia foci arising at or close to the septum. data from several studies shows that transseptal la access is crucial in determining the chamber of origin of these foci. however, numerous other studies have attempted to use endocardial activation sequence or electrogram morphology in an attempt to acquire that information before gaining la access. the results of some of these studies are promising and the techniques they use may be applied routinely in practice. however, none of them provides a definite solution for all foci locations. the exact technique or combination of techniques that is most likely to differentiate ra from la foci should be cautiously selected. | determining the chamber of origin of focal atrial tachycardias (fats) arising at or close to the septum might require biatrial mapping. this review focuses on the available tools and methods used to distinguish right atrial from left atrial origin before left atrial access is obtained. these include analysis of p wave morphology, assessing the timing of right atrial septal activation, the sequence of right atrial and/or biatrial activation and analysis of earliest electrogram morphology. the electroanatomical properties of the interatrial septum and coronary sinus that provide the basis for the above mentioned tools have also been briefly described. |
cardiovascular diseases (cvds) are the leading cause of mortality for both men and women in the united states (us), with one in every four deaths being attributed to heart disease. of the 370,000 deaths caused by coronary artery disease (cad) every year, about 20.8% are hispanics. compared with non - hispanic whites (nhw), hispanics display a worse cvd risk profile including higher rates of central obesity, physical inactivity, type ii diabetes mellitus, atherogenic dyslipidemia, lower levels of education, and lower socioeconomic status. all these factors have been associated with increased cvd morbidity and mortality in the general population. the united states census estimates that, of the 315 million people living in the us, 54 million (17%) are hispanics, becoming the largest ethnic minority in the country. however, hispanics are underrepresented in studies describing racial or ethnic disparities in the care of patients with cvds. within the us, disparities in the burden of cvd persist among racial and ethnic sub - populations and substantial heterogeneity has been observed in the trends across geographic regions. data on cad and acute myocardial infarction (ami) specific to hispanic subgroups are limited to mexican americans and lacking in other subgroups (e.g., cuban americans, dominicans and puerto ricans), as reflected in the 2010 american heart association (aha) statistics. nevertheless, the true burden of cvd in the growing immigrant populations from mexico and other spanish - speaking countries has not been adequately defined. most of the earlier studies on hispanics / latinos residing in us largely involve mexican - americans or examined hispanics / latinos residing in the us as a homogenous group with only a few studies has included other hispanic / latino background groups. usually these studies were limited in sample size or included only a small number of hispanic / latino individuals. moreover, the studies that included diverse hispanics / latinos suggested marked heterogeneity in risk factor prevalence within this population. this is particularly important because hispanics comprise a heterogeneous group that is bound by common language rather than by uniform environmental origin. puerto rican hispanics are one of the fastest growing groups in the us, currently being the second largest hispanic group only behind the mexican - american hispanics. only one previous study has described the epidemiological profile of patients admitted with an acute coronary syndrome (acs) in puerto rico. however, this study only included data for the year 2007 and was restricted to patients in the greater san juan area. in this paper, we expand the limited literature available to evaluate the epidemiological profile of patients with acs across the island and over a longer time span. the aim of this study was to describe the epidemiological profile of hispanic patients admitted with an acute coronary infarction in 19 hospitals distributed throughout puerto rico during the years 2007, 2009 and 2011. we conducted a secondary analysis of data collected in 19 puerto rican hospitals as part of the puerto rico cardiovascular surveillance study. information with international classification of diseases, ninth revision, clinical modification (icd-9-cm) from medical records was obtained by trained medical personnel. patients with ami admitted with an initial diagnosis of acs (icd 9 code : 410) or chest pain (icd 9 code : 786.5) in these hospitals during years 2007, 2009 and 2011 were included in the study. the patient information was entered into a data instrument created using a secure web - based application (redcap). variables selected for analysis included age, gender, health insurance, past medical history, mode of transportation to the hospital, ami characteristics, cardiac procedures, physiological parameters, in - hospital mortality and medications received during the hospitalization. continuous variables were presented using central tendency and dispersion measures (mean standard deviation). stratification by sex was performed to evaluate gender differences in demographics, in - hospital mortality, clinical history, risk factors, symptoms, ami characteristics, cardiac procedures performed, acute phase treatment (medications employed), physiological parameters and secondary prevention treatment. chi - squared test and t - test were used to compare proportions and means (assuming normality of data distribution) of variables of interest for gender groups. for all tests, a p - value 0.05) (table 1). during the study period, a total of 6,162 patients were hospitalized with ami in 19 participating hospitals in puerto rico. most patients were men (55.8%) and had a private insurance (79.0%). the mean age of patients diagnosed with ami was 67.0 13.6 years old, women being older than men (69.9 13.2 vs. 64.8 13.5 ; p = 0.0001) (table 1). age - group distribution suggests that men are suffering from an ami at an earlier age with almost one - third of all females being older than 75 years old at presentation (p = 0.0001) (table 1). sd : standard deviation ; cad : coronary artery disease ; ami : acute myocardial infarction ; ptca : percutaneous transluminal coronary angioplasty ; pci : percutaneous coronary intervention. significant differences in past medical history were found between genders (table 1). compared to men, women were more likely to have a history of diabetes mellitus, arterial hypertension, congestive heart failure, stroke and bronchial asthma (p 0.05) (table 1). this study provides the most recent information regarding the clinical profile and management of ami in puerto rican residents. the mean age at diagnosis of ami was 67.0 years for puerto rican patients similar to that reported by fang in 2010 (67 years) and lower than those reported in the worcester heart attack study (whas) in 2005 (71 years) [11, 12 ]. the mean ages of presentation for women and men were 69.9 and 64.8 years, respectively. a similar trend was reported by nedkoff in 2015 (67.4 and 61.7 years), whereas in fang (2010), the gap between female and male age at presentation was wider (71 and 64 years) [11, 13 ]. in terms of patient s comorbidities prior to initial ami event, our data show that women were more likely to have a history of diabetes mellitus (51.7%), arterial hypertension (87.0%), congestive heart failure (chf) (10.3%), and stroke (6.4%). on the other hand, male patients were more likely to be current smokers (16.0%) and former smokers (27.0%). in terms of hyperlipidemia and family history of heart disease, the results were not statistically significant between genders. these results are similar to those observed in the whas (2005) where female patients were more likely to suffer from diabetes mellitus, hypertension, stroke, and chf ; however, no significant difference was found in other comorbidities. in a similar way, zucker (1997) found women to be more likely to suffer from diabetes mellitus (25%) and hypertension (55%) when compared to men. nedkoff (2015) also found women to have a higher prevalence of comorbid conditions such as diabetes mellitus, hypertension, and chf. these findings warrant further research into the relationship between an increased risk factor burden for puerto rican women presenting with an ami and worse clinical outcomes, such as in - hospital mortality. the majority (65.9%) of the patients included in our study used a car or walk - in as mode of transportation with no gender differences. the reason behind this preference in unknown at this time and is a theme which needs to be deeply investigated. it could be related to unavailability of ambulance at the time of event or a decision made in search of shorter time to medical care. in 2010, song published an article comparing modes of transportation in case of an ami in terms of efficacy and patient selection. his results were very similar to ours in that 62.7% of the patients arrived in private transportation whereas only 37.3% arrived on ambulance. according to his study, if one considers emergency medical services (ems) to be part of medical care, then it was faster than arrival by private transport to a health care facility. however, if arrival to hospital is the main goal, then using a private transport is much faster and less dependent for patients and relatives. nevertheless, when using administration of reperfusion therapy as a more rigorous measure of treatment initiation, then use of ambulance was associated with a significant decrease in door - to - balloon time. this is an area to be investigated in order to assess if our population would absolutely benefit from increased implementation of ems in order to reduce mortality and complications in patients presenting with ami. when comparing the type of ami between males and females, our results reveal that women are more likely to present with non - st - segment elevation myocardial infarction (nstemi) when compared to men (p < 0.05). similar results were reported by roberts (2014) where women were significantly more likely than men to have nstemi (37.5% vs. 30.7%). nevertheless, zucker (1997) observed no difference between gender in stemi likelihood, but report a higher number of men with st elevations as compared to women (or : 1.9 ; ci : 1.5 - 2.3). therefore, the gender interaction showed an increase in likelihood of women with st elevations rather than without. in terms of the in - hospital management, our study shows that puerto rican women admitted with a myocardial infarction were less likely to receive appropriate medical therapy or a cardiac revascularization procedure. when compared to men, the women included in our study were less likely to receive ace inhibitors / arbs, aspirin, beta - blockers and lipid - lowering agents at any point in the hospitalization. there was no statistically significant difference between males and females in the use of clopidogrel. these findings are comparable to those of a study performed by jneid that also found that women presenting with ami were less likely than male counterparts to receive aspirin and beta - blockers within 24 h of arrival. a similar pattern was noticed when analyzing the data regarding the cardiac procedures performed during hospitalization, in which women were less likely to undergo a cardiac catheterization, ptca or cabg. these findings are consistent with those reported by fang s study in which he noted that even though cardiac catheterizations, cabg, ptca, and use of thrombolytic therapy have increased through time, a higher percentage of men received these procedures when compared to women. however, despite these gender differences, it is still worthwhile to note that compared to the only previously published study in puerto rico, there is a persistent underutilization of guideline - directed pharmacologic therapy among puerto rican patients with an ami. our results reveal that the mean los for patients diagnosed and admitted with an ami was 6.3 days in females and 6.9 days for men without a statistically significant difference between genders. our results contrast with those reported by fang and whas where the los was approximately 4 and 5.3 days, respectively, both lower than those seen in our data [11, 12 ]. saczynski (2010) also show decreased los (5 days) compared to our data and revealed a 30% decline from previous data obtained in 1995 (7.2 days) in the worcester area (ma). these studies show no difference in los between genders but do demonstrate an association with age where the median los increased with older females as well as development of complications such as stroke or cardiogenic shock [11, 20 ]. although gender was not an important factor determining the los in our population, we must emphasize that puerto ricans admitted with ami have an los above the national average (4.8 days). this fact should bring further attention and merits future studies to determine which factors are affecting the los in this particular population. since 1984, the number of cvd - related deaths has been greater in women than men. in 2009, 51% of the deaths attributed to cvd in us were women ; the death rate of cvd was 236 with 190.4 for white women and 267.9 for black women. puerto ricans admitted with ami show the same profile when compared in - hospital mortality rate, since women were more likely to die than men (6.5% vs. 4.55%, p < 0.001). this trend has been observed since 1768, when heberden described for the first time a gender - specific difference in cvd. in 1999, vaccarino abstracted data on over 155,000 women and 229,000 men who had been entered into the national registry of myocardial infarction. in this study, more women died than men under similar circumstances (16.7% vs. 11.5%). moreover, when examining age groups separately, he found that under 50 years of age mortality was 6.1% in women and 2.9% in men. quite the reverse, he found no significant difference in mortality between men and women after age 74. first, ami patients admitted with a different initial diagnosis other than acs or chest pain were not included. second, our data represent hispanics and consequently, our results may not be generalized to other ethnic / racial groups. however, there are important strengths to the study, including the large sample size and represented data of real - life management practices on hispanics patients hospitalized with ami in puerto rico. our study shows significant gender disparities in hispanic population as well as some deficiencies in clinical management and outcomes of ami. to our knowledge, this is the broader study conducted so far in the island that describes the epidemiological profile in patients hospitalized with ami. as part of puerto rico cardiovascular surveillance study, it represents an expandable source of information with a strong update potential over the several next years. knowledge about the epidemiological behavior of this population is critical, so hospital can establish and implement preventive measures, reduce gender disparities, and raise awareness of coronary heart disease among puerto rican populations, thus, impacting clinical management and promoting public health initiatives to improve the care provided to puerto rican patients with ami. our study shows significant gender disparities in hispanic population as well as some deficiencies in clinical management and outcomes of ami. to our knowledge, this is the broader study conducted so far in the island that describes the epidemiological profile in patients hospitalized with ami. as part of puerto rico cardiovascular surveillance study, it represents an expandable source of information with a strong update potential over the several next years. knowledge about the epidemiological behavior of this population is critical, so hospital can establish and implement preventive measures, reduce gender disparities, and raise awareness of coronary heart disease among puerto rican populations, thus, impacting clinical management and promoting public health initiatives to improve the care provided to puerto rican patients with ami. all authors take responsibility for all aspects of the reliability and freedom from bias of the data presented and their discussed interpretation. | backgrounda limited number of studies have been published about coronary artery disease in hispanics, particularly among the puerto rican population. the aim of this study was to present a clinical epidemiological profile and management practices in patients hospitalized in puerto rico with acute myocardial infarction (ami).methodsthis secondary data analysis from the puerto rico cardiovascular surveillance study included 6,162 patients at 19 hospitals in puerto rico, during years 2007, 2009 and 2011.resultsthe mean age of the patients diagnosed with ami was 67 13.6 years old, with women being older than men (p < 0.001). women had a different risk factor burden when compared to men. car / walked in was the principal mode of hospital transportation (65.9%). women received less medications and cardiac procedures when compared to men. while no significant differences in length of hospital stay (los) were observed between genders, in - hospital mortality rate was higher in females when compared with males (6.5% vs. 4.5% ; p < 0.001).conclusionprompt initiatives should be implemented to raise awareness, reduce gender disparities and improve outcomes in patients hospitalized with an ami in puerto rico. |
glycemic control is still the mainstay of type 2 diabetes (t2 dm) management, as a level of hba1c of 7% or below has been associated with a reduction in both microvascular and macrovascular complications (1). despite the wide range of antihyperglycemic drugs available, current therapeutic strategies fall short of optimal glycemic control, especially in patients with long - standing t2 dm with complications and/or multiple cardiovascular disease risk factors (2,3). in addition, current therapies have limiting side effects, such as weight gain, hypoglycemia, fluid retention, and gastrointestinal side effects (1). thus, the search for therapeutic agents with a new mechanism of action continues, and several novel drugs are on the horizon. the role of the kidney in glucose homeostasis has recently gained renewed attention. in severely hyperglycemic patients, glucose cotransporter 2 (sglt2), a low - affinity, high - capacity transporter located on the brush - border of cells in the s1-s2 segment of the proximal convoluted tubule, is the most important mediator of glucose reabsorption from the glomerular filtrate (4). several compounds acting as sglt2 inhibitors have been developed, and some of them are already available for clinical use as oral agents (510). a number of clinical trials (1125) have shown that blocking renal glucose reabsorption leads to a reduction in fasting plasma glucose (fpg) and hba1c through a generalized relief of glucose toxicity that does not depend on insulin action or secretion. furthermore, sglt2 inhibitors have proven to be generally safe and well tolerated, and they have additional clinical benefits, such as blood pressure reduction, due to their mild diuretic effect, and a decrease in body weight, caused by the attendant chronic calorie loss (26). however, in virtually all clinical trials, the observed weight loss has been consistently less than expected from the amount of urinary glucose excretion (25,26). therefore, our aim was to describe and quantify the effects of sglt2 inhibition on body weight, energy balance, and body composition in t2 dm patients on stable therapy with empagliflozin, a highly selective sglt2 inhibitor. we analyzed data from a per - protocol completer cohort of 86 patients with t2 dm (39 women and 47 men ; age 58 9 years, bmi 29.8 4.5 kg / m, hba1c 7.8 0.8% [62 9 mmol / mol ], mean sd) who received empagliflozin (25 mg / day) for 90 weeks ; these patients were one arm of a phase iib 78-week extension study of two 12-week phase ii trials (monotherapy and add - on to metformin) (27). forty - six of the patients (20 women and 26 men) were drug - nave and the other 40 patients (19 women and 21 men) were on > 1.5 g / day or maximally tolerated doses of metformin. in each patient, body weight, fpg, and serum creatinine concentration were measured at timed intervals between baseline and 90 weeks (week 4, 8, 12, 18, 30, 42, 54, 66, 78, and 90 ; body weight not recorded at weeks 4 and 8). estimated glomerular filtration rate (egfr) was calculated by the mdrd formula at each time point. the time course of glycosuria was calculated as the product of glucose filtration rate and fractional glucose excretion. the latter was estimated from the relationship between plasma glucose level and fractional glucose excretion derived from previous studies in which glycosuria was measured directly in t2 dm patients treated with empagliflozin (25 mg / day) (28). a validated mathematical model (http://bwsimulator.niddk.nih.gov) simulating the weight loss time course for a given change in calorie balance was used to estimate the relation of calorie to weight changes (29). for each patient, the simulator used sex, age, height, estimated physical activity level, and weight at baseline as model inputs. model outputs include total daily energy expenditure (tdee) and body composition (fat mass and lean body mass) at baseline and 90 weeks and the time course of energy balance (intake / expenditure). the simulator predicted the weight changes expected for a given calorie loss caused by glycosuria assuming no compensatory changes in calorie intake. the same mathematical model was then used to calculate the energy intake changes that must have taken place to result in the observed weight change (30). this model - based method has recently been validated to provide accurate calculations of energy intake changes in free - living people during a 2-year lifestyle intervention as compared with energy intake changes measured using repeated assessments of body composition and energy expenditure by doubly labeled water (31). data are given as mean sd (or median [interquartile range (iqr) ] for data with nonnormal distribution). group values were compared by mann - whitney u test, and paired group values were compared by wilcoxon signed rank test. treatment - related changes of continuous variables were tested by two - way anova for repeated measures with patient group (drug nave or metformin treated) as a fixed effect. we analyzed data from a per - protocol completer cohort of 86 patients with t2 dm (39 women and 47 men ; age 58 9 years, bmi 29.8 4.5 kg / m, hba1c 7.8 0.8% [62 9 mmol / mol ], mean sd) who received empagliflozin (25 mg / day) for 90 weeks ; these patients were one arm of a phase iib 78-week extension study of two 12-week phase ii trials (monotherapy and add - on to metformin) (27). forty - six of the patients (20 women and 26 men) were drug - nave and the other 40 patients (19 women and 21 men) were on > 1.5 g / day or maximally tolerated doses of metformin. in each patient, body weight, fpg, and serum creatinine concentration were measured at timed intervals between baseline and 90 weeks (week 4, 8, 12, 18, 30, 42, 54, 66, 78, and 90 ; body weight not recorded at weeks 4 and 8). estimated glomerular filtration rate (egfr) was calculated by the mdrd formula at each time point. the time course of glycosuria was calculated as the product of glucose filtration rate and fractional glucose excretion. the latter was estimated from the relationship between plasma glucose level and fractional glucose excretion derived from previous studies in which glycosuria was measured directly in t2 dm patients treated with empagliflozin (25 mg / day) (28). a validated mathematical model (http://bwsimulator.niddk.nih.gov) simulating the weight loss time course for a given change in calorie balance was used to estimate the relation of calorie to weight changes (29). for each patient, the simulator used sex, age, height, estimated physical activity level, and weight at baseline as model inputs. model outputs include total daily energy expenditure (tdee) and body composition (fat mass and lean body mass) at baseline and 90 weeks and the time course of energy balance (intake / expenditure). the simulator predicted the weight changes expected for a given calorie loss caused by glycosuria assuming no compensatory changes in calorie intake. the same mathematical model was then used to calculate the energy intake changes that must have taken place to result in the observed weight change (30). this model - based method has recently been validated to provide accurate calculations of energy intake changes in free - living people during a 2-year lifestyle intervention as compared with energy intake changes measured using repeated assessments of body composition and energy expenditure by doubly labeled water (31). data are given as mean sd (or median [interquartile range (iqr) ] for data with nonnormal distribution). group values were compared by mann - whitney u test, and paired group values were compared by wilcoxon signed rank test. treatment - related changes of continuous variables were tested by two - way anova for repeated measures with patient group (drug nave or metformin treated) as a fixed effect. the clinical data at baseline and week 90 are shown in table 1. as expected, hba1c improved with treatment (by 0.56 0.86 and 0.81 0.93% in women and men, respectively, p = 0.19 for the sex difference). such decreases were sharper during the first 4 weeks and remained essentially stable thereafter ; both calculated filtered glucose and glucose excretion followed the same time course (fig. 1). over the 90 weeks of drug administration, glycosuria was calculated to be 51 g / day (iqr 26) in women and 52 g / day (iqr 18) in men (p = 0.57). thus, in the whole cohort, urinary energy loss averaged 206 kcal / day (iqr 90). neither ldl - cholesterol nor serum triglycerides changed significantly, whereas hdl - cholesterol levels were significantly higher at week 90 compared with baseline (table 1). clinical characteristics at baseline and end of study data are mean sd or median [iqr ]. time course of fpg concentrations, egfr, renal glucose filtration rate, and urinary glucose excretion rate in 86 patients with t2 dm treated with empagliflozin (25 mg / day) for 90 weeks. plots are mean sem. at week 90, observed weight loss averaged 3.2 3.6 kg (range 17.0 to + 1.3) in women and 3.1 4.6 kg (range 16 to + 5.5) in men (p = 0.99 for the sex difference) ; in both groups, body weight was stable after the initial 24 weeks of treatment (fig. this weight change was estimated to consist of one - third fat - free mass loss and two - thirds fat mass loss (table 1) and was positively related to the change in hba1c (supplementary fig. 1). time course of observed and predicted (by glycosuria) weight loss in men and women with t2 dm treated with empagliflozin (25 mg / day) for 90 weeks. when accounting for glycosuria but assuming no changes in energy intake, the model - predicted weight loss was much larger than was observed, averaging 11.1 3.2 kg in women and 11.4 3.0 kg in men (p = 0.74 for the sex difference). as depicted in fig. 2, observed and predicted weight loss began to diverge at 24 weeks, when observed body weight stabilized while expected weight continued to fall. the model - calculated time course of energy intake and expenditure corresponding to the observed weight changes are shown in fig. 3 : whereas tdee was slightly, by 31 kcal / day (iqr 91 ; p = 0.03), but significantly increased over the final 6-week period, energy intake increased markedly, especially after the first 1020 weeks of treatment. over the last 6 weeks, energy intake exceeded baseline energy intake by 269 kcal / day (iqr 258). on the other hand, if the same calorie loss had been achieved by reduced calorie intake instead of glycosuria, tdee would have decreased steadily over the 90 weeks, to reach a level 100 kcal / day below baseline (supplementary fig. 2). calculated rates of energy intake and tdee in men and women with t2 dm treated with empagliflozin (25 mg / day) for 90 weeks. when searching for individual determinants of the difference between observed and expected energy expenditure (or weight loss), we found that both baseline bmi and egfr had an independent influence. thus, in multivariate analysis, the difference between expected and observed weight loss was higher in leaner patients and in individuals with a higher egfr (supplementary fig. finally, we analyzed the difference between patients on metformin and drug - nave patients. baseline clinical characteristics (including body weight) and treatment - induced changes in hba1c and fpg levels were not significantly different between these groups (data not shown). in contrast, observed weight loss was significantly greater in metformin - treated (4.4 4.4 kg) than drug - nave patients (2.0 3.6 kg), both in men and women (p = 0.024 after adjusting for sex and baseline body weight). the current results with empagliflozin confirm the benefits of sglt2 inhibition in patients with t2 dm shown in previous studies (meta - analyzed in vasilakou.). in our patient cohort (rather typical for age, bmi, and glycemic control), 90 weeks of therapy led to a significant and sustained decrease in hba1c and fpg. the initial decrease in egfr, which averaged 4 10 ml min 1.73 m and did not differ by sex or previous treatment, is likely a hemodynamic effect that has been observed with other members of this class of agents (26) and has been shown to be fully reversible upon interrupting the treatment (32,33). with regard to body weight changes, the reduction seen in these patients is in line with the findings of virtually all trials using sglt2 inhibitors : a rapid decrement, typically averaging 23 kg and made up by roughly two - thirds of fat tissue, which is maintained as long as treatment is continued (fig. 1). using the mathematical model, one can calculate that the observed weight loss corresponds to a daily energy deficit of 51 kcal (iqr 112), which is much less than the estimated daily calorie loss through glycosuria (206 kcal [iqr 90 ]). even a conservative estimate of glycosuria such as used here (based on fasting rather than day - long plasma glucose concentrations) predicts a 2.7-fold larger weight loss (11 kg). as previous studies have shown that acute or chronic (4 weeks) empagliflozin treatment does not alter resting or postprandial energy expenditure as measured by indirect calorimetry (28), the discrepancy between observed and expected weight loss implies that energy intake is increased. in fact, after about 10 weeks of treatment, the model predicts a 13% (iqr 12) increase in energy intake, which is maintained throughout (fig. 3). because an increase in water (34) and metabolizable calorie consumption leads to an increase in energy usage (i.e., diet - induced thermogenesis, associated with nutrient processing and absorption), tdee is slightly augmented (by 2% [iqr 5 ]) relative to baseline. it is interesting to reflect that if the same amount of energy deficit had been achieved with dietary restriction, energy expenditure would have decreased substantially (supplementary fig. a similar discrepancy between expected and observed weight loss has been reported in preliminary form by polidori. (35) using the same model as in the current study in patients with t2 dm on treatment with canagliflozin. an increase in calorie consumption has also been found in patients with malabsorption due to crohn disease (36). this adaptive response to glycosuria was accentuated in association with lower bmi and higher egfr (supplementary fig., leaner patients were less concerned about their weight and tended to overcompensate, and in patients with preserved renal function, sglt2 inhibition led to greater urinary glucose excretion. in addition, in patients on chronic metformin therapy, who lost significantly more weight than drug - nave patients, the increase in energy intake tended to be lower (228 kcal / day [iqr 267 ] vs. 291 kcal / day [iqr 226 ], p = 0.07). these findings indicate that marked, consistent energy loss through the urine triggers an anabolic response by which enhanced appetite and, possibly, carbohydrate craving partially offset glycosuria and defend body weight (a basic phenomenon in humans). in support of this interpretation, a study in rats treated with dapagliflozin showed that weight loss was four times larger when animals were pair - fed as compared with ad libitum diet (38). furthermore, a recent study in patients with type 1 diabetes treated with empagliflozin for 8 weeks (39) documented an increased consumption of carbohydrates, averaging 50 g / day, a figure very close to the estimated increase in energy intake of the present cohort. finally, the intrinsic restraining effect of metformin on appetite (40) likely accounts for the observed differences in weight loss and energy intake from our drug - nave patients. further investigation is needed to better understand the mechanisms whereby glycosuria signals to the central nervous system to alter appetite regulation. from the clinical standpoint, patients with t2 dm could benefit more from sglt2 treatment in terms of weight loss and glycemic control if more stringent dietary recommendations were implemented to curb appetite and overeating. | objectivesodium glucose cotransporter 2 (sglt2) inhibitors cause substantially less weight loss than expected from the energy excreted via glycosuria. our aim was to analyze this phenomenon quantitatively.research design and methodseighty - six patients with type 2 diabetes (hba1c 7.8 0.8% [62 9 mmol / mol ], estimated glomerular filtration rate [egfr ] 89 19 ml min1 1.73 m2) received empagliflozin (25 mg / day) for 90 weeks with frequent (n = 11) assessments of body weight, egfr, and fasting plasma glucose (fpg). time - dependent glucose filtration was calculated as the product of egfr and fpg ; time - dependent glycosuria was estimated from previous direct measurements. the relation of calorie - to - weight changes was estimated using a mathematical model of human energy metabolism that simulates the time course of weight change for a given change in calorie balance and calculates the corresponding energy intake changes.resultsat week 90, weight loss averaged 3.2 4.2 kg (corresponding to a median calorie deficit of 51 kcal / day [interquartile range (iqr) 112 ]). however, the observed calorie loss through glycosuria (206 kcal / day [iqr 90 ]) was predicted to result in a weight loss of 11.3 3.1 kg, assuming no compensatory changes in energy intake. thus, patients lost only 29 41% of the weight loss predicted by their glycosuria ; the model indicated that this difference was accounted for by a 13% (iqr 12) increase in calorie intake (269 kcal / day [iqr 258 ]) coupled with a 2% (iqr 5) increase in daily energy expenditure (due to diet - induced thermogenesis). this increased calorie intake was inversely related to baseline bmi (partial r = 0.34, p < 0.01) and positively to baseline egfr (partial r = 0.29, p < 0.01).conclusionschronic glycosuria elicits an adaptive increase in energy intake. combining sglt2 inhibition with caloric restriction is expected to be associated with major weight loss. |
despite the widespread use of bone - targeted agents in the care of patients with bone metastases from prostate and breast cancer, questions regarding the optimal ; choice of agent, dose, dosing frequency, and duration of therapy remain unanswered,,,,,. our research group is interested in designing and performing pragmatic de - escalation trials in both breast and prostate cancer patients. however, before undertaking this research, it is important to survey potential patients regarding this type of study with respect to their understanding of their disease and use of their bone - targeted therapies (i.e. denosumab, zoledronate, pamidronate or clodronate). the information obtained will facilitate appropriate pragmatic trial designs, which will positively impact patients potential willingness to participate. this report summarizes the conduct and findings of a survey designed to collect information from canadian patients with breast or prostate cancer regarding their views of their current treatment regimen. in addition, their opinions regarding the possibility of less frequent treatment administrations and participation in potential research studies to assess the effectiveness of this treatment option were also sought. while we are aware of similar patient surveys around the use of bone - targeted agents for osteoporosis and multiple myeloma, we are not aware of published work in patients with metastatic bone disease from solid tumors. the survey was designed to collect specific information about the use of current bone - targeted therapy and patients perception of the goals of this treatment and alternative treatment schedules. second, the survey gathered information regarding their perspectives on the concept of de - escalated bisphosphonate therapy, designed to help the study team develop an understanding of how patients would value a change in their therapy. our survey was conceived and developed using an iterative approach by the research team with backgrounds in oncology and epidemiology, including expertise in survey design. patients attending outpatient clinics at two canadian cancer centres (the ottawa hospital cancer centre in ottawa and the sunnybrook odette cancer centre in toronto, canada) who were receiving or had recently been receiving bone - targeted therapy for bone metastases from either prostate or breast cancer were approached. the survey was available in both paper and online formats (implemented via www.fluidsurveys.com), whichever the patient preferred to use, and took about 10 min to complete. the survey was open to patients seen between august 1st and october 31st, 2012. local institutional research ethics board (reb) approval for the study was granted at both participating hospitals. however, given the reb requirement that no personal patient identifiers could be collected, we were unable to collect information on the number of patients who took the survey away from clinic but then chose not to complete it. as a result, a response rate can not be calculated. the survey consisted of close - ended multiple - choice and hybrid questions (i.e. choose - one - and specify) which were analyzed using a descriptive summary of findings in the form of frequencies and percentages using microsoft excel 2010 (microsoft corporation, seattle, washington) and sas software (version 9.2, sas, cary, north carolina). this data was used to qualitatively judge the respondents views of their current treatments and to determine their willingness to be treated with de - escalated bone - targeted therapy to allow the study team to clarify the value and feasibility of a future trial around this clinical question. analyses were performed to present the overall response profile, as well as responses within the breast cancer and prostate cancer subgroups. the survey was designed to collect specific information about the use of current bone - targeted therapy and patients perception of the goals of this treatment and alternative treatment schedules. second, the survey gathered information regarding their perspectives on the concept of de - escalated bisphosphonate therapy, designed to help the study team develop an understanding of how patients would value a change in their therapy. our survey was conceived and developed using an iterative approach by the research team with backgrounds in oncology and epidemiology, including expertise in survey design. patients attending outpatient clinics at two canadian cancer centres (the ottawa hospital cancer centre in ottawa and the sunnybrook odette cancer centre in toronto, canada) who were receiving or had recently been receiving bone - targeted therapy for bone metastases from either prostate or breast cancer were approached. the survey was available in both paper and online formats (implemented via www.fluidsurveys.com), whichever the patient preferred to use, and took about 10 min to complete. the survey was open to patients seen between august 1st and october 31st, 2012. local institutional research ethics board (reb) approval for the study was granted at both participating hospitals. however, given the reb requirement that no personal patient identifiers could be collected, we were unable to collect information on the number of patients who took the survey away from clinic but then chose not to complete it. as a result, a response rate can not be calculated. the survey consisted of close - ended multiple - choice and hybrid questions (i.e. choose - one - and specify) which were analyzed using a descriptive summary of findings in the form of frequencies and percentages using microsoft excel 2010 (microsoft corporation, seattle, washington) and sas software (version 9.2, sas, cary, north carolina). this data was used to qualitatively judge the respondents views of their current treatments and to determine their willingness to be treated with de - escalated bone - targeted therapy to allow the study team to clarify the value and feasibility of a future trial around this clinical question. analyses were performed to present the overall response profile, as well as responses within the breast cancer and prostate cancer subgroups. a total of 141 patients responded, and included totals of 65 (46.1%) and 76 (53.9%) patients diagnosed with breast cancer and prostate cancer, respectively (table 1). a total of 100% of breast cancer patients and 94.7% of prostate cancer patients were aware that they had received treatment with a bone - targeted agent at some point. respondents had received bone - targeted agents for : 24 months (19.1%) ; 4.3% of patients did not respond. patients varied in the amount of time for which they expected to remain on therapy : 44.7% did not know for how long, 43.2% indicated as long as their doctor keeps prescribing it, 5.0% believed it to be as long as they remain well enough to attend their centre to receive it, and 6.4% did not respond. these response patterns were similar for breast cancer and prostate cancer patients with a few small differences noted (table 1). among prostate cancer patients, the most commonly administered agent was denosumab (63.1%, as well as an additional 5.3% who in the past had received denosumab), followed by zoledronic acid (19.7%) (fig. overall 44.7% of patients received their treatment at the hospital chemotherapy unit, while 27% received treatment via injections administered at home by a nurse or another individual (fig. a total of 31 patients (40.8% of all prostate cancer patients, or 45.6% of those responding to the related question) reported being aware of potential side effects of their treatment. according to patients descriptions, the potential side effects of bone targeted agents were ; diarrhoea, constipation, vomiting, fatigue, flu - like symptoms, osteonecrosis of the jaw, hypocalcaemia, bone pain and water retention. within the breast cancer cohort, the most commonly used agent was pamidronate (87.7%), followed by zoledronic acid (9.2% ; fig. 1). overall, 93.8% of patients reported receiving their treatments in the chemotherapy unit at their hospital (fig. 2). overall, 34 patients (52.3%) were receiving their treatment every 3 months (fig. 3). a total of 28 patients (43.1%) reported awareness of potential side effects, similar to those described by prostate cancer patients. the most commonly reported reasons were : to reduce the chance of bone fractures due to cancer (78.0%), to slow the growth of cancer (53.2%), to reduce the chance of spinal damage (49.0%), to prolong survival (46.8%), and to reduce the chance of cancer pain (46.1%) (fig. fewer patients indicated that it was related to prevent the need for radiotherapy / surgery to bones (29.8%) or to the management of hypercalcaemia (24.1%). the majority of respondents with prostate cancer (78.9% overall, or 93.5% of those that responded to the related question) and breast cancer (75.4% overall, or 87.3% of those that responded to the related question) would be willing to take a reduced dose of their medication if it were associated with the same clinical benefits as their current dose. similarly, the majority of respondents with prostate (67.1% overall, or 79.7% of those responding to the related question) and breast cancer (69.2% overall, or 88% of those responding to the related question) indicated they would be happy taking the medication less often (i.e. leaving the dose unchanged but receiving it less frequently) if the clinical benefits were unchanged. eighteen and a half percent and 30.3% of breast and prostate cancer respondents, respectively, indicated a lack of interest in changing from their current 34 week regimen. if reducing the dose would lead to fewer side effects, 64.6% of prostate cancer and 46.1% of breast cancer patients indicated an interest in taking a reduced medication schedule and dose. when asked if they would prefer a daily oral medicine or an injection in the hospital every 13 months, 21.1% of prostate cancer patients and 23.5% of breast cancer patients indicated a preference for taking oral medication daily, while 57.9% and 49.2% preferred an injection every 13 months. overall, 39.2% of prostate cancer patients and 35.4% of breast cancer patients indicated a willingness to administer their own medication via an injection. when asked if they would be willing to enter a trial randomising between monthly and 3 monthly therapy, 64.6% of prostate patients and 58.2% of breast cancer patients indicated a willingness to participate in such a study. a total of 141 patients responded, and included totals of 65 (46.1%) and 76 (53.9%) patients diagnosed with breast cancer and prostate cancer, respectively (table 1). a total of 100% of breast cancer patients and 94.7% of prostate cancer patients were aware that they had received treatment with a bone - targeted agent at some point. respondents had received bone - targeted agents for : 24 months (19.1%) ; 4.3% of patients did not respond. patients varied in the amount of time for which they expected to remain on therapy : 44.7% did not know for how long, 43.2% indicated as long as their doctor keeps prescribing it, 5.0% believed it to be as long as they remain well enough to attend their centre to receive it, and 6.4% did not respond. these response patterns were similar for breast cancer and prostate cancer patients with a few small differences noted (table 1). among prostate cancer patients, the most commonly administered agent was denosumab (63.1%, as well as an additional 5.3% who in the past had received denosumab), followed by zoledronic acid (19.7%) (fig. overall 44.7% of patients received their treatment at the hospital chemotherapy unit, while 27% received treatment via injections administered at home by a nurse or another individual (fig. a total of 31 patients (40.8% of all prostate cancer patients, or 45.6% of those responding to the related question) reported being aware of potential side effects of their treatment. according to patients descriptions, the potential side effects of bone targeted agents were ; diarrhoea, constipation, vomiting, fatigue, flu - like symptoms, osteonecrosis of the jaw, hypocalcaemia, bone pain and water retention. within the breast cancer cohort, the most commonly used agent was pamidronate (87.7%), followed by zoledronic acid (9.2% ; fig. 1). overall, 93.8% of patients reported receiving their treatments in the chemotherapy unit at their hospital (fig. 2). overall, 34 patients (52.3%) were receiving their treatment every 3 months (fig. 3). a total of 28 patients (43.1%) reported awareness of potential side effects, similar to those described by prostate cancer patients. the most commonly reported reasons were : to reduce the chance of bone fractures due to cancer (78.0%), to slow the growth of cancer (53.2%), to reduce the chance of spinal damage (49.0%), to prolong survival (46.8%), and to reduce the chance of cancer pain (46.1%) (fig. fewer patients indicated that it was related to prevent the need for radiotherapy / surgery to bones (29.8%) or to the management of hypercalcaemia (24.1%). the majority of respondents with prostate cancer (78.9% overall, or 93.5% of those that responded to the related question) and breast cancer (75.4% overall, or 87.3% of those that responded to the related question) would be willing to take a reduced dose of their medication if it were associated with the same clinical benefits as their current dose. similarly, the majority of respondents with prostate (67.1% overall, or 79.7% of those responding to the related question) and breast cancer (69.2% overall, or 88% of those responding to the related question) indicated they would be happy taking the medication less often (i.e. leaving the dose unchanged but receiving it less frequently) if the clinical benefits were unchanged. eighteen and a half percent and 30.3% of breast and prostate cancer respondents, respectively, indicated a lack of interest in changing from their current 34 week regimen. if reducing the dose would lead to fewer side effects, 64.6% of prostate cancer and 46.1% of breast cancer patients indicated an interest in taking a reduced medication schedule and dose. when asked if they would prefer a daily oral medicine or an injection in the hospital every 13 months, 21.1% of prostate cancer patients and 23.5% of breast cancer patients indicated a preference for taking oral medication daily, while 57.9% and 49.2% preferred an injection every 13 months. overall, 39.2% of prostate cancer patients and 35.4% of breast cancer patients indicated a willingness to administer their own medication via an injection. when asked if they would be willing to enter a trial randomising between monthly and 3 monthly therapy, 64.6% of prostate patients and 58.2% of breast cancer patients indicated a willingness to participate in such a study. despite the widespread use of bone - targeted agents in the care of patients with bone metastases from prostate and breast cancer, a number of important and practical clinical questions remain around optimising their use. in order to design and perform pragmatic clinical trials in these populations, it is important to obtain patient treatment information, perceptions and opinions that will help guide trial design to ensure sufficient participation. this is of particular interest for studies evaluating treatment de - escalation where it is critical to ascertain patient 's willingness to participate. to our knowledge, we are the first team to implement use of this type of survey in these patient populations, although a number of de - escalation studies are either reported,,, or are ongoing. our survey demonstrates that canadian breast cancer patients are more likely to receive pamidronate, whereas prostate cancer patients are more likely to receive either denosumab or zoledronic acid. these differences reflect not only that the drugs proven to be efficacious in these populations are different (i.e. only denosumab and zoledronic acid improve outcomes in prostate cancer), but also that the funding system for these agents in ontario differs. any trial looking at de - escalated therapy will need to take these factors into account. our findings also suggest that clinical trials evaluating single agents such as zoledronic acid or denosumab,, will not lead to broad changes in clinical practice in both disease sites. in addition, for prostate cancer patients, such a trial will need to reflect the fact that a significant number of patients have switched from zoledronic acid to denosumab when funding for denosumab became available, thus future studies may have to allow for bisphosphonate use prior to denosumab treatment in the inclusion criteria. patients in our survey population had been on bone - targeted agents for variable periods of time, again meaning that future trials will need to be reflective of this, and be flexible in the length of time a patient has been on a prior bone - targeted agent prior to study entry. to date, of the published trials,, only one did not specify the maximum length a patient could have been on a prior agent. interestingly, many patients perceived that the bone - targeted agent was slowing the growth of their cancer. given that these agents have shown no effect on either overall survival or progression - free survival in breast cancer,,,,., or prostate cancer,, there may be a need to better inform patients of this fact. preference for injectable therapy over oral therapy, contrary to our findings from a previous study, could reflect the fact that most patients have not received an oral bone - targeted agent for metastatic disease. however, the practical advantages of an injectable therapy and the lack of need for extra - care (taking the medication with water, not laying down shortly after po administration) in an elderly patient population should not be ovelooked. our sample was recruited from two cancer centres in ontario, canada and therefore reflects prescriptions practices in that province. in addition, as the reb requirement was to allow no patient identifiers on the questionnaires, we could not tell how common it was for patients to refuse participation (i.e. in place of a denominator for response rate, which we do not have). as a consequence, it is possible that our responding cohort consists of better informed, more motivated patients which may not be representative of all patients receiving treatment with bone targeted agents. this too could be reflected by the large number of breast cancer patients who are already receiving 3-monthly bone - targeted therapy. in an era of personalised medicine, involvement of patients in the design of future clinical trials is increasingly important. although our results suggest that patients still have misconceptions around the reasons for receiving bone - targeted agents, the similarities between prostate and breast cancer patients are interesting as both patient populations were aware of the main indications and toxicities of these agents. however, given that patients receive a range of agents for varying periods of time and in different locations (hospital vs. home) means that the design of future trials will need to reflect this and be very pragmatic. | backgroundin order to design studies assessing the optimal use of bone - targeted agents (btas) patient input is clearly desirable.methodspatients who were receiving a bta for metastatic prostate or breast cancer were surveyed at two canadian cancer centres. statistical analysis of respondent data was performed to establish relevant proportions of patient responses.resultsresponses were received from 141 patients, 76 (53.9%) with prostate cancer and 65 (46.1%) with breast cancer. duration of bta use was 24 months (35.2%). patients were uncertain how long they would remain on a bta. while most felt their bta was given to reduce the chance of bone fractures (77%), 52% thought it would slow tumour growth. prostate patients were more likely to receive denosumab and breast cancer patients, pamidronate. there was more variability in the dosing interval for breast cancer patients. given a choice, most patients (4957%) would prefer injection therapy to oral therapy (2123%). most patients (5864%) were interested in enrolling in clinical trials of de - escalated therapy.conclusionwhile there were clear differences in the types of btas patients received, our survey showed similarity for both prostate and breast cancer patients with respect to their perceptions of the goals of therapy. patients were interested in participating in trials of de - escalated therapy. however, given that patients receive a range of agents for varying periods of time and in different locations (e.g. hospital vs. home), the design of future trials will need to be pragmatic to reflect this. |
the residual and resistant microorganisms have been recognized as the basic reason for failures in root canal treatments. fungi, chemoorganotrophic eukaryotic microorganisms, may play a key part in periradicular diseases. while they have been found sporadically in initial root canal infections, failure of some endodontic treatments may therefore be explained by the presence of candida albicans in the oral flora. it is reported that in 20% of endodontic treatment failures, c. albicans is present. candida species, which have a tendency to invade dentin, are regarded as dentinophilic microorganisms. the presence of c. albicans in endodontic treatment failures may be the result of coronal leakage after, or contamination during, the endodontic treatment. several factors, such as an ability to survive despite a lack of nutrients or resistance to intracanalicular treatment materials, may result in c. albicans remaining in the root canal system. c. albicans has been found in some failed endodontic treatments ; a phenomenon that might be explained by the invasive nature of this fungus or its resistance to some intracanalicular medications, e.g., calcium hydroxide. if repeated treatment has either failed or is unfeasible, the only alternative is a root - end resection. apical sealing, avoiding contamination, and reducing the number of microorganisms, including bacteria and fungi, influence the success rate of such treatment. therefore, the antibacterial and antifungal properties of root - end filling materials are important to the outcome of these procedures. mineral trioxide aggregate (mta) was introduced in 1993 and, after different examinations, approved, in 1998, by the us food and drug administration as an endodontic biomaterial, used in root - end filling, vital pulp capping, apexification, and root or furcal perforation repair.[58 ] mta is marketed as gray and white. however, because of the tooth - discoloring properties of gray mta, white mta has been pushed to replace it. mta has a number of advantages, including excellent sealing ability, biocompatibility, high alkalinity, radiopacity, antimicrobial effects, and resistance to fluid and blood contamination.[911 ] in spite of having disadvantages, such as delayed setting time, poor handling properties, and high price, mta, because of its physical and chemical properties, has become the gold standard of root - end filling materials. mta - angelus particles are not as homogenous as proroot mta. in comparison to proroot mta, mta - angelus shows several advantages, such as good marginal adaptation, good sealing, sealing ability in mineralized tissues with complete closure, and has been reported to be inflammation - free in most cases.[1517 ] mta - angelus has an excellent antibacterial effect and a strong fungicidal effect against c. albicans. while both the white and gray mta - angelus have arsenic in their structures, white mta - angelus has the benefit of containing arsenic levels below the limit set by the iso 9917 - 1 standard. a limited number of researches have compared the antifungal effects of proroot mta and mta - angelus. however, such studies have been conducted using agar diffusion test (adt), a method that does not seem appropriate because of low solubility and diffusibility of mta. the aim of this in vitro study was to evaluate and compare the antifungal properties of fresh and set proroot mta and mta - angelus, in concentrations of 50 mg / ml and 100 mg / ml, against c. albicans at 1-, 24-, and 48-hour time points. the null hypothesis of this study was that there is no difference in the in vitro antifungal properties of these two materials. the antifungal activity of proroot mta (dentsply tulsa dental, tulsa, usa) as well as mta - angelus (angelus, londrina, brazil) was evaluated against c. albicans. the culture of sabouraud dextrose agar (merck, darmstadt, germany) was prepared according to the manufacturer 's instructions with a definite ratio of powder to liquid. samples of live c. albicans, (atcc 10231) provided by scientific research organization, (tehran, iran) were subcultured on the sabouraud dextrose agar plates. the agar plates, containing the fungus, were then maintained at 37c. times and conditions for the culture of c. albicans in this study were determined following the nccls standards (national committee on clinical laboratory standards). a total of 50 culture wells were divided into four experimental groups (freshly mixed mta, freshly mixed mta - angelus, 24-h set mta, 24-h set mta - angelus) and two control groups (positive control, negative control). the root canal filling materials were prepared according to their instructions and then mixed with the appropriate volume of sterile water to reach the concentrations of 50mg / ml or 100mg / ml. for the set groups, the mixture was prepared and left for 24 hours. each group consisted of 10 wells, and the control groups consisted of five wells each. suspensions were then made by the addition of several fungal colonies from the surface of the sabouraud dextrose agar plates to sabouraud dextrose broth media. a final concentration of 10 cfu / ml (colony forming unit / milliliter) was achieved, as recommended by the nccls, via microscopic cell counting. wells with 1 ml of c. albicans suspension mixed and 1 ml of sabouraud dextrose broth media without root end - filling materials served as positive control and wells with 1 ml of sabouraud dextrose broth media without c. albicans served as negative control. all wells were incubated at 37c and evaluated for 1, 24, and 48 hours by an independent observer by assessing the colonies of c. albicans in each well. this assessment was based on the turbidity of the suspension in the tubes. at each time point, 0.02 ml of each suspension was cultured on sabouraud dextrose agar plate to confirm c. albicans growth. after the culture of 0.02ml of the treated suspension on the agar plates, the fungal growth was assessed at 1-, 24-, and 48-hour time points. while fungal growth was not observed in any of the negative control groups, after 1 hour, both the mta and mta - angelus were incapable of killing c. albicans. the results were the same for both freshly mixed and set samples at both concentrations (50 and 100 mg / ml). increasing the incubation time to 24 hours and both materials were fungicidal for c. albicans at both concentrations of 50 and 100 mg / ml, and in both states of fresh and set [table 1 ]. comparison of proroot mta and mta - angelus in concentration of 50 mg / ml and 100 mg / ml at 1, 24, and 48-h time intervals there was no significant difference between using the two materials at either of the concentrations or either of the 1-hour, 24-hour, or 48-hour time points (p<0.05). in this study, the antifungal properties of proroot mta and mta - angelus against c. albicans in two concentrations and in fresh and set situations were compared by using tube - dilution test. because the two materials elicited the same reaction against c. albicans in different conditions, the null hypothesis that there is no difference in the in vitro properties of these two materials has to be accepted. the most common method for assessing the antifungal or antibacterial activity of root - end filling materials however, several parameters, such as lack of standardization of inoculum density, adequate culture medium, agar viscosity, plate storage condition, size and number of specimens per plate, time and temperature of incubation, and reading point of the inhibition zones, can affect the results of the adt. the method used in this in vitro study was the tube - dilution susceptibility test ; an efficient method for evaluating antifungal characteristics of materials. in this technique, direct contact between c. albicans and the experimental materials was allowed, allowing a more realistic interaction. because of possible changes in the chemical structure of the products, root - end filling materials should be tested in two situations ; one where the material is used immediately after mixing, and another, where it is used after having been allowed to reach its final chemical composition, or set state. in this study, proroot mta and mta - angelus were tested in freshly mixed and set states, to mimic clinical use of the materials. after setting, consequently, the perceived difference in antifungal patterns of the materials may be a result of the differences in the degree of setting. however, in our study, the results were similar in the two different states for all tested materials. based on nccls standards, the results of this study showed that c. albicans was not affected by the freshly mixed and set, proroot mta and mta - angelus, at 1 hour. however, these materials were effective in eliminating c. albicans by the 24-h time point. some studies have reported the inhibitory effect of proroot mta and mta - angelus on c. albicans by determining the diameter of the inhibition zones in millimeters, and have resulted conclusions similar to this study. the presence of calcium hydroxide, produced by a hydration reaction from the mixing of mta with water, and an increase in ph, as a result of the release of hydroxyl ions, is probably the mechanism of the antimicrobial activity of mta - angelus. they suggested that the presence of calcium hydroxide is capable of, initially, reducing and, ultimately, eliminating the fungus. it is important to mention that the results presented in this study were obtained under in vitro conditions, and may not accurately describe in vivo activity. further studies are necessary to investigate the effect of these materials on bacteria and fungi in clinical application. this study shows that mta - angelus has an effective antifungal property comparable to proroot mta at concentrations of 50 and 100 mg / ml. | background : fungi may play a key part in periradicular diseases. the aim of this study was to evaluate and compare the antifungal properties of two root - end filling materials, proroot mineral trioxide aggregate (mta) and mta - angelus, against candida albicans using tube - dilution test.materials and methods : the antifungal properties of proroot mta and mta - angelus against c. albicans was assessed at 1, 24, and 48 hours following administration of two concentrations of the antifungal agents (50 and 100 mg / ml). a total of 50 culture wells were divided into four experimental groups (freshly mixed mta, freshly mixed mta - angelus, 24-h set mta, and 24-h set mta - angelus) and two control groups. each well was prepared for one specific agent with a specific concentration. for the set groups, the mixture was prepared and left for 24 hours. one milliliter of suspension of fungal colonies with concentration of 104 cfu / ml was then added to the mixtures in each well. all wells were incubated at 37c and assessed at 1, 24, and 48 hours. this observation was based on the turbidity of the suspension in the tubes. at each time point, 0.02 ml of each suspension was cultured on a sabouraud dextrose agar plate to confirm c. albicans growth. the results were analyzed using kruskal - wallis test.results:although all fresh and set samples were incapable of killing c. albicans at 1 hour, they demonstrated fungicidal ability on agar plates at 24 and 48-hour time points.conclusion:mta-angelus proved to be an effective antifungal agent compared to proroot mta at concentrations of 50 mg / ml and 100 mg / ml. |
multiple sclerosis is a chronic inflammatory primary demyelinating disease of the central nervous system of unknown etiology ; as a result curative therapies have not yet been described [1, 2 ]. however, in the last two decades, after the discovery and approval of immunomodulators which specifically modify the natural history of the disease, as such denominated disease - modifying drugs (dmds), most ms patients have been worldwide subjected to this therapy. interferon beta (ifn) formulations and glatiramer acetate (ga) are the first - line dmd for ms. ifn is available in five distinct formulations : ifn-1b (betaferon / extavia), ifn-1a intramuscular (avonex), and ifn-1a subcutaneous (rebif). these dmds were firstly approved to the relapsing - remitting form of ms (rrms), later for clinically isolated syndromes and ifn-1b for secondary progressive ms (spms). first - line dmds for ms were introduced in portugal in 1996, after the licensing of betaferon for rrms. the other formulations of ifn for rrms were introduced later : avonex in 1997 and rebif in 1998. still, copaxone was introduced in 2001. in the last years the therapeutic armamentarium for ms was enlarged, with the approval of drugs with better efficacy yet potential limiting adverse effects, as mitoxantrone (novantrone), natalizumab (tysabri), and more recently the oral drug fingolimod (gilenya). together, these drugs constitute the second - line treatment for ms and they are usually indicated in more severe non - ifn-responder cases [1, 2 ]. first - line dmds target multiple neuroimmunological mechanisms implied in the pathogenesis of ms, yet their therapeutic effects are rather modest, mainly traduced by a 30% reduction in the relapse rate and a decrease of magnetic resonance imaging (mri) t2 lesion burden and gadolinium - enhanced lesions [1, 2 ] ; unfortunately, the benefit effects in disability at long - term remain to be settled. these drugs are usually safe and well tolerated ; however, they are not free from side effects. failure of the first prescribed dmd was found to occur in 30% of patients usually within 3 years. therefore, recommendations for escalating therapy were established and successively reviewed by international consensus. switch to another first- or second - line dmd is common and occurs in a median time of 2.9 years after the initially prescribed dmd ; overall, approximately half of ms patients discontinue the use of their dmd within 6 years. even so, ms neurologists often recognize that, in the clinical setting, some patients maintain the initial treatment for a long period with clinical stability, low disability, and nonsignificant side effects. concerning this issue, which is poorly addressed in the literature, we aim to describe the demographic and clinical characteristics of mspatients followed in our ms clinic with long - term(>12 years) maintenance of the first prescribed dmd. from the database of our ms clinic regarding patients with the definite diagnosis of rrms (revised mcdonald criteria 2005), who started a first - line dmd between 1996 and 1999 (n = 51), we identified those who were still receiving the initial therapy in march 2012 (cut - off date). inclusion criteria were definite ms diagnosis ; rr course at the moment of dmd prescription ; age 18 years ; maintain treatment with first dmd. exclusion criteria were other ms course other than rr when starting dmd ; age 12 years (mean = 14 years, range 1316) (table 1). fourteen patients (54%) were treated with betaferon, 8 (31%) with avonex and 4 (15%) with rebif (figure 1). no serious adverse effects were reported. concerning gender distribution, 18 patients (69%) were females and 8 males (ratio 2.3 : 1). first symptoms began in the mean age of 31 years (range 1648), with a mean disease duration of 18.6 years (range 1332) until march 2012. regarding clinical presentation, motor symptoms were the most common, occurring in 11 patients (42%) ; brainstem occurred in 8 (31%), sensory in 4 patients (15%), optic neuritis in 2 (8%), and cerebellar in 1 (4%) (figure 2). median edss when starting treatment and currently was 1.5 (range 06) and 4 (range 06), respectively ; present mean msss is 2.56 (range 0.105.15). regarding pregnancy, only two women get pregnant during dmd treatment, both under avonex. the remaining women, except one, already had children at the time of starting dmd. the management of patients with chronic diseases, as ms, is a major challenge to the physicians. in this sense, the growing appearance of dmd specifically developed for ms with different degrees of efficacy, represents a huge step forward in the management of these patients. however, the success of healthcare relies not only on effective therapies, but also on adherence and persistence. in ms, there is evidence that dmd must be given as early as possible, usually with a first - line drug. switch to another first - line dmd or escalating therapy to a second - line drug is common, mainly because of lack of efficacy or intolerance. open - label studies found that approximately 30% of ms patients discontinue therapy within 3years due to disease activity ; within 6 years, approximately half of the patients discontinue their dmd. in particular, some authors found that 15% and 25% of patients stopped taking the initial dmd after 6 months and 1 year of treatment, respectively ; overall, 66% were no longer taking the original dmd at some point of the disease course.. concluded that patients with a longer disease duration and higher level of disability are at higher risk for discontinuing dmd ; age, sex, and the initial dmd were not associated to discontinuation. as far as ms is better understood and new drugs with higher efficacy are regularly licensed, the clinicians are concerned with defining better escalating regimens, aiming that patients become free from disease activity (no relapses, no progression, and no imagiological activity) ; consequently, the literature data are mainly focused on those patients who experience a worse treatment response or clinical progression. on the contrary, patients that have an optimal response to the initial prescribed dmd are, apparently, subject of less interest, although this is a crucial issue in order to establish patterns of good responders. in fact, data from 260 patients showed that 30% are taking ifn-1b after a median length of exposure of almost 10 years. this heterogeneity of treatment response to dmd in ms has beenevaluated by several studies [810 ]. among dmds, responders seem to be older and to have longer disease duration at the time ifn was initiated. among patients with a rr course, also, responders seem to have a higher edss at initiation of ifn. a probable role of molecular biologic mechanisms contributing to the variable therapeutic response in ms patients has been raised. for example, cucci. demonstrated that proinflammatory cytokine and chemokine mrna blood level, in ms are related to treatment response. although dmd may reduce the clinical relapse rate, their potential benefits in terms of disability progression may be small. nevertheless, a favorable treatment response leads the patient and clinician to an expectation of a better prognosis. a multiplicity of variables have been associated with favorable disease course, such as female gender, younger age at onset, sensory symptoms or optic neuritis at onset, and monosymptomatic presentation. in contrast, male gender ; onset with motor, sphincter, or cerebellar features ; poor recovery from initial or early attacks ; higher attack rate in the first 5 years ; a progressive course were prognostic variables associated with a poor outcome [1315 ]. in this study, we found a high percentage of patients who never needed to switch or escalate therapy, some of them treated with the initial prescribed dmd as long as 16 years. interestingly, our patients had a good outcome despite preponderance of motor and brainstem symptoms at presentation, usually associated with worse prognosis as already stated. following the same reasoning, we expected a higher prevalence of sensory symptoms or optic neuritis presentation which in fact occurred only in a minority of our patients. also, as elsewhere stated, patients who maintain a rr disease course tend to be extremely well and it is only on entering the secondary progressive course of the disease that more rapid disability progression occurs. so, it is possible that our cohort had a good outcome in terms of disability, not only because of the treatment, but also because only patients with rr course (a favorable clinical form) entered the study. we also observed a low edss score and a low number of relapses, in agreement with ramsaransing and de keyser who found these clinical factors to be predictive of a good outcome at 10 years. another explanationfor ourresultscan berelatedwith a good treatment adherence, defined as the duration of time from initiation to discontinuation of therapy ; although we have not collected this data in the present cohort, prior results obtained in a larger series of our ms clinic showed a very low rate of patient 's dmd abandon (4.6%), which may be related to the organization model of our ms consultation, as stressed by castro.. this model is based on an open and multidisciplinary care, involving several ms dedicated human resources, namely, neurologists, urologists, psychiatrists, neuropsychologists, social worker, and nurses. there is a personalized training in initial drug injections, complemented with extensive verbal and written information at that time. further, our ms clinic provides a permanent (except during night) full access to a ms specialized neurologist. in case of doubts or new symptoms, patients are instructed to access this consultation, instead of emergency room, where misinterpretation of the symptoms is more frequent, particularly when neurologist is not available. our small cohort is an obvious limitation of the study ; however, it would be rather difficult to find a large number of rrms patients on a single dmd for such a long period of time. we are also aware of methodological limitations inherent to observational, retrospectivestudies, such asthe absence of standardizedprotocol. yet we found a high percentage of patients who never needed to switch or escalate therapy, who were mainly females, with low arr, edss, and msss after long disease duration. identifying characteristicsassociated with therapy persistencemay beuseful in developingstrategies to improve therapy effectiveness. | although therapy switch is common among patients with multiple sclerosis (ms), sometimes the initial prescribed treatment is maintained for a long period with clinical stability, low disability, and nonsignificant side effects. we aim to describe demographic and clinical characteristics of patients treated in our ms clinic with the same disease - modifying drug (dmd) lasting for > 12 years. from the cohort of 51 patients followed in our ms clinic with relapse - remitting ms who started an dmd between 1996 and 1999, we found a high percentage (51%) of patients who were efficiently treated with the first dmd. these patients were mainly females, with low annualized relapse rate and multiple sclerosis severity score (msss). our results may be related to the open and multidisciplinary model of our ms clinic organization. identifying characteristics associated with therapy persistence may be useful in developing strategies to improve therapy effectiveness. |
coronary artery bypass graft (cabg) surgery is one of the most commonly performed surgical procedures worldwide, intended to treat ischemic heart disease and alleviate angina pectoris.13 despite advances in the surgical procedure, neurological injury is still an important complication after cabg surgery and is found in two types.4 the first type of neurological injury occurs in 3%6% of the cases with manifestations such as stroke, transient ischemic attack, and coma. on the other hand, impairment of cognitive function including defects of attention, concentration, short - term memory, fine motor function, and speed of mental and motor responses is found in the milder type second,4,5 with an incidence of 20%80%, neurocognitive impairment after cardiac surgery has been largely attributed to the use of cardiopulmonary bypass (cpb).69 cpb increases the permeability of the blood brain barrier and generates microemboli, which may affect cognitive function.10,11 recently, cardiac stabilizer devices have been developed to facilitate cabg without using cpb (off - pump cabg surgery). although diminished cerebral embolism in off - pump cabg has been highlighted,12 it is still controversial whether the switch from on - pump to off - pump technique fully alleviates the cognitive dysfunction. minor improvements in cognitive function shortly after operation,4,12,13 better cognitive outcome,14,15 and no improvement in neurocognitive status after off - pump cabg have been hitherto reported in off - pump cabg in comparison to the on - pump method.16 due to the aforementioned controversy, the present study was designed to compare the impact of the on - pump and off - pump techniques on neurocognitive impairment in low - risk cabg groups. in a descriptive, analytic, and prospective follow - up study, 201 candidates for cabg surgery were enrolled at madani heart hospital in tabriz, iran. inclusion criteria were elective cabg, lack of concomitant cardiac arrhythmia, and left - ventricular ejection fraction (lvef) > 30%. patients undergoing emergency cabg or cabg combined with other surgical procedures such as valve replacement or carotid endarterectomy and those with history of neurological and psychiatric diseases were excluded from the study. before operation, all patients underwent neurological and neurocognitive (mini - mental state examination, mmse) examination. follow - up study was performed up to 2 months after operation by neurological and neurocognitive examination. statistical analysis was performed with spss for windows (version 13.0 ; spss inc., chicago, il) using independent samples t - test, paired samples t - test, and chi - squared test, whenever appropriate. out of 201 patients enrolled in the study, 47 patients refused or were unable to return for follow - up. data from 154 patients were therefore analyzed : 95 (61.6%) in off - pump group and 59 (38.3%) in on - pump group. out of 154 patients, 123 (79.8%) were male and 31 (20.1%) were female. two patients in on - pump group (2.5%) and nine patients in off - pump group (7.4%) had postoperative myocardial infarction (p > 0.05). in each group, there were two postoperative strokes. there was only one death in off - pump group ; the cause was severe cardiac disease and postoperative cardiac arrest, and no neurological complication was found. in on - pump group, there was no significant difference between the mean preoperative and postoperative mmse scores (23.24 5.03 versus 23.73 4.88, p = 0.16). however, in off - pump group, the mean 2-month postoperative mmse score was significantly higher than the mean preoperative mmse score (25.01 4.49 versus 24.18 4.51, p = 0.007). on the basis of the mmse results, cognitive impairment was detected in 21.2% of the cases in on - pump group and 23.1% in off - pump (p = 0.54, table 1). the results of this study showed no significant difference in postoperative neurocognitive impairment between on - pump and off - pump low - risk cabg surgery groups. this is in agreement with the findings of some previous studies that failed to detect any difference in neurocognitive impairment between on- and off - pump techniques.1620 jensen recently found similar cognitive outcome 3 months after off - pump and on - pump surgery in a group of elderly with high risk for cabg surgery.18 interestingly, ernest revealed no difference in neurocognitive state at 2 and 6 months following cabg surgery between both groups, with the exception that fewer off - pump patients showed impairment on verbal fluency at 6 months.20 in contrast to the findings of the present study, some investigations have demonstrated favorable results for patients who underwent off - pump cabg surgery. murkin and zamvar reported less neurocognitive impairment among off - pump patients at first week postoperatively, while motallebzadeh found better neurocognitive function at discharge from hospital but no difference at 6 months postoperatively.4 stroobant concluded contrarily, ie, no difference at discharge but better function at 6 months postoperatively for off - pump group.22 however, van dijk showed that low - risk patients avoiding the use of cpb had no effect on 5-year cognitive outcomes.23 altogether, such a matter of controversy in neurocognitive impairment between on- and off - pump cabg surgery has been attributed to the variations in the tests used, the time points of assessment, the definition of impairment, and the statistical methods used for comparing groups.4,24 on the other hand, factors other than cpb may be responsible for cognitive decline, such as anesthesia and the generalized inflammatory response that is associated with major surgical procedures.4,25 the present study has several limitations. first, assessment of neurocognitive function was done only by one neuropsychological test (mmse) and 1 score decline in mmse was defined as cognitive impairment. second, mmse is a simple screening instrument which might not be reliable in detection of subtle cognitive impairment. this might explain quite low preoperative mmse scores in the present study.26 fourth, the results of this investigation can not be extrapolated to older patients with more advanced coronary artery disease or higher preoperative risks. on the other hand, large sample size along with the follow - up rate of 76% which is comparable to the previous studies is among the advantages of the current study.4,7,27 on the basis of these findings, we conclude that in low - risk patients undergoing cabg surgery, avoiding the use of cpb had no effect on cognitive outcome 2 months after the procedure. | backgroundcoronary artery bypass graft (cabg) surgery is one of the most commonly performed surgical procedures worldwide, and it may be accompanied by postoperative neurocognitive impairment. although this complication has been attributed to the use of cardiopulmonary bypass, it is still a matter of debate whether the switch from on - pump to off - pump technique affects the cognitive function.objectivethe aim of this study was to compare the impact of the on - pump and off - pump techniques on neurocognitive impairment in low - risk cabg surgery groups.methodsin a descriptive and analytic study, 201 cabg patients with left - ventricular ejection fraction > 30%, and without cardiac arrhythmia were enrolled. before the elective operation, all patients underwent neurological examination and neurocognitive test, mini - mental state examination (mmse). two months following the operation, both on- and off - pump, the patients were re - examined by mmse to detect any neurocognitive impairment.resultsout of 154 patients included in the study, 95 (61.6%) and 59 (38.3%) patients were in off - pump and on - pump groups, respectively. mean age of the patients was 57.17 9.82 years. a 2-month postoperative neurocognitive impairment was detected among 17 patients of on - pump group (28.8%) and in 28 cases of off - pump group (29.4%) (p = 0.54). the mean postoperative mmse scores were not comparable between groups (25.01 4.49 in off - pump group versus 23.73 4.88 in on - pump group, p = 0.09).conclusionthe present study revealed that in low - risk patients undergoing cabg surgery, either the techniques of on - pump or off - pump did not differ regarding the neurocognitive outcome 2 months after the procedure. |
among the general population, it has been assumed that an average of 150 to 250 g of lead is ingested through food each day, five to ten percent of which is absorbed (1, 2). the other sources for lead poisoning may be lead - soldered kettles, cans, and lead - glazed pottery, which release lead when acidic fluids are stored or cooked in them (2). the lead in blood is equilibrated with that of lead in tissues, including many potential target organs such as the brain and kidney. the kinetic model of lead in the body is not defined well, but usually it consists of two pools : in the blood and in the skeleton, which may be divided further into labile and deep pools (3). thus, lead burdens have a tendency to be accumulated in the patients with renal failure (4). the skeleton acts as a reservoir of lead, and it may be mobilized by physiological and pathological states including pregnancy, lactation, and osteoporosis (5, 6). in patients with end - stage renal disease (esrd), hyperparathyroidism, abnormal vitamin d metabolism, and consequent osteoporosis have frequently been observed (7). taken together with the lack of renal excretion and stimulated bone resorption, the blood level of lead seems to increase in esrd and may aggravate uremic symptoms such as peripheral neuropathy and anemia. the classic clinical manifestations of industrial lead poisoning include colic, anemia, peripheral neuropathy, encephalopathy, renal impairment, hypertension, and reproductive disability (8). more recently, recognition has become widespread that, in addition to its clinically evident toxicity, lead also causes a spectrum of adverse effects at levels of exposure insufficient to produce obvious signs and symptoms. thus, clinically obvious manifestations of lead poisoning such as anemia, peripheral neuropathy, and renal failure lie at the upper end of the range of toxicity, while such covert effects as impaired synthesis of heme, altered excretion of uric acid, and slowed nerve conduction are their subclinical correlate (9). diseases of the peripheral nervous system are among the most prevalent neurologic conditions in esrd (10). nonetheless, differential diagnosis of peripheral nerve disease is challenging because the catalogue of disorders that can produce neuropathies is extensive in esrd. first of all, many patients with renal failure develop electrophysiologic evidence of peripheral neuropathy (11). the primary pathophysiology of uremic neuropathy remains unknown. according to one theory (12), some slowly dializable substances of intermediate molecular weight alter the peripheral nerve in some manner, resulting in axonal dysfunction and eventually degeneration. the other causes of the peripheral neuropathy in esrd are diabetes mellitus, pharmaceutic neurotoxins, alcohol - nutritional neuropathy, and industrial and environmental neurotoxins such as lead, mercury, arsenic, and thallium. although many investigators have described increased blood levels of lead in renal failure patients (13 - 16), the role of lead on the clinical characteristics remains to be elucidated. the purpose of this study is to determine the blood concentration of lead in patients with end - stage renal disease and to validate the relation with peripheral motor and sensory nerve conduction velocity as clinical correlates. one hundred ninety - eight healthy subjects (control group, 147 men, 51 women ; mean age, 43.211.5 yr) and 68 patients with esrd undergoing hemodialysis (esrd group, 31 men, 37 women ; mean age, 50.112.3 yr) were enrolled for the blood lead measurement. the control group comprised healthy volunteers recruited during a regular physical examination at soonchunhyang university health promotion center (cheonan, korea). blood samples for lead concentration were drawn from the antecubital vein in the morning after overnight fasting. the esrd patients were undergoing regular hemodialysis eight to twelve hours weekly using a cellulose acetate hollow - fiber dialyser (surface area 1.2 m ; gambro, sweden, or baxter, u.s.a.) at a hemodialysis unit of the soonchunhyang cheonan hospital. in order to avoid any influence of carpal tunnel syndrome and/or ischemic neuropathy on the result of peripheral nerve conduction study, any patients who had vascular access on the right arm and had past histories of cerebrovascular disease, with or without hemiparesis, were excluded from this study. among 124 esrd patients on hemodialysis, 68 cases were consistent with these terms. this study was approved by the institutional review board of soonchunhyang cheonan hospital (cheonan, korea), and all human subjects provided written informed consent nerve conduction was measured within two hours after hemodialysis. the upper limb temperatures were maintained above 34 and lower limb temperatures above 32, using hot packs when necessary. orthodromic sensory nerve action potentials and compound muscle action potentials were recorded in response to supramaximal constant - voltage stimulation (duration=0.1 msec ; sensory=20 ma, motor=25 - 35 ma). stimuli were delivered from a bipolar stimulator ; stimulating electrodes were 0.8 cm in diameter, and their centers separated by 1.8 cm. the amplifier band pass was 2 - 10.000 hz for all nerve conduction study. a dantec keypoint version 2.0 electrodiagnostic system (dantec com, skovlunde, denmark) was employed studies were performed by two physicians among the authors and were assisted by a technologist. for excluding idiopathic carpal tunnel syndrome, which could also be caused by diabetes and uremia, we included both the motor conduction study in the wrist - elbow segment of the median and ulnar nerves and the motor and sensory conduction studies in the radial nerve. blood samples for lead level, cbc, and general chemistry were drawn from a venous line at the beginning of hemodialysis. blood lead level was analyzed in duplicate with a zeeman background - corrected atomic absorption spectrophotometer (hitachi z-8100, japan) with niosh 's standard addition method (17) at soonchunhyang university institute of industrial medicine, a certified reference laboratory for lead in korea. data were presented as a mean (sd) for continuous variables and frequency (%) for categorical variables. multiple linear regression was used to determine associations between blood lead and nerve conduction tests, adjusting for potential confounders. for easy presentation of results, the final regression models were selected with the same set of confounding variables (i.e., age, sex, duration of hemodialysis, kt / v, and protein catabolic rate). a value of p<0.05 was considered statistically significant, and all statistical analysis was performed using a stata program (stata release 5, college station, texas, u.s.a.). one hundred ninety - eight healthy subjects (control group, 147 men, 51 women ; mean age, 43.211.5 yr) and 68 patients with esrd undergoing hemodialysis (esrd group, 31 men, 37 women ; mean age, 50.112.3 yr) were enrolled for the blood lead measurement. the control group comprised healthy volunteers recruited during a regular physical examination at soonchunhyang university health promotion center (cheonan, korea). blood samples for lead concentration were drawn from the antecubital vein in the morning after overnight fasting. the esrd patients were undergoing regular hemodialysis eight to twelve hours weekly using a cellulose acetate hollow - fiber dialyser (surface area 1.2 m ; gambro, sweden, or baxter, u.s.a.) at a hemodialysis unit of the soonchunhyang cheonan hospital. in order to avoid any influence of carpal tunnel syndrome and/or ischemic neuropathy on the result of peripheral nerve conduction study, any patients who had vascular access on the right arm and had past histories of cerebrovascular disease, with or without hemiparesis, were excluded from this study. among 124 esrd patients on hemodialysis, 68 cases were consistent with these terms. this study was approved by the institutional review board of soonchunhyang cheonan hospital (cheonan, korea), and all human subjects provided written informed consent the upper limb temperatures were maintained above 34 and lower limb temperatures above 32, using hot packs when necessary. orthodromic sensory nerve action potentials and compound muscle action potentials were recorded in response to supramaximal constant - voltage stimulation (duration=0.1 msec ; sensory=20 ma, motor=25 - 35 ma). stimuli were delivered from a bipolar stimulator ; stimulating electrodes were 0.8 cm in diameter, and their centers separated by 1.8 cm. the amplifier band pass was 2 - 10.000 hz for all nerve conduction study. a dantec keypoint version 2.0 electrodiagnostic system (dantec com, skovlunde, denmark) was employed. studies were performed by two physicians among the authors and were assisted by a technologist. for excluding idiopathic carpal tunnel syndrome, which could also be caused by diabetes and uremia, we included both the motor conduction study in the wrist - elbow segment of the median and ulnar nerves and the motor and sensory conduction studies in the radial nerve. blood samples for lead level, cbc, and general chemistry were drawn from a venous line at the beginning of hemodialysis. blood lead level was analyzed in duplicate with a zeeman background - corrected atomic absorption spectrophotometer (hitachi z-8100, japan) with niosh 's standard addition method (17) at soonchunhyang university institute of industrial medicine, a certified reference laboratory for lead in korea. data were presented as a mean (sd) for continuous variables and frequency (%) for categorical variables. multiple linear regression was used to determine associations between blood lead and nerve conduction tests, adjusting for potential confounders. for easy presentation of results, the final regression models were selected with the same set of confounding variables (i.e., age, sex, duration of hemodialysis, kt / v, and protein catabolic rate). a value of p<0.05 was considered statistically significant, and all statistical analysis was performed using a stata program (stata release 5, college station, texas, u.s.a.). in the control group, mean systolic and diastolic blood pressure were 120.517.8 mmhg and 77.112.9 mmhg, respectively. hemoglobin and hematocrit serum calcium and phosphorus were 9.90.39 mg / dl and 3.890.59 mg / dl (table 1). in esrd group, the median duration of hemodialysis was 42.5 months (range ; 1 to 210 months). the causes of renal disease were diabetes mellitus (n=22), hypertension (n=20), polycystic kidney disease (n=1), glomerulonephritis (n=20), and unknown factors (n=7). kt / v was 1.300.27. normalized protein catabolic rate (npcr) was 1.20.2. mean systolic blood pressure was 142.215.5 mmhg, and mean diastolic blood pressure was 85.49.9 mmhg. seventeen cases (25%) were current smokers and 8 patients (11.8%) were regular drinkers (table 2). serum calcium and phosphorus were 9.341.07 mg / dl and 5.131.61 mg / dl, respectively. blood lead levels were corrected by hemoglobin (15 mg / dl) in both groups, and compared. after the correction, blood lead levels were significantly higher in the esrd group than in control group (p<0.001). all other study variables were significantly different between the groups (p<0.001) (table 1). blood lead was significantly correlated with duration of hemodialysis (p<0.05) (table 3). however also, motor or sensory nerve conduction velocity did not show significant correlation with blood lead in esrd patients (table 4). the positive correlation was observed between nutritional marker, npcr, and ulnar motor nerve conduction velocity. also, the positive correlation was observed between duration and radial sensory nerve conduction velocity. in the final models, patients with diabetes mellitus showed significantly worse motor and sensory function in all three nerves than those without diabetes mellitus (p<0.01) (table 5). major forms of lead poisoning are lead colic, hypertension, and neuropathy. on the other hand, minor symptoms are common and variable : cramps, paresthesia, intermittent pain in the limbs, chronic abdominal pain, and functional digestive disturbances. it is not easy to define these symptoms in patients with esrd because uremia itself may cause of and/or aggravate these symptoms. furthermore, chronic lead poisoning may be a cause esrd (18 - 21). the design of our study does not enable us to discuss the role of lead overload in the onset of esrd. determination of such a relationship would require epidemiological studies conducted according to rigorous methodology. in our results, the blood lead levels were significantly higher, almost twice that of the control group (9.12.8 g / dl vs. 5.92.3 g / dl) in esrd patients. contrary to our expectation, there was no correlation between the blood lead levels and peripheral nerve conduction. approximately 60% of patients with renal failure develop electrophysiologic evidence of peripheral neuropathy (22). some slowly dializable substances of intermediate molecular weight have been proposed as pathogens, resulting in axonal dysfunction and eventually degeneration (12). with this point of view, but in our study, the kt / v was 1.300.27, and there was no correlation between kt / v and peripheral nerve conduction. another problem during the evaluation of peripheral nerve conduction is the possibility of carpal tunnel syndrome in patients with esrd. it is well known that patients with esrd are apt to suffer from carpal tunnel syndrome (23 - 25), which may mask the results of peripheral nerve conduction study. the carpal tunnel syndrome is related to a small 2 microglobulin that normally is catabolized by the healthy kidney (26, 27). this substance forms a type of amyloid deposit throughout the body, with particularly adverse consequences when it is deposited in the carpal tunnel about the transverse carpal ligament. on the other hand, a profound combined ischemic neuropathy, referred to as an ischemic monomelic neuropathy, may result in patients with limb ischemia secondary to shunt (22, 28). a significant combined neuropathy may be associated with loss of sensation and muscle weakness in the distal arm supplied by the shunted vessels. in order to avoid any influence of carpal tunnel syndrome and/or ischemic neuropathy on the result of our peripheral nerve conduction study, we performed the motor conduction study on the wrist - elbow segment of the median and ulnar nerves, and the motor and sensory conduction studies on the radial nerve in the right arm. any patients who have vascular access in the right arm were excluded from this study. alcohol and nutrition are also important factors in peripheral neuropathy in esrd (22). in our results, both sensory nerve action potentials and compound muscle action potentials were significantly more suppressed in patients with diabetes mellitus than in the non - diabetes mellitus group. the nutritional markers of npcr (1.030.38) and serum albumin level (3.70.4 g / dl) were relatively homogenous among the patients with hemodialysis. in our result, positive correlation was observed between npcr and ulnar motor nerve conduction. but the other nerve was not correlated. we guess that malnutrition is important factor about nerve conduction in esrd patient, but only decreased npcr may be no prognostic marker about nerve conduction. the other causes of peripheral neuropathy in esrd are pharmaceutic neurotoxins and industrial and environmental neurotoxins such as mercury, arsenic, and thallium (22). of course, there is a possibility that the blood lead levels are not high enough to produce the neuropathy in esrd patients. in the peripheral nervous system, lead - induced pathologic changes in these fibers include segmental demyelination and axonal degeneration (29). recent studies of the peripheral nerves in persons exposed to lead have used electrophysiologic probes to determine whether lead causes covert abnormalities in function. they found slowed conduction in the small motor fibers of the ulnar nerve to be the most sensitive peripheral index of the neurotoxicity of lead and the ulnar nerve conduction velocity was depressed at blood levels below 50 g / dl. in conjunction with this report, our result reveals that, even though the blood lead levels were high in esrd, they were still below the level of inducing peripheral neuropathy. | diseases of the peripheral nervous system are the most prevalent in patients with end - stage renal disease (esrd). although increased blood levels of lead in esrd have been reported, the role of lead remains to be elucidated. the purpose of this study was to determine the connection of blood lead concentration with peripheral nerve conduction velocity. one hundred ninety - eight healthy subjects (control group) and 68 patients with esrd undergoing hemodialysis (esrd group) were enrolled. nerve conduction was measured within two hours after hemodialysis. orthodromic sensory nerve action potentials and compound muscle action potentials were recorded on the median, ulnar, and radial nerves. hemoglobin - corrected blood lead was significantly higher in esrd patients than in controls (9.12.8 g / dl vs. 5.92.3 g / dl, p0.05). our result suggested that even though the blood lead levels were high in esrd, they were not associated with the decline of peripheral nerve function. diabetes mellitus is a primary independent risk of neuropathy in esrd patients. |
in early january 2005, the french yersinia national reference laboratory (ynrl) received 3 y. pseudotuberculosis strains isolated by the same laboratory in dijon over a 1-week period : 2 from fecal samples of 2 children attending the same daycare center and 1 from the blood of a 65-year - old woman. during the same month, 8 additional strains were isolated from persons in other parts of the country by the yersinia surveillance network (based on voluntary participation of 88 hospital - based and private - sector medical laboratories throughout france). in view of this unusually high number of y. pseudotuberculosis isolations over a short period, the ynrl alerted france s national disease surveillance network, the institut de veille sanitaire, which thereafter performed an epidemiologic investigation. in early february, a request was mailed to all member laboratories in the yersinia surveillance network and all 95 microbiology laboratories in university medical centers, asking them to report any recent cases of y. pseudotuberculosis infection. a reminder letter was mailed to all laboratories that had not replied within 1 month of the initial communication. moreover, from february through april 2005, a total of 76 general medical center laboratories were contacted by telephone and asked to provide the ynrl with any relevant information and/or isolates. overall, 27 cases of culture - confirmed y. pseudotuberculosis infections were spontaneously reported or actively retrieved (table 1). this patient s signs / symptoms began after hospitalization ; all other patients signs / symptoms began before hospitalization. case reports of y. pseudotuberculosis infection peaked in january 2005. a food - exposure analysis for the first 10 patients did not identify a potential common food source, so a food survey was not performed for subsequent cases. the pseudotuberculosis cases occurred in 19 different counties throughout france, not necessarily the most populated ones (figure 1). data on lifestyle and living conditions were obtained for all but 7 patients, of whom 3 had died and 4 (including 2 children) were lost to follow - up. of the 22 adults, 13 lived in small towns (50,000 inhabitants), compared with only 3 of the 22 adults ; 4 of the children lived in apartments. map of france, showing spatial distribution of yersinia pseudotuberculosis infections during the winter of 200405. black circles, patients ' residences ; open circles, cities with medical laboratories that stated that they had not isolated any y. pseudotuberculosis from clinical specimens. of the 27 strains isolated, 25 all but 4 strains belonged to serotype i, the most common serotype in france. pulsed - field gel electrophoresis after spei digestion of genomic dna showed that (with the exception of the isolates from the 2 children attending the same daycare center) the dna fingerprints of the 16 other isolates sent to the ynrl during the peak period were all distinct, even when the strains were isolated from the same county (data not shown). this episode of increased case numbers differs from episodes reported in the literature by the nationwide distribution of cases, the absence of a locally defined cluster, the genetic diversity of the isolates, the predominance of rural residence for patients, and the dominant clinical presentation of septicemia (38). because the cases were not related to the consumption of a food product sold nationally (e.g., by a supermarket chain), the unknown origin of this phenomenon raises the question of an emerging risk in a new epidemiologic situation. of the 27 patients, 19 lived in a strip of land that stretches from northern france to the atlantic coast and corresponds to the flyways of small migratory birds. hence, the avian introduction of strains into the country would have been a possible scenario, as has already been suspected for an epizootic of pseudotuberculosis in an american wildlife park (9). indeed, during the winter of 200405, france witnessed a large and unexpected invasion of bohemian waxwings (bombycilla garrulus) (10), a species known to migrate from circumpolar areas where pseudotuberculosis is endemic. at that same time, 3 pseudotuberculosis outbreaks were reported in siberia (11). however, the genetic diversity of the strains isolated from the patients in france and the absence of pcr amplification of the superantigen - encoding gene ypm gene (12) (which is highly prevalent in far eastern strains) do not support bird - borne arrival in france of a y. pseudotuberculosis clone from russia or the far east. these cases occurred in areas where other human cases of y. pseudotuberculosis septicemia had been diagnosed (albeit at a much lower rate) in the past 16 years (figure 2). most previous cases of y. pseudotuberculosis septicemia also concerned inhabitants of low - altitude plains (figure 2), mainly in rural areas with extensive agriculture zones, which provide favorable habitats for small mammals. cases were frequently (27.1%) reported in 5 central - western counties of france, where just 4.6% of the population live and incidence of francisella tularensis infection (tularemia) is high (27.3%) (www.invs.sante.fr/surveillance/tularemie/donnees.htm). moreover, 40 cases of tularemia (with incidence peaks in summer and autumn) were reported to the national surveillance system in 2004 (notification of the disease has been obligatory since 2002), whereas only 8 to 19 cases per year had been reported over the preceding and following periods. like y. pseudotuberculosis, f. tularensis is known to have a rodent reservoir. hence, the spatial and temporal correlations between human tularemia and pseudotuberculosis in france over recent years suggest the sudden expansion of a common reservoir in 2004. county distribution, france, of yersinia pseudotuberculosis isolated from human blood and reported to the yersinia national reference laboratory over the 16 years preceding the winter of 200405. the number of isolates is represented by proportionally sized circles arbitrarily located at the center of the counties. our analysis of the temporal distribution of human y. pseudotuberculosis septicemia cases over the past 16 years showed a peak every 5 years (table 2). this finding is reminiscent of human hantavirus infections, which have been linked to cyclical oscillations in the vole population (the virus reservoir). taken as a whole, these data suggest that a rodent reservoir, mainly in rural areas, could have suddenly increased in the spring of 2004, thus increasing the risk for human transmission of y. pseudotuberculosis and f. tularensis over the following months. received by the french yersinia national reference laboratory since september 1988. to encompass the whole cold season, numbers in parentheses are mean monthly value over the 17 years in which case numbers were not increased. rodent populations tend to increase with ongoing changes in agricultural practices, e.g., removal of farmland hedges (which provide shelter for the rodents predators) and reduction in pesticide use. hence, the dynamics of the wild rodent population and reduction in pesticide use may represent a useful predictive marker for the occurrence of new outbreaks. the surveillance and control of the small mammal population might help limit the incidence of pseudotuberculosis and other wild rodent borne diseases of humans in france. | cases of yersinia pseudotuberculosis infection increased in france during the winter of 200405 in the absence of epidemiologic links between patients or strains. this increase represents transient amplification of a pathogen endemic to the area and may be related to increased prevalence of the pathogen in rodent reservoirs. |
in the early days of laparoscopic surgery, many surgeons used clips and staplers to avoid suturing. however, laparoscopic suturing requires advanced skills, which are mastered during advanced laparoscopic training. laparoscopic surgery requires many small and precise movements to manipulate and suture tissue in a confined space ; it has shown applicability in pancreatic tumor for staging the disease and at the time of biliary bypass. in 1997, robotically assisted surgery was proposed for cardiovascular applications. robotic surgery can increase precision, accuracy, and filter out hand tremor in a micro - anastomosis. now, sophisticated robot devices can reproduce the surgeon 's movements with the advantage of increased range of motion. the robot can reproduce a surgeon 's moves rapidly, precisely, and can filter out the tremor of the human hand caused by fatigue. this study evaluates the feasibility and safety of robotically assisted laparoscopy to perform a roux - en - y hepaticojejunostomy. pigs received humane care in compliance with the guide for care and use of laboratory animals published by the national institutes of health. eighteen female yorkshire pigs, weighing 18 kg to 31 kg, underwent a common bile duct (cbd) ligation to create a jaundice model. with the pigs under general anesthesia, 3-ports were placed with mini - laparoscopic instruments (2 mm to 3 mm). pneumoperitoneum was performed, a window behind the cbd wall was created, and a distal duct ligation with silk was made. transabdominal ultrasound was performed 3 to 5 days later, and the cbd diameter was measured and documented for follow - up. once the cbd distention was confirmed, the animals were randomly assigned to 3 groups for the roux - en - y hepaticojejunostomy (hj) : open (n=6), standard laparoscopy (lap) (n=6), and robotically assisted laparoscopy (zeus) (n=6). one surgeon, who had clinical experience in the assistant position in laparoscopic hepaticojejunostomy, performed all 3 approaches with the help of 1 assistant. neither the surgeon nor the assistant had experience with robotics in surgery. in the open group, under general anesthesia, the abdomen was prepped and draped in sterile fashion for ventral midline laparotomy. once the common bile duct dilation was confirmed, the roux - en - y was performed as a side - to - side enteroenterostomy at 40 cm from the angle of treitz by using a 3.0 running suture. a 1-cm incision at the common hepatic duct and small bowel was made, and the side - to - side hepaticojejunostomy was performed with a double running 4 - 0 suture. starting with the superior vertex, the posterior wall was initially made with a running suture and later tied with the separate suture from the anterior wall, tying both sutures at the inferior vertex. closure of the abdominal wall was accomplished in 2 layers. in the standard laparoscopic group, pneumoperitoneum with a veress needle was made at the umbilicus and maintained with a 12 mm hg pressure. the liver was retracted with cephalic gallbladder traction with an endograsp through the right lower port to expose the common bile duct. port placement in standard laparoscopy group and robotically assisted group (zeus system, computer motion, inc) ; surgeon 's instruments (a, b) ; endocamera (aesop) (c) ; assistant (d, e, f). the roux - en - y was performed by exposing the small bowel through the umbilical port, and side - to - side enteroenterostomy was performed outside the peritoneal cavity. after completion, it was reintroduced into the peritoneal cavity, pneumoperitoneum was reestablished, and the roux - en - y was retracted cephalic close to the cbd. the hj was performed in the same fashion as described for the open group, with a double running suture using intracorporeal knots with laparoscopic needle drivers. voice control of the aesop camera allows the surgeon to simultaneously manipulate the laparoscopic camera and 2 laparoscopic surgical instruments from a remote location. the surgeon operated seated in an armchair in front of a console including a monitor and control instruments. movements of robotic handles are transmitted to a computer controller that filters the moves, reduces the tremor, and then translates the surgeon 's movements to the robotic arms. the scale can be managed in a way that a robot reproduces in smaller scale the surgeon 's movements for more precision (figure 2). the zeus system (computer motion, inc.) while voice control of the aesop (b) manipulates remotely the laparoscopic camera, movements of robotic handles are transmitted to a computer controller translating the surgeon 's movements to the robotic arms. in the zeus approach, all the surgical steps were reproduced as in the standard laparoscopy group until the time of hj when the zeus sterile devices were set up. the surgeon operated from the console while the assistant stood between the robotic arms for tissue traction. the time required for the hj for the 3 groups, number of stitches on the posterior and anterior wall, and the total time of surgery were analyzed. the animals were recovered from the anesthesia with proper care after surgery and were sacrificed 2 weeks later for anastomosis inspection. to analyze the data regarding the differences between the groups, we used 3 types of tests : multiple comparison tests, 2-sample t tests, and paired t tests. three to 5 days after creation of the jaundice model with mini - laparoscopic cbd ligation, ultrasound was performed prior to the biliary bypass. the cbd was measured, confirming dilatation with a mean of 17, 15, and 16.8 mm for the open, lap, and zeus groups, respectively. one of the 18 pigs in the lap group developed a small ischemic hole on the anterior wall of the cbd after ligation. no free collections were shown in the ultrasound ; this was probably an ischemic consequence of the ligation. this case was diagnosed at the time of surgery, and the cbd wall was sutured (4.0 running suture) adding 20 minutes with a laparoscopic approach before performing the hj. analyzing roux - en - y time, a statistical difference was found between the open (20.2 min) and the lap and zeus (39.2 and 40.5 min) procedures. summary of surgical time for open, standard laparoscopy, and robotically assisted laparoscopy groups before the hepaticojejunostomy, the robot set up for sterilized procedures included covering 3 arms with a sterile plastic cover required a mean of 30.16 minutes (range, 18 to 45) with a fast learning curve. no difference was noted in the total number of stitches used in hj among the 3 groups. when comparing zeus to lap (2-sample t test), the former required fewer stitches on the posterior wall of the hj (p=0.0083). the time needed for the posterior wall of the hj between lap and zeus showed zeus was faster. the total time required for hj and surgery compared with that in lap and zeus were similar (table 2). hepaticojejunostomy for the open, standard laparoscopy, and robotically assisted laparoscopy groups postoperative complications related to roux - en - y and hj included 2 small bowel obstructions (11.1%) and 2 instances of ischemia / necrosis in the small bowel (11.1%) in the lap group. only 1 pig (5.5%) developed a bile leak at the anastomosis due to a broken suture (first case of zeus group). one pig developed a subcapsular hepatic hematoma found at necropsy (5.5%) (zeus group), probably due to instrumentation. the roux - en - y hepaticojejunostomy by a laparoscopic approach is a technically demanding procedure that requires knowledge of advanced laparoscopic suturing and the intracorporeal knot technique. significant suture practice is required, especially when smaller instruments are used to master the technique. trainees usually spend many hours practicing before competency is achieved. a strong limitation while suturing with laparoscopic instruments is the angle of work between the direction of the needle drivers and the direction of the anastomosis line. in standard and robotically assisted laparoscopy, the most time - consuming and critical factor in performing the hepaticojejunostomy was establishing the proper working angle in the anastomosis and the instruments. robotics appears to provide some advantages to the surgeon, such as improved range of motion and filtering out hand tremor to allow micro suturing. still, no proven benefits to the patient have been shown with these models in general surgery. the laparoscopic instruments are fixed to the port and the tip is rigid. the robotic device used at duke university was an initial prototype with a fixed tip of the instrument similar to that in laparoscopic instruments. today, new robots have a micro - wrist in the instrument tip that allows the surgeon to have a complete flexible tip like a little hand inside the abdominal cavity with wrist mobilization through a small access port. robotics may have an application in laparoscopic suturing, the learning curve is mainly to achieve the suturing task, and minimizing work angle difficulty. garcia - ruiz compared manual with robotically assisted laparoscopic maneuvering for suturing and found that the laparoscopic manual approach was faster when the size of the suture increased. we did not find differences in the total time of anastomosis (hj) in these 2 groups ; we performed all anastomoses with the same suture size. the posterior wall of the hj took less time for the zeus group than for standard laparoscopy group (table 2) even after excluding one laparoscopic case (l4) which presented with a difficult angle of work (2-sample t test, p=0.02049). when using the zeus at the first robotically assisted anastomosis, this animal developed a postoperative bile leak due to a broken suture at the superior vertex of the anastomosis. this complication was caused by a loss of tactile perception with the robot instruments and a mesh in the suture, feedback that is important when tying the suture knot. a surgeon must learn how to handle the suture while maintaining visual control through the monitor. we found that once the surgeon has some training in laparoscopic intracorporeal suturing, the learning curve for the robotic device is short. the basic concept of intracorporeal suturing is the same in laparoscopy and robotics because the instruments are similar. we believe that when the technique is mastered, the laparoscopic or the robotic - assisted approach does not make a difference, especially in this non - microwrist device. the critical factor of the angle may be minimized with more advanced robotic prototypes, such as flexible tip instruments. this brings a new concept, hands inside the peritoneal cavity through mini - invasive access, and it may show a difference for intracorporeal suturing techniques. robotically assisted laparoscopic roux - en - y hepaticojejunostomy is a feasible procedure that requires the same operative time as that of standard laparoscopy. | introduction : this study evaluates the feasibility and safety of using robotically assisted laparoscopy to perform a roux - en - y hepaticojejunostomy. this new method was compared with the open and standard laparoscopic approaches.methods:eighteen pigs underwent a needlescopic common bile duct ligation to create a jaundice model. three to 5 days later, transabdominal ultrasound was performed, and the common bile duct diameter was documented. for the roux - en - y hepaticojejunostomy, the pigs were randomly assigned to the open group (n=6), standard laparoscopy group (n=6), or robotically assisted laparoscopy group (zeus) (n=6). one surgeon performed all 3 approaches with 1 assistant. operative times, techniques, and complication rates were documented.results:the open approach was faster in all instances. at the hepaticojejunostomy, no difference was noted between the groups with the total number of stitches used. the robot required fewer stitches and less time in the posterior wall of the hepaticojejunostomy (p=0.0083 and p=0.02049, respectively). the hepaticojejunostomy time was similar for the laparoscopy and robotically assisted groups.conclusion:robotically assisted laparoscopic roux - en - y hepaticojejunostomy is a feasible procedure. when compared with standard laparoscopy, operating time is similar. |
a 57-year - old woman visited our hospital with right flank discomfort of two - month duration. the serum ca 19 - 9 level was slightly elevated at 45.2 u / ml (normal range : 0 - 37 u / ml). urine analysis demonstrated three to four white blood cells per high - power field, but the patient denied dysuria or other urinary symptoms. transabdominal us showed a 67-cm, well - marginated, retroperitoneal mass with isoechogenicity relative to the liver parenchyma, and this was adjacent to the pancre atic head. while color doppler us demonstrated the hypervascularity of the tumor (fig. dynamic pancreas ct revealed a 7-cm, well - demarcated, low attenuating mass in the pancreatic head. on the arterial phase ct, this mass showed strong enhancement and also non - enhancing tubular - shaped portions ; several intratumoral vessels that were connected to the peritumoral vessels were also noted. there was early contrast filling of the main portal vein and several prominent draining veins surrounding the mass. on the portal venous phase ct, the mass was still well - enhancing and the extent of the non - enhancing portions within the mass was reduced compared with that seen on the arterial phase. there was diffuse dilatation of the pancreatic duct that was most likely secondary to mass compression or invasion. given the mass compression of the distal main portal vein, the possibility of main portal vein invasion could not be ruled out (figs. ercp demonstrated superior displacement of the head portion of the main pancreatic duct and diffuse mild dilatation of the main pancreatic duct (fig. endoscopic us showed a well - marginated, echogenic mass with several anechoic portions, and this represented cystic degeneration or hemorrhagic necrosis in the pancreas head. it also demonstrated several large vessels within and around the mass and mild dilatation of the main pancreatic duct. based on these imaging findings, we radiologically diagnosed the tumor as being a non - functioning islet cell tumor of the pancreatic head. the gross specimen revealed a 6.566-cm, well - circumscribed ovoid soft - tissue mass with multifocal hemorrhagic portions and no cystic degeneration (fig. 1f). the tumor had displaced the common bile duct and the main pancreatic duct without any evidence of invasion. microscopic examination showed a zell - ballen pattern composed of mild pleomorphic chief cells and sustentacular cells within the tumor (fig. immunohistochemistrical staining was positive for synaptophysin, chromogranin and s-100 protein, but negative for cytokeratin (figs. paragangliomas or extraadrenal pheochromocytomas are rare, affecting about one in 2,000,000 people (6). although most paragangliomas are solitary and they arise sporadically, they can be multicentric or hereditary. paragangliomas of the abdomen predominantly arise from paraganglia that are symmetrically distributed along the abdominal aorta in the retroperitoneum. the most prominent collection is near the origin of the inferior mesenteric artery (the organ of zuckerkandl), which is where the majority of abdominal paragangliomas originate. other less common locations of abdominal paragangliomas include the gallbladder, urinary bladder, prostate, spermatic cord, uterus and duodenum (2). although paragangliomas occur in a variety of anatomic locations, they have nearly identical imaging features, namely a homogeneously or heterogeneously hyperenhancing, soft - tissue mass with cystic areas on ct scanning and multiple areas of signal void interspersed with hyperintense foci (a salt - and - pepper appearance) within the tumor on the mr imaging (2). the mean age of these eight patients was 67 years (range : 42 to 85 years) with a male to female ratio of 1/7. six of these eight tumors were located in the pancreatic head, whereas the remaining two originated from the body of the pancreas. although the available radiologic images were limited in the previously reported cases of pancreas paraganglioma (3 - 5), the imaging findings were generally characterized as a well - defined mass with frequent areas of hypoechogenicity on us, a well - marginated, hypervascular tumor with cystic areas of low - attenuation on contrast - enhanced ct, and tumor displacement of the main pancreatic duct on ercp. although the present case is characterized by similar findings, it is unique because the dynamic ct demonstrated robust enhancement of the mass that was comparable to that of the greater abdominal vessels, prominent intratumoral vessels and early contrast filling of the main portal vein and draining veins from the mass during the arterial phase. the differential diagnosis of a hypervascular pancreatic mass should include islet cell tumor (ict), which can be functioning or nonfunctioning according to their clinical and laboratory manifestations (7). functioning icts are usually less than 3 cm in size and they are homogeneously hyperenhancing during the arterial phase of contrast - enhanced ct (7). on the other hand, nonfunctioning icts tend to be larger than functioning icts, they have a greater predilection for cystic change or necrosis, and they are heterogeneously enhancing (7). the radiologic differentiation of pancreas paragangliomas from nonfunctioning icts can be difficult. yet to the best of our knowledge, there is no report regarding early contrast filling of the prominent draining veins of nonfunctioning icts. therefore, early contrast filling of the prominent draining veins of this tumor and the portal vein too may be a clue for differentiating pancreas paragangliomas from nonfunctioning icts of the pancreas. in conclusion, we report here on a patient with a rare diagnosis of primary paraganglioma of the pancreas, and this tumor was characterized by hypervascularity with prominent intratumoral vessels and early contrast filling of the draining veins from the mass. despite its rarity, paragangliomas should be a part of the differential diagnosis of a hypervascular pancreatic mass, and especially in the setting of early contrast filling of the prominent draining veins from the mass. | paragangliomas rarely originate from the pancreas and they are characterized on imaging studies as well - marginated, hypervascular masses with cystic areas. we herein report on a case report of pancreatic paraganglioma in a 57-year - old woman, which was confirmed on pathology. color doppler ultrasonography and dynamic ct demonstrated a well - demarcated, extremely hypervascular mass with prominent intratumoral vessels and early contrast filling of the draining veins from the mass. endoscopic retrograde cholangiopancreatography showed that the main pancreatic duct was displaced and mildly dilated. |
micronodules may be found during neck examination for non - nodular thyroid diseases, as well as in nodular thyroid disease when a clinical thyroid nodule is found to be associated with other non - clinically detected thyroid micro - nodules. generally, most patients with thyroid nodules lacked symptoms, with 5~10% of nodules having potential for malignancy. therefore, early diagnosis and adequate treatment are urgently needed to decrease the incidence of thyroid carcinoma. in 2009, the thyroid imaging reporting and data system (ti - rads), modeled on the breast imaging reporting and data system (bi - rads) and the initial ti - rads scores, classified and determined the risk of thyroid nodule malignancy with ultrasound scanning. ti - rads scores range from 1 (normal gland) to 6 (proven malignancy) ; ti - rads 3 and ti - rads 4 correspond to highly probable benign nodule and high suspicion for malignancy, respectively. multiple diagnostic tools are currently available for thyroid nodules, including ultrasonography (us), thyroid nuclear scan, and fine - needle aspiration cytology (fnac). differentiation between benign and malignant lesions is difficult in many cases because of the overlap of grey - scale and color doppler us findings. as a major improvement in ultrasound, contrast - enhanced ultrasonography (ceus) with ultrasound contrast agents (ucas) in addition, ceus has several advantages over traditional imaging techniques, including cost - effectiveness, portability, lack of radiation, non - invasiveness, and better patient compliance, which may be why ceus performs better than us in the diagnosis of renal cystic lesions. ceus plays an important role in the identification of malignant lesions in various organs, such as colon, gallbladder, pancreas, and kidney [1315 ]. although the improvement in diagnostic performance of conventional us combined with ceus in the differentiation capability for benign from malignant breast masse has been elucidated in previous data, there is limited data on conventional us combined with ceus in differentiating solitary thyroid nodule (stn) and micronodules from normal thyroid tissue. we performed the current study to investigate the diagnostic performance of conventional us combined with ceus in micronodules with categories of ti - rads 3 and ti - rads 4. the study was conducted according with the protocols proposed by the ethics committee of sichuan provincial people s hospital. written informed consent was obtained from each subject before the study. all procedures in this study were in compliance with the declaration of helsinki. from september 2012 to july 2014, patients with thyroid disease who underwent conventional us or ceus in sichuan provincial people s hospital were considered as candidates for inclusion. the inclusion criteria were : nodules with a hypoechoic aspect, normal thyroid tissue with homogeneous echogenicity, maximum diameter for thyroid nodules 10 mm, and category 3 or 4 in ti - rads. the exclusion criteria were : (1) patients with neck deformity, (2) patients with cystic nodules or mixed nodules with less parenchymal nodules, (3) solid components that can not be revealed in nodules due to macrocalcification ; (4) patients with severe allergies ; and (5) patients who were pregnant. after applying the inclusion and exclusion criteria, a total of 102 patients (29 males and 73 females) were included, with age range 1980 years. the thyroid lesions collected from included subjects had maximum diameter of 2.80~9.65 mm (mean, 6.691.70 mm). among the included lesions, lesions with maximum diameter 0.05) (table 1). conventional us showed that the micronodules in ptc had the following characteristics : irregular shape, unclear margins, a / t 1, microcalcification in nodules, majority 0i blood flow signals, and presence of suspicious lymph gland (figures 1, 2). there were significant differences in the above characteristics between the malignant group and benign group (all p0.05) (table 3). binary logistic regression analysis was conducted with pathological diagnosis as the dependent variable and statistically different indexes in convention us, ceus, and conventional us combined with ceus as independent variables. logistic regression analysis was conducted with or > 1 indicating risk factors and or 1) (table 5). logistic regression analysis of conventional us combined with ceus demonstrated that a / t 1, microcalcification, suspicious lymph gland, slow enhancement time, and absence of rim - like enhancement are risk factors (all p 1) (table 6). the roc curve showed that us had an area under the roc curve (auc) of 90.0% and ceus had an auc of 90.7%, in contrast to the auc of 99.0% for combined use of conventional us and ceus (figure 5), which indicated that the combination of these 2 methods had a superior diagnostic value compared with conventional us and ceus alone. among the 102 subjects, 52 patients were included in the malignant group, among which 22 patients had preoperative fine - needle aspiration (fna) and 52 patients received surgery. the remaining 50 patients were included in the benign group, among which 36 patients received preoperative fna and 14 patients received surgery. in the malignant group, age range was 30~50 years. a significant difference was detected in age between the 2 groups (p0.05) (table 1). conventional us showed that the micronodules in ptc had the following characteristics : irregular shape, unclear margins, a / t 1, microcalcification in nodules, majority 0i blood flow signals, and presence of suspicious lymph gland (figures 1, 2). there were significant differences in the above characteristics between the malignant group and benign group (all p0.05) (table 3). binary logistic regression analysis was conducted with pathological diagnosis as the dependent variable and statistically different indexes in convention us, ceus, and conventional us combined with ceus as independent variables. logistic regression analysis was conducted with or > 1 indicating risk factors and or 1) (table 5). logistic regression analysis of conventional us combined with ceus demonstrated that a / t 1, microcalcification, suspicious lymph gland, slow enhancement time, and absence of rim - like enhancement are risk factors (all p 1) (table 6). the roc curve showed that us had an area under the roc curve (auc) of 90.0% and ceus had an auc of 90.7%, in contrast to the auc of 99.0% for combined use of conventional us and ceus (figure 5), which indicated that the combination of these 2 methods had a superior diagnostic value compared with conventional us and ceus alone. the current study investigated the diagnostic value of conventional us combined with ceus in ti - rads 3 and ti - rads 4 thyroid micronodules. we demonstrated the combination of these 2 technologies showed superior performance compared with single use of convention us or ceus. our results were further enhanced by the use of both logistic regression analysis and roc curve. although data show that the incidence of ptc has been decreasing significantly and the prognosis for this disease has greatly improved, the early detection on this disease still needs to be emphasized because it easily spreads to the lungs and bones [2022 ]., our study investigated the diagnostic value of conventional us combined with ceus in ti - rads 3 and 4 thyroid micronodules. according to the definition of ti - rads, patients with ti - rads 3 presented with hyper-, iso-, or hypoechoic nodules, while ti - rad4 has a suspicious neoplastic pattern or malignant pattern. thyroid micronodules with maximum diameter 10 mm were selected because nodules larger than 1 cm have high potential to develop into clinically significant cancers. with the high sensitivity and specificity of ti - rads, diagnosis of thyroid nodules has shown great improvement using ultrasound contrast. as an important diagnostic tool in predicting thyroid malignancy and selecting thyroid nodules, the characteristics of suspicious thyroid nodules include marked hypo - echogenicity, irregular margins, microcalcifications, and a taller than - wide shape, which contribute to better diagnostic accuracy. consistent with these features, our study, using conventional 2-dimensional and color doppler contrast, demonstrated that the micronodules in ptc had irregular shape, unclear margins, a / t 1, microcalcifications, blood flow 0-i, and suspicious lymph gland. another meta - analysis, which showed that ultrasound features are important predictors of malignancy for thyroid nodules, reported a similar result regarding the accuracy of conventional us. a meta - analysis by brito found that the value of diagnostic us features, such as internal calcifications, echogenicity, infiltrative margins, and solid vs. cystic content of the nodule, were often overestimated, which is slightly different from the results of the present study. the ready repeatability, lack of risk, and low cost of conventional us make it a particularly attractive modality. in this same setting, our study revealed that ceus had significantly better sensitivity, specificity, and accuracy than conventional us. a previous study found that real - time ceus showed remarkably different images in benign vs. malignant thyroid nodules ; therefore, ceus has clinical value use for differential diagnosis of stn. evidence supports that ceus has a better visualization of the microcirculation and details of stenosis compared with conventional us and power doppler. our study demonstrated that ptc tissues had hypo - enhancement and normal thyroid tissues had hyper - enhancement using ceus. most malignant nodules have fibrosis, calcification, focal necrosis, and aberrant blood vessels, which may lead to hypo - echogenicity on us and hypo - enhancement on ceus. however, research also found that the differential diagnosis between benign and malignant thyroid nodules using conventional imaging methods lacks reliability, and that ceus enhancement patterns varied between benign and malignant lesions. therefore, the combined use of conventional us and ceus may improve accurate diagnosis of benign and malignant lesions compared with convention us or ceus alone, which is reflected in the improved specificity and accuracy and higher auc in our results. our findings are consistent with a previous study describing the value of ceus combined with conventional us in the diagnosis of thyroid micro - carcinoma using a logistic regression model, which found that ceus and conventional us were very effective in thyroid nodule diagnosis. the findings of our study suggest that the combined use of conventional us and ceus has superior diagnostic performance in ti - rads category 3 and 4 thyroid micronodules, compared with conventional us and ceus alone. therefore, the combined use of conventional us and ceus should be advocated in thyroid micronodule diagnosis. | backgroundthe present study was conducted to investigate the diagnostic performance of conventional ultrasonography (us) combined with contrast - enhanced ultrasonography (ceus) in thyroid micronodules with thyroid imaging reporting and data system (ti - rads) category 3 and 4.material/methodsthe features of conventional us and ceus ion 102 case of thyroid micronodule samples, which were diagnosed based on pathological and clinical examination, were retrospectively analyzed. logistic regression analysis was used to analyze the diagnostic accuracy in malignant thyroid micronodules. receiver operator characteristic (roc) curve was used to assess the performance of those 2 technologies.resultsa significant difference in age was found between the benign and malignant groups. the benign and malignant groups showed significant differences in shape, margin, aspect ratio (a / t) 1, microcalcification, suspicious lymph gland, enhancement time, enhancement pattern, enhancement intensity, nodule sizes, enhancement margins, and rim - like enhancement. logistic regression analysis of conventional us showed that a / t 1, irregular shape, microcalcification, and suspicious lymph glands are risk factors for thyroid micronodules, while logistic regression analysis of ceus showed that slow enhancement time and absence of rim - like enhancement are risk factors for thyroid micronodules. logistic regression analysis of conventional us combined with ceus demonstrated that a / t 1, microcalcification, suspicious lymph gland, slow enhancement time, and absence with rim - like enhancement are risk factors. the roc curve for conventional us, ceus, and conventional us combined with ceus were 90.0%, 90.7%, 99.0%, respectively.conclusionsour results show that conventional us combined with ceus had superior diagnostic performance for ti - rads 3 and 4 thyroid micronodules compared with conventional us and ceus alone. |
the action of phospholipase a2 (pla2) on membrane phospholipids produces free fatty acids such as arachidonic acid (aa) and the phospholipid backbone. to the former belongs eicosanoids (such as prostaglandins, prostacyclin, thromboxane, and leukotrienes) through the cyclooxygenase (cox) and lipoxygenase (lox) pathways ; and to the latter, platelet - activating factor (paf) (figure 1) [1, 2 ]. while countless studies have highlighted the actions of eicosanoids and paf on normal human mature myeloid and lymphoid cells (from hematopoietic progenitors to mature blood cells), their effects on leukemic blasts are poorly documented, and furthermore, their putative involvements during leukemic diseases remain almost speculative. this paper focuses on new results about lipid mediators and human leukemic blast cells from acute myeloid (aml) and acute lymphoid (all) patients. the vast majority of results reported previously have been obtained with aml blasts without maturation according to the classification system of the world health organization, thus corresponding to the past aml m0 - 2 nomenclature. pla2 catalyzes the hydrolysis of the sn-2 position of membrane glycerophospholipids to liberate the eicosanoid precursor aa (figure 1) [3, 4 ]. three distinct families are documented : low molecular weight soluble forms of pla2 (spla2) ; ca - dependent high molecular weight pla2 (cytoplasmic pla2, cpla2) ; cytoplasmic ca - independent high molecular weight pla2 (ipla2). in addition, the spla2 family is implicated in several biological processes such as inflammation and host defence [3, 4 ]. the cpla2 family consists of four members, with cpla2-iva being the central regulator of the stimulus - coupled cellular aa release [3, 4 ]. freshly isolated leukemic blasts from aml and all patients express mrna from four out of five cpla2 (pla2-iva, pla2-ivb, pla2-ivc, and pla2-vi) and six out of nine spla2 (pla2-ib, pla2-iia, pla2-iid, pla2-v, pla2-x, and pla2-xii) and that transcript levels exhibit wide variations as compared to control blood mononuclear cells. one of the most notable findings is that aml and all blasts express high amounts of pla2-vi and pla2-x. this could be extremely significant as these two enzymatic activities play a major role in aa release for the generation of cox- and lox - derived lipid mediators. thus, aml and all blasts have the potential to express multiple isoforms of cpla2 and spla2 which could be of importance given the role of these enzymes in inflammation, generation of lipid mediators, anticoagulant activity, and bacterial infection. biological activities of pla2 are attributed to their enzymatic capacity to hydrolyze membrane phospholipids. however, in addition spla2 exerts various biological proinflammatory responses through the binding to the cell surface pla2 receptor (pla2-r). of interest is the functional membrane pla2-r found on aml and all blasts strengthening a role for pla2 signalling in these cells (denizot and coll. the concept of anti - inflammation is currently evolving with the discovery of endogenous inhibitory circuits, such as the annexin (anx) system, that are important in the control of the host inflammatory response. anx-1 (also termed lipocortin) is a well - known cpla2 inhibitory protein produced by and acting on several blood cell types such as monocytes / macrophages and polymorphonuclear leukocytes. the anx-1 protein level is markedly elevated in aml blasts, where anx-1 is not only considered as an anti - inflammatory and tumor suppressor molecule (through its inhibiting cpla2 activity) but also as one of the eat - me signals on apoptotic cells to be recognised and ingested by phagocytes. it is, thus, tempting to speculate that pla2-r and anx-1 might take an important place in the yin and the yang of the inflammatory reaction occurring in aml blasts. during the past decade, considerable research has been directed towards the identification of new biological targets for aml treatment. it is tempting to suggest that pla2-r antagonists might be one of them especially with respect to the emerging roles for pla2 enzymes in cancer. in the cox pathway, aa is converted to pgh2 by cox-1 or cox-2 enzymes. pgh2 is subsequently metabolised to generate different prostanoids, depending on the enzymes expressed in the cell. cox-1 and cox-2 transcripts are documented in aml and all blasts, but only the cox-1 protein is found. similarly cox-1, but not the cox-2 protein, is detected in human primary promyelocytic blasts during differentiation. in fact, the aml and all blasts can express the cox-2 protein in response to lipopolysaccharide (lps) but only in the subsets of patients. the ability of all blasts to express cox-2 is consistent with its presence in stimulated normal b - cells and in chronic lymphocytic leukaemia (cll) b - cells [14, 15 ]. the production of cox-2 in response to lps by aml blasts is consistent with data reporting that lps is a potent inductor of cox-2 in mature monocytes / macrophages and that stimulated hl-60 cells (an aml cell line with an m2/3 subtype) express cox-2. the heterogeneity in the lps - stimulated cox-2 expression by aml blasts is not linked to a different toll - like receptor (tlr2 or tlr4) expression and remains an open question that requires further evaluation. following the action of the cox pathway, pgh2 is subsequently metabolized to generate different prostanoids, depending on the enzymes expressed in the cell. prostanoids include prostacyclin (pgi2), thromboxane a2 (txa2), and prostaglandin e2 (pge2), synthesized by a pgi2 synthase, a txa synthase, and a pge synthase, respectively. three pge synthase isoforms exist : inducible membrane - bound pge synthase-1 (mpges-1), constitutive membrane - bound pge synthase-2, and cytosolic pge synthase. in addition, the ability of pge2 to regulate the immune system has been widely explored. data reporting the ability of pge2 to modulate several functions in mature blood cells such as monocyte - macrophages, dendritic cells, and t and b lymphocytes can be readily found. human aml and all blasts spontaneously release pge2, with pge2 synthesis being inhibited by indomethacin. transcripts for mpges-1 are detected in aml and all blasts suggesting its role in pge2 synthesis (denizot and coll., unpublished results). pge2 effects are well known and are mediated through interactions with four distinct membrane - bound g - protein - coupled ep receptors : ep1, ep2, ep3, and ep4. ep2 and ep4 are coupled to gs and stimulate camp production which leads to gene regulation. ep1 is coupled to gq / p, and ligand binding induces intracellular calcium level variations. functional ep2 receptors are present on aml and all blasts [19, 20 ]. in contrast to ep2 receptors, no functional ep1, ep3, and ep4 receptors are found. in view of the potentially important role of pge2 in processes of cancer and leukocyte maturation and function, pge2 enhances the spontaneous and lps - stimulated growth of aml blasts without affecting their apoptosis. in summary, aml and all blasts secrete pge2. a role for pge2 as a compound contributing to aml cell proliferation (via an ep2 receptor - mediated pathway) can be hypothesized. txa2 is produced abundantly by platelets upon exposure to injured blood vessels and thus exhibits potent platelet - aggregating and vessel - contracting activities. pgi2 is the major cox - derived product of aa formed in the macrovascular endothelium and is a potent inhibitor of platelet aggregation activity and vessel vasodilatation activity. aml and all blasts express low levels of tx synthase transcripts compared to normal blood mononuclear cells (denizot and coll., unpublished results) and additionally produce very low amounts of txa2 in response to a calcium ionophore stimulation. hl-60 cells have also been shown to release txa2, but only after induction of differentiation [23, 24 ]. pgi synthase transcripts are absent in aml and all blasts, a result similar to that found in control blood mononuclear cells (denizot et coll., unpublished results). in accordance with the absence of pgi transcripts in aml and all blasts, calcium ionophore - stimulated blasts do not release pgi2 (denizot et coll. txa2 and pgi2 act through membrane receptors (namely txa2r and ip for txa2 and pgi2, resp.) [25, 26 ]. as to whether aml and all blasts release txa2 and pgi2, they express levels of transcripts for txa2r and ip equal or higher than those found in control blood mononuclear cells. txa2r and ip receptors belong to the class of gs - protein - coupled receptors [25, 26 ]. stimulation of leukemic blasts with u-46619, the txa2 receptor agonist u-46619, and pgi2 stimulate in a dose - dependant manner camp synthesis from leukemic blasts showing the presence of functional txa2r and ip receptors, respectively. however, simulation of leukemic blast with u-46619 and pgi2 has no effect on their growth and apoptosis rate. at the present time the physiological meaning of functional txa2r and ip receptors on leukemic blasts remains an open question. in conclusion, among the various cox - derived metabolites of aa only pge2 has, thus, a significant effect on the growth of aml blast cells, and none of them affect their apoptosis rate. the lox pathway involves the conversion of aa to 5-, 12-, or 15-hydroperoxyeicosatetraenoic acids (hpete) by 5-, 12-, or 15-lox, respectively, hpetes being rapidly metabolized to 5-, 12, or 15-hydroxyeicosatetraenoic acids (hete). 5-hpete could be dehydrated into leukotriene a4 (lta4), which was enzymatically hydrolyzed to ltb4 (figure 1). the ability of ltb4, 12-hete, and 15-hete to regulate important functions of the immune system has been widely explored. these compounds activate various blood cell types and stimulate their proinflammatory cytokine productions [2830 ], indicating an ability of ltb4, 12-hete, and 15-hete to augment and prolong tissue inflammation. leukemic blasts express 5-lox, 12-lox, and 15-lox transcripts, their expression being in general lower than in blood mononuclear cells from a healthy donor [22, 31, 32 ]. leukemic blasts produce in vitro lower amounts of ltb4 than healthy donors [22, 31, 33 ]. this reduced capacity of aml blasts to produce ltb4 the various lox - derived metabolites of aa regulate a wide spectrum of cellular processes including cell proliferation and apoptosis. however, ltb4, 12-hete, and 15 hete have no effect on the growth and apoptosis rate of aml and all blasts in vitro. as to whether receptors for 12-hete and 15-hete remain to be molecularly identified, two g - protein - coupled seven transmembrane domain receptors for ltb4 were identified : blt1 and blt2. amounts of blt1 transcripts are similar in aml and all blasts as well as control blood mononuclear cells, while amounts of blt2 transcripts are markedly higher. at this time the physiological meaning (if any) of blt1 and blt2 transcripts in aml and all blasts remains an open question. a similar question exists for the significance of ltb4- and 12-hete - derived leukemic blasts. one might suggest that these compounds could initiate, augment, and prolong tissue inflammation and damages by affecting the cytokine network, but currently no studies have provided evidences in support of this. paf is a phospholipid mediator that sparks off a wide range of immunoregulatory activities on blood cells such as polymorphonuclear neutrophils, eosinophils, monocytes, macrophages, and lymphocytes. paf is released in vitro from several leukemic cell lines of b and t origin as well as from freshly isolated neoplastic cells of leukemic patients. however, in spite of experimental evidence reporting its in vitro release from leukemic cells, no clinical studies provide evidences to support this view in vivo. in contrast, decreased levels of paf are found in the blood of patients with lymphoid and nonlymphoid hematologic malignancies. blood paf levels are regulated by an acetylhydrolase activity (aha, also named pla2-viia) found in serum and plasma. plasma aha is not altered in leukemic patients suggesting a lowered paf production by leukemic cells rather than an increased paf catabolism. paf acts through membrane and nuclear paf receptors (pafr) that belong to the g - protein - coupled family [40, 41 ]. as to whether membrane pafr is found on aml and all cells [42, 43 ] studies report that mature monocytes, macrophages, polymorphonuclear leukocytes, and b lymphocytes produce camp in response to paf [44, 45 ]. the gq proteins mediate their effects by activating phospholipase c and thus, generating second messengers, inositol-1,4,5-triphosphate (ip3) and diacylglycerol, thereby leading to the activation of protein kinase c and the mobilisation of intracellular calcium. paf stimulates in a receptor - dependent process ca flux from aml and all blasts showing the presence of functional pafr and highlighting that pafr signals via the gq instead of the gi / gs protein pathways. paf has no significant effect on growth and apoptosis rate in these cells suggesting that paf is not an important modulator of blast cell physiology. the lack of paf effect is linked to low levels of pafr in aml and all blasts compared to those found in mature leukocytes. further, strengthening this issue, the differentiation of hl-60 cells towards the macrophage phenotype is associated with the induction of pafr gene expression. thus, pafr mrna accumulation is correlated to the induction and development of specific paf responsiveness. recently web-2170, a pafr antagonist, has been reported to induce apoptosis in aml cells [51, 52 ]. in fact, web-2170 does not behave as a pure pafr but instead as an inverse agonist leading to a marked cytoplasmic increase of pten proteins (pten is a protein / phosphoinositide phosphatase regulating the pi3k / akt signaling pathway). consequently, these recent results [49, 51, 52 ] support the view that paf has probably no significant role in the growth and apoptosis of leukemic blasts. data reporting our knowledge concerning the enzymatic activities (such as pla2, lox, cox) implicated in lipid mediator synthesis and their receptors on aml and all blasts are schematised in figure 2. aberrant expression of several pla2 enzymes is common place in tumors derived from many different organ sites. numerous studies report that altered aa metabolism in a solid tumor microenvironment has a profound impact on the pathogenesis of tumor development. a multitude of biological activities of paf are evidenced both on the normal cell as well as on their cancer counterpart. there is evidence, however, that it is not the case for leukemic blast cells. among the various pro - inflammatory lipid molecules so far tested (paf, pge2, pgi2, txa2, ltb4, 12-hete, 15-hete), none of them exhibit any role on leukemic blast apoptosis despite the expression of functional receptors (pafr, ep2, ip, txa2r). among the various compounds so far tested only pge2 clearly demonstrated a potential role in aml cell growth in vitro. however, it is difficult to compare the m amounts of pge2 used in most of the in vitro studies with the fm amounts of pge2 found in the blood at steady state conditions. studies showing the effects of continuous addition or infusion of low doses of pge2 (which seems to be a more relevant protocol of stimulation to obtain information for the in vivo effects of pge2) are extremely rare., there is absolutely no evidence that pge2 is implicated in the growth of aml blasts in vivo. thus, in conclusion the biological effects of eicosanoid and paf are particularly important in immunity and inflammation. though their roles are well known in numerous pathology and cancers, no such role is currently known for leukemic blast growth. | some of the most potent inflammatory mediators share a lipid origin. they regulate a wide spectrum of cellular processes including cell proliferation and apoptosis. however, the precise roles and ways (if any) in which these compounds impact the growth and apoptosis of leukemic blasts remain incompletely resolved. in spite of this, significant advances have been recently made. here we briefly review the current knowledge about the production of lipid mediators (prostaglandins, leukotrienes, platelet - activating factor) by leukemic blasts, the enzymatic activities (phospholipase a2, cyclooxygenases, lipoxygenases) involved in their productions and their effects (through specific membrane bound receptors) on the growth, and apoptosis of leukemic blasts. |
the increasing popularity of esthetic restorations has drawn attention to long - term durability and bond success of these restorations. adequate isolation and contamination control must be considered before bonding procedures. however, the difficulty of achieving moisture control is a potential problem encountered in clincal situations, especially when rubber dam isolation is unfeasible. saliva contamination more probably occurs in regions near or at the gingival margin and many carious lesions are found in these areas isolated difficulty [15 ]. some studies have suggested that saliva contamination could reduce the bond strength of adhesive systems [69 ]. others have reported that saliva contamination could not significantly affect modern adhesives when compared to the previous generation of bonding agents [1014 ]. moreover, effects of saliva contamination were not the same in different stages of bonding when modern adhesives were used. reduction of bond strength as a result of saliva contamination may relate to the type of resin adhesive and the stage of bonding procedures. the bond strengths may be restored by different contaminant - removing treatments depending on the stages of bonding process contaminated such as re - conditioning, washing with water and re - application of the adhesive [310,12,13,16,17 ]. there are a few studies evaluating the effect of saliva contamination without any treatment on bond strength of different adhesive systems when moisture control means very difficult achievement. self - etch adhesives contain non - rinse acidic monomers that simultaneously condition and prime, and vinyl groups that co - polymerize with resin composite. this was followed by development of a so - called self - etching primer that can etch and prime in one step. finally the one self - etch or so called all - in - one adhesive was introduced which conditions, primes and bonds in a single step. the self etching adhesive provides decreased clinical application time and reduces the risk of saliva contamination, especially when the carious site is near or at the gingival margin and maintaining a dry field may be impossible. in addition, the technique sensitivity of this adhesive which bonds to a dehydrated collagen matrix is eliminated as a result of its water component. the null hypothesis of this study was that saliva contamination would not affect the dentin bond strength of self - etch adhesive. in order to test this hypothesis, the present study evaluated the effect of saliva contamination without eliminating saliva during different bonding steps of these adhesives. in this experimental interventional study, seventy - two extracted human first molars were cleaned, stored in 0.5% chloramines - t solution for 7 days, and then immersed in 04c stilled water for a maximum of 6 months until further processing in the laboratory. teeth were mounted in cylindrical molds using self - curing acrylic resin up to their cervical areas. buccal enamel of mounted teeth was then eliminated by diamond disc (d&z, diamant, germany) and the superficial dentin was exposed in a depth of 1 mm. these surfaces were polished using 600-grit silicon carbide paper in order to prepare a uniform surface and a smear layer. the specimens were randomly divided into three groups according to the materials, and for each group the adhesive was placed on the prepared surfaces according to the manufacturer s recommendations (table 1). a thin saliva layer collected from a single individual was applied on the surfaces with brush during the different steps of bonding mentioned in the following subgroups and left undistributed for 5 seconds. the three adhesives were applied on prepared surfaces according to the manufacturer 's instructions (table 1) as follows : subgroup a1 : specimens were contaminated with saliva after etching. subgroup b2 : specimens were contaminated with saliva after the adhesive application before its polymerization. subgroup b3 : specimens in this group were contaminated with saliva after polymerization of the adhesive. after the bonding procedure, the resin composite (z-100, 3 m dental products, st. paul, mn, usa) was built up in two increments using plastic mold (inner diameter : 3 mm and height : 3 mm) and individually light - cured for 40 seconds (coltolux 75, coltene / whaledent, mahwah, nj, usa, 500 mw / cm measured by digital radiometers). all prepared specimens were thermocycled for 500 cycles between 555c with a 30 second dwell time. after storage of specimens in distilled water at 37 c for 24 hours, shear test was performed by universal testing machine (zwick / roellz020, zwick gmbh & co, kg, germany) at the crosshead speed of 0.5 mm / min. the mechanical loading was applied to the interface of composite and dentin until debonding of the composite and the data were registered in mpa. finally, the mode of failures which occurred during debonding were determined by stereomicroscopic (smz 1500, nikon, kanagawa, japan)(20). the collected data were statistically analyzed using two - way anova for three adhesives in order to compare among the subgroups which were not contaminated (a5, b4, c3), contaminated before (a3, b2, c1) and after (a4, b3, c2) adhesive polymerization. consequently, because the interaction of saliva contamination and the adhesive type was significant, one - way anova and duncan s post hoc test were conducted for each adhesive in different contaminated bonding steps and for each contaminated bonding step with different adhesives. subgroup b2 : specimens were contaminated with saliva after the adhesive application before its polymerization. subgroup b3 : specimens in this group were contaminated with saliva after polymerization of the adhesive. after the bonding procedure, the resin composite (z-100, 3 m dental products, st. paul, mn, usa) was built up in two increments using plastic mold (inner diameter : 3 mm and height : 3 mm) and individually light - cured for 40 seconds (coltolux 75, coltene / whaledent, mahwah, nj, usa, 500 mw / cm measured by digital radiometers). all prepared specimens were thermocycled for 500 cycles between 555c with a 30 second dwell time. after storage of specimens in distilled water at 37 c for 24 hours, shear test was performed by universal testing machine (zwick / roellz020, zwick gmbh & co, kg, germany) at the crosshead speed of 0.5 mm / min. the mechanical loading was applied to the interface of composite and dentin until debonding of the composite and the data were registered in mpa. finally, the mode of failures which occurred during debonding were determined by stereomicroscopic (smz 1500, nikon, kanagawa, japan)(20). the collected data were statistically analyzed using two - way anova for three adhesives in order to compare among the subgroups which were not contaminated (a5, b4, c3), contaminated before (a3, b2, c1) and after (a4, b3, c2) adhesive polymerization. consequently, because the interaction of saliva contamination and the adhesive type was significant, one - way anova and duncan s post hoc test were conducted for each adhesive in different contaminated bonding steps and for each contaminated bonding step with different adhesives. table 2 summarizes the mean shear bond strengths and standard deviations of different groups and subgroups. the effect of each contaminated bonding step on different adhesives shear bond strength is revealed as follows : a) in the uncontaminated condition, shear bond strength of prompt l - pop was significantly lower than sbmp s (p sb > prompt l - pop) (p<0.05). the effect of each adhesive in different contaminated bonding steps on the shear bond strength is as follows. there was significant difference between the shear bond strength of contaminated and uncontaminated specimens in groups a and b (p<0.05) ; whereas, in group c there was no such difference (p=0.411). in addition, there was no significant difference between subgroups a2 and a3 (p=0.714), subgroups a1, a2 and a4 (p=0.054), and subgroups b1, b2 and b3. stereomicroscope observation showed that the specimens fracture types contained i) adhesive failure ii) cohesive failure and iii) mixed failure (table 2). this study evaluated the effect of saliva contamination during bonding steps without removing saliva on the shear dentin bond strength of three adhesives. the present results showed that saliva contamination and the type of contaminated adhesive could be effective on the bond strength. in contrast to sbmp and sb, saliva contamination did not affect the bond strength of prompt l - pop. in self - etching adhesive systems, all three basic steps (etching, applying primer and adhesive) occur simultaneously. thus, at the same time these adhesives demineralize dentin while infiltrating it with monomers to the same depth, and then polymerization in situ is disclosed. therefore, no gaps would be left between the resin surface and the demineralized dentin surface. simplicity, time saving and fewer time points for probable contamination during bonding procedures are the advantages of self - etching adhesive, especially in the saliva contamination condition. in the present study, prompt l - pop was not affected by saliva contamination which may result from its water componant and simultaneous bonding steps. thus, the hydrophilicity of this adhesive may allow its diffusion through the salivary film. for the polymerized self - etch adhesive which was not significantly affected by saliva, it may be speculated that the chemical property of its poorly polymerized oxygen - inhibited surface may be responsible. shear bond strenght of total etch adhesives used in the present study are affected by saliva contamination. when steps of bonding were accomplished separately, some regions of the demineralized dentin may not be penetrated by the resins. in addition, there are longer time points during placement of these adhesives when contamination can occur. when surfaces are contaminated with saliva after etching, water and glycoproteins of saliva may interfere with the proper adhesion. when surfaces are contaminated with saliva after application of primer and adhesive before light curing, saliva can affect the degree of conversion and bond strength ; because hydroxyethyl methacrylate (hema) molecules with their hydrophilic nature may retain water within the adhesive layer and they dispersed in water, thus they become unable to participate in chain growth during polymerization. when surfaces are contaminated with saliva after light curing, absorption of glycoproteins to the poorly polymerized, air - inhibited adhesive surface may cause reduction of bond strength. these glycoproteins may prevent complete infiltration of the next resin layer and sufficient copolymerization [3,1214,17 ]. saliva or blood contamination is a major clinical problem in restorative procedures, especially when the caries site is near or at the gingival margin. in the preceding clinical situation, sulcular fluid and saliva contamination can not be controlled sufficiently. therefore, in the present study unlike numerous previous reports, the specimens were not treated at all after contamination with saliva in order to evaluate the effect of contamination on bond strength when proper isolation is not possible. previous researchers have evaluated the effect of dried or rinsed - off saliva contamination [25,9,1214,16 ]., saliva - contaminated specimens were not blotted, dried or rinsed in order to study the effects of an unobservedsaliva - contaminated surface for total - etch and self - etch ahesive. in contrast to the results of our study, johnson showed that there was no significant difference in the mean shear bond strength of scotch bond mp plus between control and contaminated groups, and lowest shear bond strength belongs to a group contaminated after primer application. contrary to johnson s report in which the excess of saliva was gently shaken off and dried, in the present study saliva was not removed and the lowest shear bond belongs to the additional group contaminated with saliva after adhesive application before light curing (a3). abdalla evaluated the effect of blood and saliva contaminations on shear dentin bond strength, and demonstrated that saliva could not affect the shear bond strength of one - bottle significantly. unlike abdalla s study in which saliva contamination was removed only after etching, in the present study saliva contamination was examined during all bonding steps (after etching, after bonding, before caring, after bonding and after curing) without any treatment. few reports evaluated the effect of saliva contamination on shear bond strength of uncured adhesive between composite and dentin during bonding procedures. contrary to fritz who showed that saliva contamination blot - dried before adhesive polymerization (acetone as a solvent) could not affect the shear bond strength, in our study this value was reduced may be due to the remaining saliva and different adhesive solvents (water and ethanol). the results of our study demonstrated that the self etch adhesive with hydrophilic feature may be less sensitive to salivary contamination compared to previous generations of adhesive systems which is in agreement with the findings of previous studies for modern adhesives. however, in these investigations contaminated surfaces were treated by decontaminated methods such as blot - dry or wash off. yoo reported that saliva contamination could significantly affect the bond strength of all - in - one adhesive systems to dentin. because in their study, the adhesive layer was removed during washing and drying of unpolymerized resin, and the demineralized surface remained without infiltration of monomers. although limited studies compared shear bond strength of different generations of adhesives, abdalla stated that the shear bond strength of the fourth generation adhesive (sbmp) was higher when compared to the fifth generation, but this difference was not significant. this report is in accordance with the result of our study which stated that there are no significant differences between shear bond strengths of the fourth and fifth generation (p=0.09), and also the fifth and seventh (p=0.366). however, significant differences are observed between the shear bond strength of the fourh and seventh generation (p<0.05). in this in vitro investigation, prompt l - pop (self - etch adhesive) appears more tolerant to unnoticed saliva contamination compared to sbmp and sb (total etch adhesive). in contrast to the self etch adhesive, the total etch adhesive groups displayed a significant decrease in mean shear bond strength when contaminated with saliva, but the difference between contaminated subgroups was not significant. however, mean shear bond strengths of sbmp in contaminated or uncontaminated subgroups were higher in comparison to the other two groups. | objective : this study evaluated the effect of saliva contamination during bonding procedures without removing saliva on shear dentin bond strength of three adhesive generations when rubber dam isolation is not feasible.materials and methods : flat superficial dentin surfaces of seventy - two extracted human molars were randomly divided into three groups (a : scotch bond mp plus (sbmp), b : single bond (sb), c : prompt l - pop) according to the applied adhesives and twelve subgroups (n=6) according to the following saliva contamination applied in different bonding steps. the specimens were contaminated with saliva after etching (a1 and b1), after primer application (a2), after adhesive application before polymerization (a3, b2 and c1), and after adhesive polymerization (a4, b3 and c2). three subgroups were not contaminated as controls (a5, b4 and c3). resin composite was placed on dentin subsequently followed by thermocycling. shear test was performed by universal testing machine at 0.5 mm / min crosshead speed. the collected data were statically analyzed using one and two - way anova and tukey hsd.results:in contrast to sbmp and sb, the mean shear bond strength of promote l - pop was not significantly different between contaminated and uncontaminated subgroups. mean shear bond strengths of sbmp subgroups contaminated after adhesive polymerization or uncontaminated were significantly higher compared to the other two groups (p<0.05).conclusion : unlike promote l - pop, saliva contamination could reduce shear bond strength of the total - etch adhesives. furthermore, the step of bonding procedures and the type of adhesive seems to be effective on the bond strength of adhesive contaminated with saliva. |
increasingly important polylactide (pla), a main biodegradable and biobased candidate for replacement of petrochemical polymers, which can be produced from annually renewable resources, has the glass transition temperature tg of 5560 c. as a consequence, pla is stiff and brittle at ambient conditions, which limits its applications demanding high toughness and drawability. both optically pure poly(l - lactide) and poly(d - lactide) are crystallizable polymers, but a decrease of the optical purity lowers crystallizability of pla. slowly crystallizing plas could be quenched below tg without crystallization and cold - crystallized during heating from the glassy state. crystallinity, if developed, increases slightly the stiffness but further decreases the drawability of pla. to improve its flexibility and ductility, pla has been plasticized with various plasticizers of low and high molar mass (m), including citrate esters [3, 4 ], triacetine, poly(ethylene glycol) (peg) [511 ], and poly(propylene glycol) [12, 13 ], block copolymers of ethylene glycol and propylene glycol, poly(ethylene adipate) and poly(diethylene adipate), diethyl bishydroxymethyl malonate oligoester and oligoesteramide [16, 17 ]. by increasing pla 's chain mobility, plasticization decreases tg, yield stress and elastic modulus, and improves elongation at break. apart from the desired mechanical properties, there are other requirements like non - volatility and biodegradability of a plasticizer. since peg fulfils that prerequisite, plasticization of pla with peg was widely investigated in the past [e.g., 811 ]. however, the key aspect is stability of plasticized pla. owing to their high mobility, plasticizers with low m migrate within pla matrix. even peg with m of 1,000 g mol migrated from the bulk of plasticized pla and accumulated on its surface. the use of pegs with even higher molar masses creates other problems phase separation and crystallization of peg in pla / peg blends can occur in a degree dependent on concentration and also on m of peg [810, 18 ]. hence, although plasticization led to improvement of flexibility and ductility of pla, it usually did not allow reaching low modulus elastomeric - like behavior stable over time. for instance, hu. [9, 10 ] demonstrated that unaged pla / peg blends, containing 30 wt% of peg with m of 8,000 g mol, were low modulus elastomeric - like materials with tg at 7 c, but they aged via either crystallization of peg and pla or phase separation and, as a result, their tg increased and behavior changed over a relatively short time to higher modulus thermoplastic - like. after 30 h, the yield stress and 2 % secant modulus of elasticity of the blend reached 2 and 100 mpa, respectively whereas, after 75 days, these values further enlarged four times and tg increased to 27 c from 7 c. recently, grafting peg on maleated pla chains was explored to improve stability and compatibility between the components. however, the elastomer - like behavior was not reached ; the blend with 20 wt% of peg exhibited a relatively high yield stress of nearly 16 mpa, even though tested at a low rate of 0.06 min. although grafting of tributyl citrate on maleated pla permitted reaching a yield stress of about 7 mpa, after 6 months of aging, dynamic mechanical thermal analysis evidenced an increase of tg and signs of phase separation in this blend. plasticization is unavoidably associated with a decrease of tg, yield strength, and stiffness. toughening of pla by blending with immiscible polymers [e.g., 2123 ] allows maintaining its tg and diminishes less the yield strength and elastic modulus. it is long recognized that multiple crazing initiated by dispersed rubber phase is one of the main toughening mechanisms acting in systems with a glassy matrices, for instance, in high - impact polystyrene. the second well - known phenomenon is cavitation in dispersed inclusions of the minor component, which facilitates shear yielding of the glassy matrix [e.g., 25 ]. in pla - based blends, the cavitation either inside the inclusions or at the inclusion matrix interface was observed. it was also demonstrated that the poly(1,4-cis - isoprene) particles dispersed in pla initiated crazes, but cavitation inside the particles promoted the change of deformation mechanism of matrix to shear yielding. in this study, in order to obtain stable pla - based ductile materials, pla was melt - blended with hybrids of ethylene glycol derivatives and polyhedral oligomeric silsesquioxane (poss). posss are a relatively new class of materials, which are used for modification of polymers. poss molecules have the general formula rn(sio1.5)n, where r can be hydrogen, an organic group or polymer chain ; for the most common octameric structure, n = 8. poss cages grafted with polymer chains are multiarm polymers, which can exhibit properties and miscibility with other polymers different from their linear counterparts [e.g., 26, 27 ]. moreover, although the thermal properties of such hybrids are chain - length - dependent, they can have lower tgs than their linear analogs and decreased, or even entirely suppressed, crystallizability. we hypothesized that blending pla with multiarm polymers obtained by grafting of peo or peg arms on posss would allow obtaining a ductile material stable over time. poss polymers can be obtained through formation of direct urethane linkage between hydroxy end groups of peg and isocyanate groups of poss macromer [28, 29 ]. peg - substituted octasilsesquioxanes, prepared by the hydrosilylation of unsaturated ethylene glycol monoallyl ethers (allyl - peg) of various chain lengths (two to six repeating units) with both octakis(dimethylsiloxy)octasilsesquioxane and octahydridosilsesquioxane, were also reported. the respective allyl ethers can be synthesized through the reaction of hydroxyl end groups with allyl bromide in the presence of a base (naoh [27, 3133 ], nah, or alkali metals [30, 35 ]). in general, poss is non - biodegradable and, owing to its hydrophobic nature and inertness to hydrolysis, can slow down hydrolytic degradation of a material in which it is dispersed [e.g., 36 ]. however, poss has been demonstrated to be nontoxic and cytocompatible [37, 38 ]. recently, the use of peg poss in biomedical applications was explored, for instance for drug delivery or to obtain hydrogels designed for scaffolds for bone repair. in the present study for modification of pla two poss hybrids were used, with shorter and longer arms, being derivatives of ethylene glycol. in order to avoid crystallization - induced phase separation, pla with low stereoregularity, containing 18 wt% of d - lactide, was used. especially peg methyl ether grafted poss exhibited very good miscibility with pla, despite its relatively large molar mass of 9,500 g mol and acted as an efficient plasticizer, which allowed us to obtain transparent, elastomer - like low modulus material with excellent drawability retaining its good properties during 6 months. the study utilized pla 4060d purchased from natureworks llc (minnetonka, mn), with density of 1.24 g cm, weight average molar mass mw of 120 kg mol and polydispersity mwmn = 1.4 as determined by size exclusion chromatography (sec)with multi - angle laser light scattering detector in dichloromethane. d - lactide and l - lactide contents were 18 and 82 mol%, respectively, as determined by measurements of specific optical rotation. rn(sio1.5)n with r = ch2ch2(och2ch2)moch3, being a cage mixture with n of 8, 10, 12 and average m of 13.3 (p1), was purchased from hybrid plastics inc. density and peg content were 1.2 g cm and 92 wt%, respectively. molar mass of a single arm calculated based on m of 13.3 was equal to 644.2 g mol. mw and mwmn of p1 were 9,500 g mol and 1.3, respectively, as determined by a sec method in aqueous solution, with triple detection (ldc ri detector and viscotek t60a dual detector) on the chromatograph (knauer k-501 hplc pump) with a set of tsk - gel columns (g5000 pwxl + 3000 pwxl + 2500 pwxl) at 26 c. r8(sio1.5)8 with r = osi(ch3)2ch2ch2ch2(och2ch2)2och2ch3 (p2) was synthesized by hydrosilylation of 3-[2-(2-ethoxyethoxy)ethoxy]-propene-1 with octakis(dimethylsiloxy)octasilsesquioxane [hsi(ch3)2o]8(sio1.5)8. monoallyl ether of di(ethylene glycol) ethyl ether was obtained according to the slightly modified literature procedure. the reaction with allyl bromide was exothermic, and there was no need to increase the temperature after the addition of allyl bromide. the hydrosilylation reaction was carried out in the presence of a platinum catalyst (karstedt s catalyst). the stoichiometry of the reaction was controlled in order to obtain octasubstituted poss and not to leave any remnant of unreacted 3-[2-(2-ethoxyethoxy)ethoxy]-propene-1 in the reaction mixture. both the formed 3-[2-(2-ethoxyethoxy) ethoxy]-propene-1 and octasubstituted product were characterized by h nmr spectroscopy. the resonance signals in h nmr spectrum were attributed to fragments of side groups, and the number of repetitive glycolic units was estimated. m of octakis{3-[2-(2-ethoxyethoxy)ethoxy]-propyldimethylsiloxy}-octasilsesquioxane, calculated on the basis of h nmr spectrum was 2,409 g mol, whereas that of a single arm was 249 g mol. for clarity, further details of the synthesis of p2 tgs of p1 and p2 measured by differential scanning calorimetry (dsc) during heating at 10 c min were at 81 c and 84 c, respectively. the melting enthalpy was 76 j g, which corresponded to peg crystallinity level of 56 wt% for the peg content of 92 wt% and melting enthalpy for 100 % crystalline peg of 146.7 j g. during cooling p1 prior to blending, the components were vacuum - dried for 4 h : pla at 100 c and both poss hybrids at 90 c. melt - blends containing from 5 to 20 wt% of poss (selected compositions) were prepared using a brabender batch mixer (duisburg, germany) operating at 180190 c for 10 min at 60 rpm, under the flow of dry gaseous nitrogen. the blends will be referred to as, for example, pla / p1 - 15, where 15 stands for the p1 content in weight percent. the 0.5- and 1-mm - thick films of all the materials were prepared by compression molding at 180 c for 3 min in a hydraulic hot press followed by quenching between thick metal blocks kept at 15 c. the films were then stored in dry atmosphere (relative humidity of 10 %) in desiccators at room temperature. films of pla / p1 - 20 were stored for 6 months, and their properties were examined over this period of time. in addition, specimens of this blend were held for 4 weeks at 35 c in a sand bath in order to determine the influence of aging at elevated temperature on their thermal properties. the films were examined by dsc carried out with a ta instrument 2920 dsc (new castle, de) at a heating rate of 10 c min. tg was taken as a temperature corresponding to the midpoint of the heat capacity increment. aged pla / p1 - 20 after the first heating to 195 c was cooled to zero celsius and heated again at 10 c min. dynamic mechanical thermal analysis (dmta) was carried out in a dual - cantilever bending mode with a dmta mk iii, rheometric scientific ltd. apparatus (epsom, uk) at a frequency of 1 hz and a heating rate of 2 c min on rectangular samples, 10 mm 30 mm, cut from 1-mm - thick films. for tensile tests, oar - shaped specimens conforming to iso 5272, with a gauge length of 25 mm and a gauge width of 5 mm, were cut from 0.5-mm - thick films. strain measurements were performed on an instron tensile testing machine (high wycombe, uk) at a rate of 0.4 min (40 % min) at 25 c in a temperature chamber with circulating air. at least three specimens of each material were tested. to have an insight into the structure, cryo - fracture surfaces of the blends were studied under a jeol 5500lv and jeol 6010la (tokyo, japan) scanning electron microscopes (sem) after sputtering with gold. in addition, the blends were cryo - microtomed to produce specimens with flat and smooth surfaces, which were coated with carbon using electric arc spraying, and analyzed by sem with energy - dispersive spectroscopy (eds). the heating thermograms of the quenched films of the pla and pla - based blends are collected in fig. 1, whereas their tgs determined from dsc are listed in table 1. the heating thermograms evidenced that pla in all the materials was amorphous and unable to cold - crystallize during heating at 10 c min. all the blends exhibited tgs below that of neat pla, 57 c. while for pla / p2 blends the decrease of tg was rather moderate, by 89 c for both compositions, tg of pla / p1 blends decreased gradually with increasing p1 content and reached 16 c for pla / p1 - 20. no evidence of melting of p1 in the blends was detected, indicating that it did not form a separate phase.fig. 1dsc heating thermograms of pla, pla / p1, and pla / p2 blends. heating rate of 10 c min. thermograms shifted vertically for claritytable 1 t g of pla, pla / p1, and pla / p2 blends : t dsc measured by a dsc method and temperatures of loss modulus e peaks, t 1e, and t 2e sample code t dsc (c) t 1e (c) t 2e (c)pla5755pla / p1 - 54849pla / p1 - 103939pla / p1 - 152930pla / p1 - 201616pla / p2 - 15497750pla / p2 - 20487949 dsc heating thermograms of pla, pla / p1, and pla / p2 blends. thermograms shifted vertically for clarity t g of pla, pla / p1, and pla / p2 blends : t dsc measured by a dsc method and temperatures of loss modulus e peaks, t 1e, and t 2e temperature dependencies of loss modulus e '' and storage modulus e ' of the materials are compared in figs. 2 and 3. the e peak temperatures, which are listed in table 1, correlated closely with tgs from dsc. the e '' temperature dependencies of pla / p1 blends were also featured by single peaks, but the peak temperature decreased with increasing p1 content, from 49 c for pla / p1 - 5 to 16 c for pla / p1 - 20, which evidenced good miscibility of the components and plasticizing effect of p1 on pla. in addition, the e '' peaks of pla / p1 widened with increasing p1 content reflecting broadening of the spectrum of relaxation times ; e '' of pla / p1 - 20 started to rise even below zero celsius. contrary to that, the two separate e '' peaks indicated phase separation in pla / p2 blends. the e '' peaks at 77 and 79 c reflected the glass transitions in p2-rich phase whereas those at about 50 c corresponded to the glass transition in the pla - rich phase. the temperature of the latter, below tg of neat pla, indicated partial miscibility of the components.fig. 2temperature dependencies of loss modulus e '' of pla, pla / p1 (a), and pla / p2 (b) blendsfig. 3temperature dependencies of storage modulus e ' of pla, pla / p1 (a), and pla / p2 (b) blends temperature dependencies of loss modulus e '' of pla, pla / p1 (a), and pla / p2 (b) blends temperature dependencies of storage modulus e ' of pla, pla / p1 (a), and pla / p2 (b) blends e ' of neat pla behaved in a typical way, decreasing with increasing temperature and falling below 10 mpa in the temperature range of glass transition. below zero celsius, e ' of pla / p1 blends was close to that of neat pla for small contents of p1 but enlarged with increasing content of the modifier. e ' of each blend diminished with increasing temperature and finally dropped rapidly in the glass transition temperature range. as a result, from 10 to 70 c e ' strongly decreased with increasing p1 content and was the smallest for pla / p1 - 20. below 90 c e ' of pla / p2 - 15 and especially of pla / p2 - 20 exceeded e ' of neat pla, but, in the range of low - temperature glass transition, from approximately 90 c to approximately 60 c, it dropped below that of pla. the second and much more pronounced rapid fall of e ' occurred in the temperature range of glass transition in the pla - rich phase of the blends.. nevertheless, sem examination of cryo - fracture surfaces confirmed the phase separation in the pla / p2 blends. 4a and b. the inclusions were larger in pla / p2 - 20 than in pla / p2 - 15 with maximum size of 90 and 30 m, respectively. contrary to that, no evidence of the phase separation was found in the pla / p1 blends, even at the highest p1 content of 20 wt%, as shown in fig. 4sem micrographs of cryo - fracture surfaces of blends : pla / p2 - 15 (a), pla / p2 - 20 (b), pla / p1 - 20 (c), and x - ray si mapping of : pla / p2 - 15 (d), pla / p1 - 20 (e). magnification in (d) the same as in (e) sem micrographs of cryo - fracture surfaces of blends : pla / p2 - 15 (a), pla / p2 - 20 (b), pla / p1 - 20 (c), and x - ray si mapping of : pla / p2 - 15 (d), pla / p1 - 20 (e). magnification in (d) the same as in (e) tg of binary miscible blends is often expressed by the empirical fox equation : 1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { t_{\mathrm{g}}}^{-1}\kern0.5em = \kern0.75em { w}_1{t_{\mathrm{g}1}}^{-1}\kern0.5em + \kern0.75em { w}_2{t_{\mathrm{g}2}}^{-1 } $ $ \end{document}tg1=w1tg11+w2tg21where w1 and w2, tg1 and tg2 are the weight fractions and tgs of the blend components. figure 5 shows that the tg of homogeneous pla / p1 blends followed the fox equation. assuming the same for the continuous phase of pla / p2 blends and taking into account that tg of pure p2 was at 84 c, it can be calculated based on eq. 1 that the continuous phase of pla / p2 - 15 and pla / p2 - 20 contained about 3 wt% of p2 whereas the rest, that is, 12 wt% and 17 wt%, respectively, formed the droplets.fig. 5dependence of the glass transition temperature of pla / p1 blends determined from the dsc thermograms on blend composition. dashed line : prediction based on the fox equation. symbols : experimental data dependence of the glass transition temperature of pla / p1 blends determined from the dsc thermograms on blend composition. dashed line : prediction based on the fox equation. symbols : experimental data the stress strain dependencies of the blends are plotted in fig. 6, whereas the average values of yield stress y, stress at break b, elongation at break b, and 2 % secant modulus of elasticity are collected in table 2. neat pla yielded at y of 58 mpa and fractured early at b of 0.15 and b of 48 mpa. y and b of pla / p1 blends, ranging from 0.4 to 50 mpa, and from 25 to 36 mpa, respectively, were below those of neat pla and decreased with increasing p1 content. the stress strain dependencies of pla / p1 - 5 and pla / p1 - 10 were featured by distinct yield points at relatively high y above 40 mpa. crazing in the gauge regions of deformed specimens was visible even to the naked eye. increase of p1 content in the blends to 1520 wt% caused a dramatic improvement of ductility reflected in a drop of y and improvement b to 910. pla / p1 - 20 specimens deformed uniformly without necking and any signs of localized plastic deformation or cavitation related phenomena, in an elastomer - like manner. owing to the lack of a distinct yield point, y of pla / p1 - 20 had to be determined with 2 % offset and was equal to 0.4 mpa. the intense strain hardening in pla / p1 - 15 and pla / p1 - 20 resulted in b of about 29 and 25 mpa, respectively. appearance of strain hardening is a sign of very significant plastic deformation of the amorphous phase and straining of the chain entanglement network.fig. 6tensile stress strain behavior of pla, pla / p1, and pla / p2 blendstable 2tensile properties of pla, pla / p1, and pla / p2 blends : average and standard deviation (in brackets) values of yield stress, y, stress, b, and elongation, b, at break and 2 % secant modulus of elasticity e s sample code y (mpa) b (mpa) b e s (mpa)pla57.8 (0.8)48.2 (0.8)0.15 (0.05)1,200 (30)pla / p1 - 550.4 (0.5)36.1 (0.7)0.11 (0.02)1,130 (20)pla / p1 - 1042.7 (0.4)32.4 (5.7)0.08 (0.04)1,010 (30)pla / p1 - 1511.6 (0.3)28.8 (2.7)8.8 (0.6)330 (18)pla / p1 - 200.4 (0.1)25.1 (1.1)10.0 (0.4)30 (5.5)pla / p2 - 1528.8 (0.220.1 (0.9)2.3 (0.3)730 (30)pla / p2 - 2022.5 (0.8)14.5 (0.5)1.0 (0.4)490 (22) yield stress determined with 2 % offset tensile stress strain behavior of pla, pla / p1, and pla / p2 blends tensile properties of pla, pla / p1, and pla / p2 blends : average and standard deviation (in brackets) values of yield stress, y, stress, b, and elongation, b, at break and 2 % secant modulus of elasticity e s yield stress determined with 2 % offset es of pla / p1 blends was lower than that of neat pla, 1,200 mpa, and diminished with increasing plasticizer content to 30 mpa for pla / p1 - 20. such low value of es is consistent with the elastomeric - like behavior of this blend. pla / p2 blends exhibited a pronounced yield at y being approximately half of that of neat pla. b was for 2.3 pla / p2 - 15 but diminished to 1 for pla / p2 - 20. es values of the blends were lower than that of neat pla but exceeded es of pla / p1 blends with the same plasticizer contents. although the decrease of tg of pla / p2 blends to 4850 c undoubtedly increased segmental mobility of pla chains, it can not be the only reason of the improved ductility of these materials because plasticized pla becomes ductile when its tg decreases to at least 35 c. pla / p1 - 5 blend with tg at 48 c exhibited relatively high y, and b even smaller than that of neat pla. during drawing, an intense stress whitening in both pla / p2 blends sem and plm micrographs of pla / p2 - 15 specimen, which fractured at elongation of 2.6 and for sem was cryo - fractured parallel to the drawing direction, in fig. 7, demonstrate a heavily crazed matter with a network of crazes. the crazes were obviously originated and terminated by inclusions, although empty holes where p2 was accumulated are seen rather than the inclusions. the holes are elongated in the drawing direction and have undulant surface, resulting most probably from the localization of plastic deformation in crazes and also from the strong post - break shrinkage of the tensile specimen, which reduced the strain to about half of that at break. as reported so far, toughening of pla by blending with immiscible polymers was associated mainly with cavitation either in the dispersed particles or at the particle matrix interface, which facilitated shear yielding of pla matrix. in kowalczyk and piorkowska, poly(1,4-cis - isoprene) particles initiated crazes in pla matrix, but cavitation inside the particles promoted shear yielding. in the present study, neither sem nor plm provided evidence of shear bands in the deformed pla / p2 - 15 blend ; all crazes were well aligned perpendicular to the drawing direction. moreover, the plastic deformation, although concentrated in the gauge region, proceeded without necking. it was also noticed that crazing occurred at the very beginning of the deformation process. according to refs. [43, 44 ], pools of plasticizing diluents dispersed in glassy polymer can promote crazing because the diluent from pools that are tapped by an advancing craze spreads along the craze border between the craze fibrils, driven by capillary forces, and sorption of the diluent into plastically deforming base region of the craze fibrils occurs, enhanced by the plastic flow of polymer. the resulting local plasticization decreases the craze flow stress and therefore facilitates propagation of crazes. in pla / p2 blends, the liquid inclusions of p2 not only initiated crazes but also promoted their propagation in matrix via such local plasticizing effect. this mechanism could act at the very beginning of craze formation because, unlike in argon and piorkowska., the inclusions initiating crazes were liquid pools themselves. the drainage of liquid modifier into crazes left emptied holes in matrix which could easily elongate in the drawing direction. observed that to toughen polystyrene, liquid pools of low - molar - mass polybutadiene had to be of submicron size. however, an increase of p2 content from 15 to 20 wt% had a detrimental effect on b, most possibly because of an excessive increase in the inclusion size, similarly as reported for other pla - based blends, e.g., as too - large inclusions can promote early fracture. nevertheless, pla / p2 - 15 blend exhibited good drawability while maintaining the relatively high tg, yield strength and stiffness, typical of a rubber toughened pla rather than of a plasticized pla. in contrast to other phase - separated pla - based blends, both pla / p2 blends were transparent.fig. 7sem (a, b) and plm (c) micrographs of the gauge zone of pla / p2 - 15 tensile specimen, which fractured at a strain of 2.6 and for sem was cryo - fractured parallel to the drawing direction. the drawing direction, horizontal sem (a, b) and plm (c) micrographs of the gauge zone of pla / p2 - 15 tensile specimen, which fractured at a strain of 2.6 and for sem was cryo - fractured parallel to the drawing direction. [9, 10 ], the most intense aging phenomena occur in pla / peg blends with tg below ambient temperature and slow down when their tg increases. only the homogeneous pla / p1 - 20 blend had tg below room temperature, at 16 c. the temperature dependencies of e '' measured after 1 day, 15 days, and 6 months of aging at ambient temperature are plotted on a linear scale in fig. the curves did not exhibit any marked differences ; after 6 months of aging, the e '' peak temperature was 16 c, the same as for unaged blend. nevertheless, as illustrated in fig. 8b, aging caused gradual changes in the tensile behavior. after 6 months, y of pla / p1 - 20 increased to 1.4 mpa, whereas b and b decreased to approximately 23.5 mpa and 8, respectively. the dsc heating thermogram recorded after 6 months of aging presented in fig. 8c exhibited a small melting peak at 60 c and a broad endotherm ending near 140 c. a complex melting behavior of low optical purity plas, with multiple melting peaks, was reported previously by others. the melting enthalpy of the aged pla / p1 - 20 was about 7 jg, which corresponds to the crystallinity level of 7 wt% if the enthalpy of fusion for the alpha orthorhombic form of pla, 106 j g, is assumed. the presence of that amount of stiff crystalline phase of pla was obviously responsible for the change in mechanical properties of pla / p1 - 20. in general, crystallization increases plasticizer content in the amorphous phase and can therefore affect tg, but the crystallinity level in aged pla / p1 - 20 was too small to cause a noticeable change in tg of the blend. it can be also seen that the second heating thermogram of the aged blend does not differ from the first heating thermogram of the unaged blend, as shown in fig. 1, indicating that no irreversible changes in the thermal behavior of the material occurred upon aging.fig. 8effect of aging at room temperature on loss modulus e '' temperature dependence (a), tensile properties (b), and thermal properties (c) of pla / p1 - 20 blend. c shows the first and the second dsc heating thermograms of the blend after aging for 6 months at room conditions (solid line) and after aging for 28 days at 35 c (dotted line). thermograms shifted vertically for clarity effect of aging at room temperature on loss modulus e '' temperature dependence (a), tensile properties (b), and thermal properties (c) of pla / p1 - 20 blend. c shows the first and the second dsc heating thermograms of the blend after aging for 6 months at room conditions (solid line) and after aging for 28 days at 35 c (dotted line). thermograms shifted vertically for clarity despite the small morphology changes upon aging, the tensile behavior of pla / p1 - 20 remained elastomer - like with the low tg, small y, large b, and strong strain - hardening leading to high value of b. it can be concluded that, after 6 months of aging, the blend retained very good ductility, flexibility, and also transparency. during aging at 35 c pla in pla the first heating thermogram recorded after 7 days (not shown) and 4 weeks (fig. the melting enthalpy after 7 days of aging was about 13 j g which corresponds to the crystallinity of 12 wt%. longer aging did not increase the crystallinity of the blend any further ; after the next 21 days, it was at the same level. it can be seen that the first heating thermogram of the blend aged at 35 c differs from that of the blend aged at room temperature only above 50 c, where the melting of the crystalline phase occurs. the second heating thermogram of pla / p1 - 20 aged at 35 c differs neither from that of the blend aged at room temperature nor from that of the unaged blend. it appears that, although aging at 35 c accelerated crystallization of pla, it did not result in any irreversible changes in the thermal behavior of the blend, similar to aging at room conditions. novel blends of polylactide with ethylene glycol derivatives engrafted poss were prepared by simple blending and studied. the first of them, p1, was a commercial poss with peg methyl ether arms, whereas the second, p2, was synthesized by hydrosilylation reaction of 3-[2-(2-ethoxyethoxy)ethoxy]-propene-1 with octakis(dimethylsiloxy) octasilsesquioxane [hsi(ch3)2o]8(sio1.5)8. both types of hybrids efficiently modified the mechanical properties of pla. poss substituted with peg methyl ether arms, ch2ch2(och2ch2)moch3 (average m of 13.3), was well miscible with pla, despite its relatively large molar mass (mw of 9,500 g mol), and acted as a plasticizer, efficiently decreasing tg ; the blends with up to 20 wt% of the plasticizer were transparent and exhibited single glass transitions. the blends with 15 and 20 wt% of the plasticizer having tg of 29 and 16 c, respectively, were ductile. pla / p1 - 20 was transparent and elastomeric - like material with low y of 0.4 mpa and b of 10 (1,000 %), and es of only 30 mpa, two orders magnitude lower that that of neat pla. although during 6 months aging at room temperature a small crystallinity level of 7 wt% developed in this blend, resulting in an increase in y and es, the elastomeric - like behavior was retained. the blend remained homogeneous and retained its good drawability as well as flexibility after 6 months of aging at room temperature : es well below 100 mpa, y below 2 mpa, and a large b of 8 (800 %). increase of aging temperature to 35 c accelerated crystallization of pla in the blend, but the crystallinity level reached was small, about 12 wt%. we note that using pla with a higher d - lactide content, unable to crystallize, will allow achieving even better stability. miscibility of poss with relatively short arms of osi(ch3)2ch2ch2ch2(och2ch2)2och2ch3 comprising osi with pla was worse. pla / p2 blends contained distinct inclusions of p2-rich phase, exhibiting a separate glass transition below 70 c. tg of the continuous phase in pla / p2 - 15 and pla / p2 - 20 was only several degrees below that of neat pla. it fractured at b of about 2.3 (230 %) and exhibited y and es approximately two times smaller than those of neat pla. such mechanical behavior is typical of rubber - toughened pla rather than of plasticized pla. the liquid inclusions initiated crazes and also promoted their growth, thus toughening the material. unlike majority of pla - based phase - separated blends, pla / p2 blends were transparent. synthesis of octakis{3-[2-(2-ethoxyethoxy)ethoxy]-propyldimethylsiloxy}-octasilsesquioxane (p2) commercially available octakis (dimethylsiloxy)octasilsesquioxane [hsi(ch3)2o]8(sio1.5)8 was obtained from hybrid plastics inc. and used without further purification. allyl bromide (99 % reagent plus, aldrich), sodium hydroxide (micropills, pure p.a. poch s.a.), and di(ethylene glycol) ethyl ether (> 99 %, safc) were used as received. the hydrosilylation catalyst, a platinum tetramethyldivinyldisiloxane complex (karstedt s catalyst) (2 % solution in xylene, low color), was purchased from abcr gmbh. toluene, which was used as a solvent in the hydrosilylation reaction, was carefully dried according to the literature procedures and distilled prior to use. dichloromethane and methanol (both of analytic grade) were used as received from poch s.a. liquid - state h nmr spectra of precursors and condensed soluble materials were recorded in cdcl3 as a solvent on drx-500 mhz spectrometer. gas chromatography (gc) analysis was performed on hewlett - packard 6890 chromatograph fitted with a 30-m capillary column hp-1 hp 190592023 and equipped with a thermal conductivity tdc detector. the carrier gas was helium, and the flow rate was 5 ml min. the temperature was changed from 60 to 240 c at the rate of 10 c min. the 40.0 g (1.0 mol) of sodium hydroxide and 90 ml (0.67 mol) of 2-(2-ethoxyethoxy) ethanol (etoch2ch2och2ch2oh) were placed in a three - necked flask equipped with a dropping funnel, a thermometer, a condenser, and a tube with cacl2. the mixture was stirred vigorously for 15 min at room temperature with a magnetic stirrer, and it turned yellow after that time. allyl bromide (102 ml, 1.17 mol) was placed in the dropping funnel and added slowly to the mixture. once the addition of allyl bromide was finished, the reaction mixture was stirred at room temperature for another 16 h. the mixture was filtered to remove solids (nabr and unreacted naoh). the aqueous fractions were collected and washed with dichloromethane (4 100 ml). after filtration, the fractions were combined, and the volatiles were removed under reduced pressure. the residue was dried at room temperature under vacuum (1 10 mm hg), to give 98.6 g of pure 3-[2-(2-ethoxyethoxy) ethoxy]-propene-1 as indicated by gc analysis (the reaction yield 84.5 %). [hsi(ch3)2o]8(sio1.5)8 (20.05 g, 0.0179 mol) and 3-[2-(2-ethoxyethoxy)ethoxy]-propene-1 (25.0 g, 0.1435 mol) were placed in a flask equipped with a condenser, an inlet of argon, and a magnetic stirrer and dissolved in dry toluene (170 ml). the solution of pt(0)-divinyltetramethyldisiloxane (0.5 ml, 2 % pt) was added to the stirred mixture at room temperature. after 1 h, the temperature was increased to 80 c, and the mixture was kept at this temperature for 48 h. the progress of addition of sih to alkene bonds was followed with h nmr spectroscopy. the polymeric product was collected and dried to constant weight at room temperature under vacuum (0.1 mm hg), to give 42.83 g of pure octakis{3-[2-(2-ethoxyethoxy)ethoxy]-propyldimethylsiloxyl}-octasilsesquioxane (the reaction yield 95.1 %). h nmr (cdcl3) (ppm)0.15 s sime2 (48h), 0.5 m och2ch2ch2 (16h), 1.2 t ch3ch2o (24h), 1.6 m och2ch2ch2 (16h), 3.4 m och2ch2ch2 (16h), 3.5 m ch3ch2o (16h), 3.6 m och2ch2o (64h). | polylactide (pla), a main biodegradable and biobased candidate for the replacement of petrochemical polymers, is stiff and brittle at room conditions. it is therefore of high interest to formulate new pla - based materials suitable for applications demanding flexibility and toughness. in this work, novel blends of pla with polyhedral oligomeric silsesquioxanes (poss) grafted with longer (p1) and shorter (p2) arms of ethylene glycol derivatives were prepared and studied. it was hypothesized that, owing to their architecture with the central poss cage grafted with arms, miscibility and stability of the blends could be improved. indeed, pla / p1 blends were homogeneous despite p1 relatively high mw of 9,500 g mol1. the blend with 20 wt% of p1, having tg at 16 c, was transparent and flexible, elastomer - like material with excellent drawability. the blend remained homogeneous and retained its good drawability as well as flexibility during 6 months of aging at room temperature : a 2 % secant modulus of elasticity well below 100 mpa, a low yield stress below 2 mpa, and and a large strain at break of 8 (800 %). contary to that, pla / p2 blends were only partially miscible. nevertheless, owing to the liquid state of the dispersed phase, the blend with 15 wt% of p2 was transparent and ductile, with tg at 49 c, a relatively high yield strength of 29 mpa, and a large strain at break of 2.3 (230 %). the toughening mechanism involved the initiation of crazes and facilitation of their propagation by the liquid inclusions via the local plasticization effect. |
roughly 3040% of patients with pancreatic ductal adenocarcinoma (pdac) have locally advanced disease. their prognosis is not really different from that of patients with metastatic pdac. in about one third of the patients, if complete (r0) resection is possible, patients have a similar overall survival (os) as those with an initially resectable pdac. these are the results of a meta - analysis of 111 trials with a total of 4,394 patients and mainly 5-fluorouracil- or gemcitabine - based neoadjuvant chemoradiotherapy. initially nonresectable patients reached an estimated median os of 20.5 months (range 962 months) following resection. recently, 2 large phase iii trials in patients with metastatic pdac demonstrated encouraging improvements in clinical benefit for 2 combination therapies over single - agent gemcitabine [2, 3 ]. in one trial, the combination of oxaliplatin, 5-fluorouracil, leucovorin and irinotecan (folfirinox) significantly improved median os to 11.1 months as compared to 6.8 months for gemcitabine [hazard ratio (hr) 0.57 ; 95% confidence interval (ci) 0.450.73, p 180) by the pancreatic tumor. thus, the locally advanced pdac could be classified as pt4 nx cm0 g2. during surgery, the patient received a biliodigestive anastomosis using roux - en - y anastomosis and a central venous port system. three weeks after surgery, intensified neoadjuvant chemotherapy was started with the objective to achieve secondary resectability. the patient was given 2 cycles of a combination of nab - paclitaxel plus gemcitabine. nab - paclitaxel was given at a dose of 125 mg / m on days 1, 8 and 15, gemcitabine at a dose of 1,000 mg / m on days 1, 8 and 15, both repeated on day 29. chemotherapy was tolerated well. the follow - up pet - ct showed no more pet - avid lesions (fig. oxaliplatin was administered at a dose of 85 mg / m on day 1, leucovorin at a dose of 400 mg / m on day 1, and irinotecan at a dose of 180 mg / m on day 1 followed by continuous infusion of 5-fluorouracil over 46 h at a dose of 2,400 mg / m. side effects were grade 1 neutropenia, grade 2 fatigue, grade 1 diarrhea and grade 1 peripheral neuropathy. the follow - up pet - ct confirmed the complete metabolic remission ; yet, it also showed a persistent tumor abutment of the celiac trunk, formally still unresectable according to nccn guidelines (fig. no vital tumor cells were found in the biopsies taken intraoperatively (frozen sections) of the tissue surrounding the celiac trunk and the dhc stump. thus, a pylorus - preserving resection of the pancreatic head, regional lymphadenectomy, pancreaticojejunostomy and antecolic duodenojejunostomy were performed. thus, the postoperative pathology result was ypt3 (2.2 cm) ypn1 (2/33 ; 1 mm) l0 less than 10% vital tumor cells were found in the resection specimen, corresponding to evans regression score grade iii. subsequently, the patient was given 2 cycles of adjuvant chemotherapy with gemcitabine from july to september 2013. the patient was alive 18 months after the primary diagnosis at the last follow - up visit in april 2014. our case shows that secondary r0 resection can be achieved with neoadjuvant chemotherapy utilizing modern combination regimens. this is relevant as patients with an initially unresectable locally advanced pdac have a similar prognosis to patients with a primarily resectable pancreatic cancer if r0 resection can be achieved. however, it needs to be stressed that the concept of neoadjuvant therapy in locally advanced unresectable pdac and its actual clinical benefits still have to be proven, since evidence from prospective, randomized trials is still lacking. after a follow - up of 18 months, the neoadjuvant chemotherapy we chose was actually a sequence of the 2 most active regimens for metastatic pdac known today. however, she already showed complete metabolic remission after 2 cycles of nab - paclitaxel plus gemcitabine. in the pivotal trial in metastatic pdac, patients with a biochemical response after nab - paclitaxel plus gemcitabine had a better outcome than patients without a pet - response. there are also data suggesting that a metabolic pet response may be of predictive value for patients with locally advanced pancreatic carcinoma treated with definitive concurrent chemoradiotherapy. it is unclear to what extent the sequential treatment with folfirinox improved clinical outcome or whether neoadjuvant chemotherapy with the combination of nab - paclitaxel plus gemcitabine alone would have sufficed. this will be the objective of the randomized phase ii trial neolap that will start in september 2014. in this trial, patients with locally advanced, initially unresectable or borderline - resectable pdac will either receive neoadjuvant chemotherapy with 4 cycles of nab - paclitaxel plus gemcitabine or 2 cycles of nab - paclitaxel plus gemcitabine followed by 4 cycles of folfirinox the primary tumor of our patient showed extensive stromal desmoplasia which is a typical feature of pdac. the concept of stromal depletion has been identified as a promising therapeutic route in the treatment of pancreatic cancer. preclinical data show that the treatment with nab - paclitaxel results in depleting desmoplastic stroma and reducing stromal density of pancreatic tumor tissue [10, 11 ]. the effect of stromal depletion was highest with the combination of nab - paclitaxel and gemcitabine. there is also clinical evidence from a neoadjuvant trial in 16 patients with resectable pancreatic cancer treated with 2 cycles of nab - paclitaxel plus gemcitabine before surgical resection. of the 12 patients who completed treatment, histology showed a change in the architecture of the tumor stroma as indicated by markedly disorganized collagen and a very low density of cancer - associated fibroblasts, which was not observed in the untreated or conventionally treated control groups. a preclinical, co - clinical study in a mouse model showed that these effects were specific of nab - paclitaxel and not of gemcitabine. the precise mechanism of stroma depletion by nab - paclitaxel is currently not known and has to be defined in further preclinical and clinical studies. among the multitude of stromal components, the matricellular glycoprotein secreted protein acidic and rich in cysteine (sparc) has attracted particular interest. however, despite encouraging data from previous phase i / ii trials, extensive sparc analysis within a phase iii trial (mpact trial) failed to demonstrate any prognostic or predictive role of stromal and/or tumor sparc expression in the metastastic setting. in conclusion, chemotherapy with nab - paclitaxel plus gemcitabine followed by folfirinox was an effective and well - tolerated neoadjuvant treatment in a patient with initially irrresectable, locally advanced pdac which resulted in secondary resectability (r0 resection). not only the clinical benefit of sequencing both combination regimens as neoadjuvant therapy for patients with locally advanced pdac, but also the general concept of neoadjuvant treatment in locally advanced pancreatic cancer needs to be assessed in future prospective clinical trials. theyare fully responsible for the entire content and all editorial decisions relating to this paper. | the prognosis of patients with locally advanced pancreatic cancer can be improved if secondary complete (r0) resection is possible. in patients initially staged as unresectable this may be achieved with neoadjuvant treatment which is usually chemoradiotherapy based. we report the case of a 46-year - old patient with an unresectable, locally advanced pancreatic cancer (pt4 nx cm0 g2) who was treated with a sequential neoadjuvant chemotherapy regimen consisting of 2 cycles of nab - paclitaxel plus gemcitabine followed by 4 cycles of folfirinox. neoadjuvant chemotherapy resulted in secondary resectability (r0 resection). after 2 cycles of nab - paclitaxel plus gemcitabine, the patient already had a complete metabolic remission as measured by integrated fludeoxyglucose (18f) positron emission tomography and computerized tomography. after a follow - up of 18 months the patient is alive without progression of disease. we propose to assess the clinical benefit of sequencing the combinations nab - paclitaxel plus gemcitabine and folfirinox as neoadjuvant therapy for patients with locally advanced and initially unresectable pancreatic cancer in a controlled clinical trial. |
duodenal tumors are extremely rare, with only 0.1% prevalence according to upper gastrointestinal endoscopy screenings [1, 2 ]. duodenal tumors may be adenomas or adenocarcinomas ; however, most are adenomas in the descending portion. patients with ampullary tumors typically present with jaundice or pancreatitis due to obstruction of the common bile duct, sometimes even in the early stages.. they may present as an obstruction of the duodenum or bleeding at advanced stages. it is, therefore, important to obtain biopsy specimens from portions of adenocarcinoma to ensure their accurate diagnosis. we diagnosed a case of duodenal adenocarcinoma with a biopsy specimen obtained from a depressed lesion on the duodenal tumor. the findings of our case suggest that depressed lesions may be suitable targets for biopsy. an 82-year - old woman regularly visited national hospital organization shimoshizu hospital for rheumatoid arthritis. she underwent abdominal ultrasonography screening (ssa-700a ; toshiba medical systems, otawara, japan) with a 3.75-mhz curved - array probe (pvt-375bt ; toshiba medial systems). 1a). to investigate the possibility that the pancreatic duct was stenotic or obstructed, she underwent magnetic resonance cholangiopancreatography (achieva, software version 3.2.2. ; philips medical systems, best, the netherlands). this imaging indicated that the dilated pancreatic duct was not stenotic or obstructed (fig. a duodenoscopy (jf-260v ; olympus, tokyo, japan) revealed an elevated lesion around the papilla of vater (fig. upper gastrointestinal series showed that the lesion had an irregular surface and was located in the descending portion of the duodenum (fig. endoscopic ultrasonography (gf - uct260 ; olympus) was performed to investigate the depth of the tumor invasion of the common bile and pancreatic ducts. a 20-mhz mini - probe (um-3r-3 ; olympus) was used to examine the depth of invasion of the wall of the duodenum. the border between the mucosa and submucosa was not clear ; however, the border between the submucosa and muscularis propria was clear (fig. this finding suggested that the tumor was restricted to the mucosa and submucosa, and that the muscularis propria was intact. a magnifying endoscopy was used to examine the surface of the tumor (gif - h260z ; olympus). another biopsy specimen obtained from a non - depressed portion of the elevated lesion (fig. no additional symptoms developed, such as jaundice, bleeding, or obstruction of the duodenum. it is, however, difficult to target a particular portion of the adenocarcinoma with upper gastrointestinal endoscopy. random biopsy of duodenal tumors is not practical for the efficient differentiation of adenomas and adenocarcinomas. magnifying endoscopy narrow - band imaging has been proposed as a method to investigate duodenal tumors to diagnose adenomas or adenocarcinomas [12, 13 ]. reported that pinecone / leaf - shaped villi or irregular / non - structured duodenal tumor surfaces strongly correlate with adenomas or adenocarcinomas. classified tumor surfaces as monotype or mixed - type on the basis of the presence of a single pattern. however, despite these efforts, it remains difficult to differentiate adenomas from adenocarcinomas by observation with upper gastrointestinal endoscopies. it is also difficult to target a portion of adenocarcinoma for biopsy. in our patient, a biopsy specimen from the depressed lesion on the tumor surface was diagnosed as an adenocarcinoma. however, a biopsy specimen from a non - depressed lesion was diagnosed as an adenoma. however, they did not analyze the diagnoses of biopsy specimens obtained from depressed lesions of duodenal tumors. to our knowledge, no reports have compared the diagnoses of biopsy specimens from depressed and non - depressed lesions. in our case, the depressed lesion was very clearly an adenocarcinoma. our case also suggests that depressed lesions should be targeted for a biopsy for the diagnosis of adenocarcinomas. based on the findings of this report, biopsy specimens should be obtained from depressed lesions on elevated lesions in the duodenum. this report was approved by the ethics committee of the national hospital organization shimoshizu hospital. the patient 's records were anonymously and retrospectively analyzed. written informed consent was obtained for magnetic resonance cholangiopancreatography, upper gastrointestinal endoscopy with duodenoscopy and magnifying endoscopy, endoscopic ultrasonography, and mini - probe. | biopsies are necessary for the management of duodenal tumors. however, the most suitable targets for biopsy are not known. an 82-year - old woman who regularly visited our hospital for rheumatoid arthritis underwent abdominal ultrasonography. this screening revealed a dilated pancreatic duct. magnetic resonance cholangiopancreatography was performed, and dilatation of the pancreatic duct was confirmed. the patient underwent duodenoscopy to investigate the possibility of obstruction of the papilla of vater. the examination revealed an elevated lesion around the papilla of vater. endoscopic ultrasonography and a 20-mhz mini - probe were used to investigate the depth of the invasion. the common bile and pancreatic ducts were intact. the mucosal and submucosal borders were indistinct ; however, the border between the submucosa and muscularis propria was clear, suggesting that the muscularis propria was intact. magnifying endoscopy was used to examine the surface of the elevated lesion, which revealed a depressed lesion. a biopsy specimen of the depressed lesion was taken, and the tumor was diagnosed as an adenocarcinoma. another biopsy specimen from a non - depressed lesion was diagnosed as an adenoma. the patient was diagnosed with duodenal adenocarcinoma, and was recommended surgery. she declined surgery and was followed up for 34 months. because it is possible for depressed lesions of duodenal tumors to be adenocarcinomas, biopsy specimens should be obtained from depressed lesions of duodenal tumors. |
malignant melanoma (mm) is a skin malignant tumor which derives from melanocytes and can originate in any part of the body that contains these cells. primary extracutaneous mm is rare and can present in ocular, rectal, mucosal, under the nail, conjunctival, vaginal, urogenital, esophageal, and meningeal locations. in the respiratory system most cases are metastases. treatment strategies of mm include surgery, radiotherapy, chemotherapy and the latest management option is biological treatment. surgical excision of the primary lesion can be curative only for patients with localized disease. in patients who present with disseminated disease, the treatment aims to extend survival and improve quality of life. in this article we present 3 cases of the respiratory system malignant melanomas, two primary lung melanomas and one metastatic melanoma originated from vaginal mucosa. a 69-year - old female was admitted to the department of pulmonology with a history of persistent cough and exertional dyspnea. chest x - ray showed atelectasis of the right upper lobe and solid tumor in the same area. computed tomography (ct) revealed a large solid and cystic lesion in the upper right lobe expanding from the apex along the anterior chest wall. additionally paratracheal, inferior tracheobronchial, sub- and supraclavicular lymph nodes were enlarged (fig. 1). positron emission tomography (pet) scan showed no other malignancy except for findings in ct. bronchoscopy exposed a darkly pigmented, endobronchial mass obstructing the right upper lobe bronchus, visually mimicking thrombus. cytological examination revealed non - small cell cancer, but full histopathological examination resulted in the diagnosis of malignant melanoma. immunohistochemistry of tissue biopsy tested positive for s100 protein, hmb45, melanin a and negative for panck, ttf1, ck7, p63 and ck5/6 (fig. 2). the patient was qualified to adjuvant chemotherapy, unfortunately progression of the disease was observed and the patient died 6 months after diagnosis. solid cystic mass in the upper pole of the right hilum a) he stain : bronchial mucosa infiltrated by epithelioid tumor cells. a 63-year - old male with a medical history of hypertension and diabetes mellitus presented in the emergency department with right - sided hemiparesis lasting for 2 days. computed 3), chest x - ray revealed a round shadow in the middle zone of the left lung. for further assessment the patient was admitted to the department of internal medicine. physical examination was unremarkable except for a right - sided hemiparesis, no skin lesions suggestive of skin cancer were found. computed tomography of the chest showed solid tumor in the upper lobe of the left lung and one similar smaller lesion in the same lung (fig. due to disseminated lesions in the central nervous system and the right lung the patient was disqualified from surgery. at that moment, because of hemorrhagic changes in the brain radiotherapy was not indicated either. treatment with systemic corticosteroids was introduced and improvement of hemiparesis was observed although the general condition of the patient gradually deteriorated. due to no available effective management further examinations were not performed. with the diagnosis of stage iv malignant melanoma in the stable clinical condition lesion in the left parietal lobe with hyperdense fluid - fluid level, surrounded with white matter edema that could be suspicious for the brain metastasis with the hemorrhage male, 63-year - old, right - sided hemiparesis. computed tomography examination of the chest after i.v. large rounded mass in the 3 segment of the left lung surrounded with ground - glass opacity. a 50-year - old female after surgically treated mm of vaginal mucosa 7 years before and reoperation for local recurrence 4 years later presented with persistent cough. computed tomography of the chest revealed 3 circular solid tumors (0.6 cm in diameter) in the right lung and 2 similar lesions in the left lung (fig. 5). positron emission tomography scan showed increased glucose metabolism in the same locations and additionally in the iliac lymph nodes. in poland, mm is responsible for 1.7% cancer morbidity in men and 1.9% in women but these numbers have been still growing for the past three decades. at the time of diagnosis in approximately 80% of patients melanoma is a localized skin lesion, 15% of patients present with regionally advanced disease and 5% with disseminated cancer [3, 4 ]. no data regarding occurrence of primary extracutaneous mm in our country are available. in the literature only 4 - 5% of all primary melanomas most frequently they originate from the mucous membranes lining the respiratory, digestive, and genitourinary tracts or in the eyes as well as in the cerebral meninges. among lung tumors, frequently cited criteria for diagnosing primary melanoma of the lung proposed by allen and darsh and others in the 1960s include pathological (1 junctional change with a dropping off or nesting of malignant cells containing melanin just beneath the bronchial epithelium ; 2 invasion of the bronchial epithelium by the melanoma cells in an area where the bronchial epithelium is not ulcerated ; 3 an obvious melanoma beneath the above described changes) as well as clinical features (1 no history of a cutaneous, mucous membrane or ocular melanoma ; 2 solitary lung tumor ; 3 absence of any other tumor at the time of diagnosis) [2, 4, 5 ]. presented case 1 meets the above criteria. the patient had no history of neoplasm, pet scan excluded other malignancy locations except for the respiratory system, the lesion presented as solitary, dark pigmented tumor obstructing bronchus, histopathological staining was typical for melanoma cells. case 2 can not be so unfailingly considered as primary lung melanoma because of lack of examinations certainly excluding other origin of mm, however solitary lung tumor with no pigmented skin lesions suggests that it could be primary disease. a complete endoscopic or imaging diagnostic procedure in this case was not performed due to severe and gradually worsening general condition of the patient. we included this case study in this article because of primary origin in vaginal mucosa which is quite rare. it occurs in less than 1% of all malignant melanomas and less than 3% of all primary malignant tumors of the vagina. contemporary treatment of malignant melanoma includes surgery, radiotherapy and systemic therapy with chemo- and immunotherapy. as there are no specific guidelines for primary lung mm recommendations for cutaneous melanoma the first line treatment is surgical excision of the primary lesion with an oncologically adequate margin. radiotherapy can be beneficial in particular locations (head and neck) as palliative management in order to prevent or delay hemorrhagic complications and neurologic symptoms. in presented case 2, the patient already has hemorrhagic changes in central nervous system which disqualified him from this type of treatment. adjuvant therapy with interferon -2b should be considered on a case - by - case basis. although data show prolonged median survival [3, 7, 8 ] these drugs are still investigated. according to polish guidelines in 2013 patients with advanced melanoma unfortunately most of these clinical trials exclude patients with brain metastases which require steroids, as in our case 2. results of treatment of disseminated mm also remain unsatisfactory, median survival is about 6 - 10 months. primary lung melanoma should be considered in cases with no extrapulmonary disease which presents as a solitary lesion especially when bronchoscopy reveals dark pigmented and endobronchial mass. | extracutaneous locations of primary malignant melanoma are rare. in the respiratory system most melanomas present as metastatic tumors. for the diagnosis of primary lung melanoma, strict histopathological and clinical criteria should be met. in this paper we present three cases of malignant melanoma which showed in the respiratory system. the first 2 case studies present primary lung melanomas, while the last one shows late lung metastasis of tumor originated from vaginal mucosa. the treatment of choice for localized disease as well as single metastasis is surgical excision. |
to generate -cell specific pkckn transgenic mouse lines, the rip - i / pkckn chimeric gene containing the k376r mutation was excised from the plasmid, purified, and microinjected into fertilized eggs as described previously (10,11). two transgenic mouse lines (# 179 and # 162) on a c57bl/6 background were selected and analyzed. for in vivo experiments control mice (wild type) were littermates of transgenic mice. all animal experiments were done in accordance with the accepted standard of human care of animals and were approved by the local animal care and use committee. ins-1e cells were infected with a retrovirus containing the wild - type or pkckn construct. transfected cells were selected by geneticin (g418) and subcloned by separation of single cells. mice were fed either a standard diet or hfd and kept under a light / dark cycle of 12/12 h. hfd consisting of 45% kcal fat from lard was fed to 4-week - old mice for 8 weeks (d12451 ; research diet, new brunswick, nj). blood glucose was determined in overnight - fasted mice using a glucometer elite (bayer, elkhart, in). glucose tolerance tests (intraperitoneal glucose tolerance tests [ipgtts ]) were performed in overnight - fasted mice. animals were injected intraperitoneally with a single dose of d - glucose (2 g / kg body wt), and blood glucose concentrations were detected at the indicated times. for glucose - stimulated insulin release, a d - glucose dose of 3 g /kg body wt was used. to determine insulin tolerance, a bolus of human insulin (1 unit / kg body wt) was injected intraperitoneally into fed mice and glucose concentrations were determined. mouse islets were isolated and cultured as described previously (12). in brief, after culture islets were preincubated for 1 h at 37c in modified krebs - ringer bicarbonate buffer containing (in mmol / l) 140 nacl, 5.6 kcl, 1.2 mgcl2, 2.6 cacl2, 10 hepes, 2.8 glucose, and 4 g / l bsa (ffa free ; sigma, deisenhofen, germany), ph 7.4. thereafter, batches of 5 islets/0.5 ml were incubated for 30 min at 37c in the presence of test substances as indicated for each experiment. insulin released into the supernatant and insulin content after extraction with acid ethanol (hcl 1.5% [vol / vol]/ethanol 75% [vol / vol ]) were measured by radioimmunoassay. activation of caspase-3 was examined by immunohistochemical staining against cleaved caspase-3 in pancreatic slices of mice fed standard diet or hfd. sections of frozen pancreatic tissue were fixed with 4% paraformaldehyde in pbs for 20 min, permeabilized with 0.2% triton x-100 for 2 min on ice, and then blocked with 10% fcs in pbs for 45 min. primary antibody against cleaved caspase-3 (1:200 ; cell signaling technology, danvers, ma) was applied overnight in pbs supplemented with 10% fcs. after washing with pbs supplemented with 10% fcs, the slices were incubated for 1 h with an anti - rabbit secondary antibody (1:400 ; alexa - fluor546 igg, invitrogen, paisley, u.k.). transferase - mediated dutp nick - end labeling (tunel) staining was performed in isolated islet cells that were prepared as described previously and cultured in rpmi-1640 supplemented with 10% fcs, 10 mmol / l hepes, 1 mmol / l na pyruvate, 2 mmol / l l - glutamine, 100 iu / ml penicillin, and 100 g / ml streptomycin (13). prior to the addition to the culture medium palmitate from a 200 mmol / l stock solution dissolved in dmso was coupled to fcs at a concentration of 6 mmol / l. the palmitate - to - albumine ratio was 10:1, with the final concentration of dmso of 0.3%. after 3 days of culture in the presence of 0.6 mmol / l palmitate, apoptosis was quantified in islet cells by tunel staining using a commercial kit (roche diagnostics, mannheim, germany). nuclei were stained with 1 mol / l to - pro3 in pbs for 1 h (invitrogen, karlsruhe, germany). the fluorescence was examined with a confocal microscope (leica, wetzlar, germany) using a 40 objective and excitation wavelengths of 546 nm (for cleaved caspase-3 staining), 488 nm (for tunel staining), and 633 nm (for nuclei staining). mitochondrial potential () was measured in isolated and cultured islet cells loaded with rhodamine-123 as described previously (13). briefly, cultured islet cell clusters were treated for 3 days with 0.6 mmol / l palmitate. thereafter the cells were loaded with 10 mg / l rhodamine-123 in modified krebs - ringer bicarbonate buffer solution containing 0.5 mmol / l glucose for 10 min at 37c. the fluorescence was measured using a device provided by till photonics (grfelfing, germany). mitochondrial hyperpolarization induced by increasing glucose concentration is expressed as percentage of maximal increase of fluorescence induced by carbonylcyanide - p - trifluoromethoxyphenylhydrazone (1 mol / l fccp). sections of frozen pancreatic tissue, cultured isolated islet cells, and ins-1e cells, control or stably transfected with rip - i / pkc constructs, were fixed with 4% paraformaldehyde in pbs, permeabilized with pbs containing 0.2% triton x-100, and blocked with 10% fcs in pbs for 45 min. primary antibodies against pkc (1:500 ; bd transduction laboratories, heidelberg, germany), insulin (1:150 ; dako denmark, denmark), and forkhead box class o (foxo)-1 (1:200 ; santa cruz biotechnology, santa cruz, ca) were applied overnight in pbs containing 10% fcs. after 30 min washing with pbs supplemented with 10% fcs, the samples were incubated for 1 h with the appropriate secondary antibodies (1:400 in 10% fcs - pbs) : alexa - fluor488 anti - mouse igg (for pkc), alexa - fluor546 anti - rabbit igg (for foxo1), and alexa - fluor546 anti - guinea pig igg (for insulin). mol / l to - pro3 in pbs for 1 h. for morphometric estimation of -cell mass, insulin antibody binding on pancreatic sections was visualized with a second antibody coupled to horseradish peroxidase. the islet sizes (insulin - stained areas) of every 10th cryosection of a mouse pancreas were measured with the axiovision le documentation program (axiovs40 le v 4.4.0.0. ; isolated islets, excised mouse tissues, and ins-1e cells were lysed in buffer containing 125 mmol / l nacl, 1% (v / v) triton x-100, 0.5% sodiumdeoxycholate, 0.1% sds, 10 mmol / l edta, 25 mmol / l hepes ph 7.3, 10 mmol / l napp, 10 mmol / l naf, 1 mmol / l na - vanadate, 10 g / ml pepstatin a, 10 g / ml aprotinin, and 0.1 mmol / l phenylmethylsulfonyl fluoride. cytosolic and nuclear fractions of ins-1e cells were prepared using a commercial kit (# 78833 ; pierce biotechnology, rockford, il). the 10,000 g supernatant of homogenates or the cell fractions were subjected to a sds - page (812%) and blotted onto nitrocellulose membranes (schleicher and schuell, dassel, germany). the membranes were incubated overnight with primary antibodies against foxo1, pkc, pkc, pkc, histone h1, and glyceraldehyde-3-phosphate dehydrogenase (each 1:1,000 in tris - buffered saline [tbs ] containing 5% milk, santa cruz biotechnology) ; p - ser256-foxo1 and tubulin (each 1:1,000 in tbs containing 5% bsa, cell signaling technology) ; followed by incubation with a secondary antibody (horseradish peroxidase linked anti - rabbit igg ; 1:2,000 in tbs containing 5% milk). insulin was extracted by acid ethanol from homogenized whole pancreata of 9-month - old mice. data are expressed as means sem ; p 9 months) control () and pkckn - overexpressing (# 179,) mice fed standard chow. c : mean islet size assessed in pancreatic slices of 3-month - old control () and pkckn - overexpressing (# 179,) mice fed hfd. results are presented as means sem of n = 4 pancreata (a) and of the number of islets as indicated in each column from two mice (b and c). d : representative pictures of insulin (red) and cleaved caspase-3 (green) staining in pancreatic slices of control and pkckn mice after either chow (cd, upper panels) or hfd (lower panels) feeding. e : means sem of cleaved caspase-3positive cells in islets (n = 3040) of control () and pkckn () mice. f : isolated islet cells were cultured for 3 days under control culture conditions and in the presence of 0.6 mmol / l palmitate as indicated. the percentage of apoptotic tunel - positive cells is expressed as means sem of n = 3 independent experiments. (a high - quality color digital representation of this figure is available in the online issue.) as apoptosis modulates -cell mass, the effect of pkckn overexpression on cell death was examined next. in pancreatic slices from wild - type but not from pkckn mice, inhibition of apoptosis by overexpression of pkckn was further confirmed in cultured isolated islet cells pretreated with palmitate. in cells of pkckn mice, palmitate did not change the percentage of apoptotic cells, whereas in control cells palmitate doubled the amount of tunel - positive cells (fig. therefore, the inhibition of palmitate - induced cell death in pkckn - overexpressing cells suggests that pkc indeed transmits palmitate - mediated -cell dysfunction. apoptotic activation of caspase-3 implies cytochrome c release and mitochondrial depolarisation. to examine the integrity of mitochondria, glucose - induced hyperpolarization was assessed. the rise of glucose from 0.5 to 16.7 mmol / l hyperpolarized mitochondria in isolated islet cells from control and pkckn mice to the same extent. after pretreatment with palmitate, the effect of glucose was reduced by 60% in islet cells of control but not of pkckn mice (fig. isolated islet cells were cultured for 3 days under control culture conditions and in the presence of 0.6 mmol / l palmitate as indicated. b : the hyperpolarizing effect of 16.7 mmol / l glucose expressed as percent of the maximal increase in fluorescence measured after addition of carbonylcyanide - p - trifluoromethoxyphenylhydrazone (1 mol / l fccp) is given as means sem of n = 1830 cells of three independent experiments. p 9 months) control () and pkckn - overexpressing (# 179,) mice fed standard chow. c : mean islet size assessed in pancreatic slices of 3-month - old control () and pkckn - overexpressing (# 179,) mice fed hfd. results are presented as means sem of n = 4 pancreata (a) and of the number of islets as indicated in each column from two mice (b and c). d : representative pictures of insulin (red) and cleaved caspase-3 (green) staining in pancreatic slices of control and pkckn mice after either chow (cd, upper panels) or hfd (lower panels) feeding. e : means sem of cleaved caspase-3positive cells in islets (n = 3040) of control () and pkckn () mice. f : isolated islet cells were cultured for 3 days under control culture conditions and in the presence of 0.6 mmol the percentage of apoptotic tunel - positive cells is expressed as means sem of n = 3 independent experiments. (a high - quality color digital representation of this figure is available in the online issue.) as apoptosis modulates -cell mass, the effect of pkckn overexpression on cell death was examined next. in pancreatic slices from wild - type but not from pkckn mice, inhibition of apoptosis by overexpression of pkckn was further confirmed in cultured isolated islet cells pretreated with palmitate. in cells of pkckn mice, palmitate did not change the percentage of apoptotic cells, whereas in control cells palmitate doubled the amount of tunel - positive cells (fig. therefore, the inhibition of palmitate - induced cell death in pkckn - overexpressing cells suggests that pkc indeed transmits palmitate - mediated -cell dysfunction. apoptotic activation of caspase-3 implies cytochrome c release and mitochondrial depolarisation. to examine the integrity of mitochondria, glucose - induced hyperpolarization was assessed. the rise of glucose from 0.5 to 16.7 mmol / l hyperpolarized mitochondria in isolated islet cells from control and pkckn mice to the same extent. after pretreatment with palmitate, the effect of glucose was reduced by 60% in islet cells of control but not of pkckn mice (fig. isolated islet cells were cultured for 3 days under control culture conditions and in the presence of 0.6 mmol / l palmitate as indicated. b : the hyperpolarizing effect of 16.7 mmol / l glucose expressed as percent of the maximal increase in fluorescence measured after addition of carbonylcyanide - p - trifluoromethoxyphenylhydrazone (1 mol / l fccp) is given as means sem of n = 1830 cells of three independent experiments. p < 0.05 against control ; p < 0.05 against islet cells of control mice cultured under the same condition. because it has been described that the dominant - negative transcription factor foxo1 inhibited ffa - dependent -cell death (14), experiments were performed to examine whether nuclear translocation of foxo1 is altered by pkckn. in control palmitate triggered foxo1 accumulation into the nucleus, and this effect was inhibited in cells overexpressing pkckn (fig. is further confirmed by experiments performed with the insulin - secreting cell line ins-1e overexpressing either wild - type pkc (pkcwt) or pkckn (fig. 6b ; supplemental fig. s2), whereas other novel pkcs (pkc and pkc) remained undetectable (data not shown). in control ins-1e cells foxo1 accumulated in the nucleus after palmitate treatment (fig. 6b and 7a c). overexpression of pkcwt resulted in an increased nuclear staining of foxo1 already under control conditions, whereas overexpression of pkckn inhibited nuclear accumulation of foxo1 (fig. 6b and 7a c). because jun nh2-terminal kinase (jnk) inhibition by sp600125 did not affect foxo1 accumulation induced by palmitate in either control ins-1e cells or pkcwt ins-1e cells, it is suggested that foxo1 accumulation does not depend on jnk activity (supplemental fig. because phosphorylation of foxo1 at ser256 stimulates its nuclear extrusion and degradation, the amount of foxo1 protein and its phosphorylation were examined in control and transfected ins-1e cells. indeed, increased phosphorylation of foxo1 and significantly lower protein levels were detected in pkckn ins-1e cells, whereas reduced phosphorylation of foxo1 and increased foxo1 protein levels were found in pkcwt ins-1e cells (fig. 7d, these data suggest that overexpression of pkckn in -cells inhibits palmitate - mediated -cell dysfunction by protecting against mitochondrial dysfunction and apoptosis while counteracting nuclear accumulation of foxo1. palmitate - mediated nuclear accumulation of foxo1 was inhibited by overexpression of pkckn in insulin secreting cells. a : islet cells from control and pkckn mice and b : control ins-1e cells and ins-1e cells overexpressing pkcwt or pkckn were cultured under control conditions or in the presence of 0.6 mmol / l palmitate for 1 day. (a high - quality color digital representation of this figure is available in the online issue.) overexpression of pkcwt and pkckn changes foxo1 cellular distribution, phosphorylation, and protein concentration in insulin - secreting cells. ins-1e cells and ins-1e cells overexpressing pkcwt or pkckn were cultured under control conditions and in the presence of 0.6 mmol / l palmitate for 1 h and 1 day. b and c : representative western blot for foxo1 detection in nuclear fractions with means sem of n = 4 independent experiments. d f : representative western blot for foxo1, p - ser256-foxo1, and tubulin (as loading control) of cell homogenates with means sem of n = 4 independent experiments of relative amounts of foxo1 and phosphorylation of foxo1 (p - foxo1/foxo1) by setting the respective bands of control ins-1 cells to 100%. significant difference to ins-1e under control condition ; significant difference to the respective condition of ins-1e ; # significant difference to pkckn at the same condition. the observation that overexpression of pkckn in -cells protects against hfd - induced glucose intolerance strongly supports the idea that pkc plays a central role in lipotoxicity. indeed, significantly lower blood glucose and increased plasma insulin concentrations during hfd were observed in pkckn - overexpressing mice, whereas animals fed standard chow displayed no significant difference. our study, therefore, differs from observations in whole - body pkc knockout (pkcko) mice that displayed glucose intolerance (9). the directly opposed results may be explained first by the difference between whole - body knockout versus -cell specific inhibition of pkc because, especially in brain tissue, large amounts of pkc are expressed. to exclude alterations in other tissues than - cells, we therefore restricted expression of the transgene to insulin - secreting cells. moreover, the most significant effect of pkckn overexpression was observed after high - fat feeding in our study, a condition that was not studied in pkcko mice. the conclusion that pkckn protects against lipotoxicity in vivo was further substantiated by the fact that the percentage of islet cells that stained positive for cleaved caspase-3 after hfd was not increased and that palmitate - induced apoptotic cell death was inhibited in pkckn - expressing -cells. because we were unable to detect proliferation, i.e., brdu incorporation or ki67-positive cells in pancreatic slices of hfd - fed mice (data not shown), it is anticipated that pkckn inhibits cell death in insulin - secreting cells ; this effect contributes to the increase in islet size and the increase in pancreatic insulin content. this is in contrast to studies that demonstrated that pkc exerts stimulatory or inhibitory effects on cell proliferation. these opposing effects of pkc may depend on the cell cycle status of the respective cell (15). because -cell mass depends on proper insulin / igf-1 receptor (5) reported that pkc stimulation by pma inhibits protein kinase b (pkb)/akt activation in insulin - secreting cells and described a reduced binding of insulin receptor substrate (irs)-2 to p85 after pma stimulation of the cells. this observation may be explained by putative pkc-mediated serine / threonine phosphorylations of irs-2 that inhibit irs-2 activation in parallel to known pkc phosphorylation sites in irs-1 (16). further experiments are needed to clarify whether pkc mediates changes of irs-2 activity via serine phosphorylation that reduce pkb / akt activation. in -cells, the mitochondrial potential plays a central role in normal glucose responsiveness of insulin secretion, whereas the collapse of mitochondrial potential is an essential step of the intrinsic pathways of apoptotic cell death (17,18). the protective effect of pkckn on mitochondrial potential strengthens the idea that pkc interferes with mitochondrial function. in line with this concept, salivary epithelial cells overexpressing pkckn were resistant to the loss of mitochondrial potential and apoptosis induced by etoposide, ultraviolet irradiation, bredfeldina, and paclitaxel (19). this complex transfers the endoplasmic reticulum (er) stress to mitochondria, which further leads to apoptotic cell death (20,21). although we can not exclude direct effects of pkc in mitochondria and in er, our data favor the idea that pkc leads to nuclear accumulation of foxo1 with palmitate treatment. in fact, it has been previously demonstrated that ffa - induced er stress and apoptosis are inhibited in insulin - secreting cells expressing the dominant - negative form of foxo1 (14). activation of foxo1 leads to multiple cellular changes and plays a major role in stress resistance and survival of -cells (2227). in contrast, inhibition of foxo1 is necessary for adaptive -cell proliferation during insulin resistance (28,29). our study therefore provides strong evidence that ffas stimulate nuclear sequestration of foxo1 through the activation of pkc because the nuclear accumulation of foxo1 is inhibited in cells overexpressing pkckn. moreover, phosphorylation of foxo1 at the pkb / akt phosphorylation site ser256 is reduced in cells overexpressing pkcwt but increased in cells expressing pkckn. the signaling pathway through pkc that leads to reduced ser256 phosphorylation of foxo1 may involve activation of inhibitory pathways such as jnk and protein phosphatase 2a (30,31). however, jnk inhibition did not affect nuclear foxo1 accumulation induced by palmitate, suggesting a pkc-dependent but jnk - independent mechanism. our study delivers in vivo evidence that activation of pkc by ffas mediates -cell failure. because pkckn inhibited ffa - induced nuclear accumulation of foxo1, mitochondrial dysfunction, and apoptosis, it is anticipated that the underlying mechanism involves pkc-dependent chronic activation of foxo1. in fact, a gene variation of foxo1 associates with impaired glucose tolerance and type 2 diabetes in humans (32). | objectivein vitro models suggest that free fatty acid induced apoptotic -cell death is mediated through protein kinase c (pkc). to examine the role of pkc signaling in vivo, transgenic mice overexpressing a kinase - negative pkc (pkckn) selectively in -cells were generated and analyzed for glucose homeostasis and -cell survival.research design and methodsmice were fed a standard or high - fat diet (hfd). blood glucose and insulin levels were determined after glucose loads. islet size, cleaved caspase-3, and pkc expression were estimated by immunohistochemistry. in isolated islet cells apoptosis was assessed with tunel / to - pro3 dna staining and the mitochondrial potential by rhodamine-123 staining. changes in phosphorylation and subcellular distribution of forkhead box class o1 (foxo1) were analyzed by western blotting and immunohistochemistry.resultspkckn mice were protected from hfd - induced glucose intolerance. this was accompanied by increased insulin levels in vivo, by an increased islet size, and by a reduced staining of -cells for cleaved caspase-3 compared with wild - type littermates. in accordance, long - term treatment with palmitate increased apoptotic cell death of isolated islet cells from wild - type but not from pkckn mice. pkckn overexpression protected islet cells from palmitate - induced mitochondrial dysfunction and inhibited nuclear accumulation of foxo1 in mouse islet and ins-1e cells. the inhibition of nuclear accumulation of foxo1 by pkckn was accompanied by an increased phosphorylation of foxo1 at ser256 and a significant reduction of foxo1 protein.conclusionsoverexpression of pkckn in -cells protects from hfd - induced -cell failure in vivo by a mechanism that involves inhibition of fatty acid mediated apoptosis, inhibition of mitochondrial dysfunction, and inhibition of foxo1 activation. |
retrospektywna analiza dokumentacji szpitalnej pacjentw leczonych z powodu urazw drg oddechowych obejmowaa okres midzy 1990 a 2012 r. uraz wystpi u 16 pacjentw podczas intubacji, u 1 podczas sztywnej bronchoskopii, u 1 podczas sztywnej ezofagoskopii, u 1 podczas mediastinoskopii i u 5 podczas zabiegw chirurgicznych. u 1 pacjenta stwierdzono uraz tchawicy w szyjnym odcinku, a u pozostaych 23 w czci piersiowej. zabieg chirurgiczny obejmowa napraw boniastej czci tchawicy z dostpu poprzez praw torakotomi u 10 chorych (u 1 do pokrycia uyto mini midzyebrowych), samorozpralny metalowy stent zaoono u 1 pacjenta. i przeyku, u 1 sleeve - lobektomi lew grn, u 1 praw grn lobektomi, u 3 zaoono stent silikonowy y, u 1 przeprowadzono minitracheostomi, a u 5 chorych wczono leczenie zachowawcze.. most often they result from tracheal intubation, with frequency being reportedly 0.05 - 0.37% [13 ]. risk factors are : emergency intubation, extra - hospital intubation and use of double - lumen tracheobronchial tubes. other causes of iatrogenic airway injuries include : percutaneous tracheostomy, airway patency restoration using rigid bronchoscopy, mechanical ventilation, surgical procedures on the thyroid gland, oesophagus and lung. percutaneous tracheostomy, airway patency restoration using rigid bronchoscopy, mechanical ventilation, surgical procedures on the thyroid gland, oesophagus and lung. most published papers are case reports or small case series, and very few present collective reviews [47 ]. diagnostic work - up includes history and physical examination with particular attention to subcutaneous emphysema in the neck, face and chest, coughing, haemoptysis, respiratory distress, pneumothorax and pneumomediastinum. the most important tool in confirmation of the diagnosis is bronchoscopy, enabling precise assessment of location, size and type of injury. the mainstay of treatment is surgical repair, although conservative treatment is increasingly popular in selected patients [812 ]. retrospective analysis of hospital records of all patients treated for main airway injuries between 1990 and 2012. in the study period, 24 patients were treated for iatrogenic injuries to the trachea or main bronchi. there were 21 women (87.5%) with the mean age of 61 years (range : 17 - 84) and 3 men with the mean age of 52 years (range : 49 - 56). in 1 patient there was an injury in the cervical trachea and in the remaining 23 in the thoracic part of the airway. in 16 patients the injury occurred during tracheal intubation : in 7 it was located in the upper 2/3 of the trachea and in 9 in the lower segment. injury resulted from rigid bronchoscopy in 1 patient, from oesophagoscopy in 1, from mediastinoscopy in 1 and was a complication of open surgery in 5 patients (4 oesophagectomies and 1 resection of oesophageal diverticulum). in 4 patients the laceration was located in the right main bronchus and in 2 in the left main bronchus. all 16 injuries related to tracheal intubation happened in other hospitals and included intubation for gynaecological procedures, mastectomy, laparoscopic cholecystectomy and large bowel resection. in 13 patients injury occurred during elective intubation and in 3 during emergency extrahospital intubation. mean time between injury and bronchoscopic confirmation of the diagnosis was 12 hours (range : 2 - 48). the most frequent sign was subcutaneous emphysema, followed by haemoptysis, coughing, and neck or chest pain. in all patients chest radiogram and ct were obtained on admission to check for pneumomediastinum and mediastinitis, pneumothorax and/or hydrothorax and pneumonia, as well as to gain more detailed information on anatomy. next, bronchoscopy was performed with assessment of the location, size and depth of injury. the mechanism was rigid bronchoscopy in 1 patient, rigid oesophagoscopy in 1, mediastinoscopy in 1 and open surgery in 5 patients (oesophagectomy in 4 and oesophageal diverticulum resection in 1). in all cases injury was diagnosed during the procedure and time elapsed until the decision regarding further treatment was 50 years, physician 's limited experience and re - positioning of the tube with the inflated balloon [6, 10 ]. also, steroid therapy and any pathology within the trachea or in its surroundings, i.e. tumours, adenopathy, tracheitis, bronchomalacia, tracheal stenosis, chronic obstructive pulmonary disease, kyphosis or kyphoscoliosis, may increase the risk of tracheal injury [6, 14, 15 ]. the clinical picture includes subcutaneous emphysema in the neck, face and chest, dyspnoea, coughing, haemoptysis, pneumomediastinum and pneumothorax [4, 6, 810, 1618 ]. there is no consensus regarding the treatment of iatrogenic airway injuries [68, 11, 17, 19, 20 ]. however, several authors agree on the following criteria of conservative treatment : good general condition, haemodynamic and respiratory stability, limited tear in the upper trachea, no signs of mediastinitis, and no progressive subcutaneous emphysema or pneumomediastinum [6, 8, 10, 21 ]. other authors consider the length and depth of injury as the most important criteria of conservative treatment [5, 17, 22 ]. however, this is not contradictory, as deep lacerations are usually associated with progressive pneumomediastinum, mediastinitis and respiratory failure. cardillo. proposed a 4-grade morphological classification of intubation - related tracheal injuries : grade i : tear limited to mucosa or submucosa, no pneumomediastinum or mediastinitis; grade ii : tear of the membranous part with subcutaneous emphysema and/or pneumomediastinum, no oesophageal injury or mediastinitis; grade iiia : full disruption of the tracheal wall, no oesophageal injury or mediastinitis; grade iiib : any tracheal injury associated with oesophageal injury or mediastinitis. grade i : tear limited to mucosa or submucosa, no pneumomediastinum or mediastinitis ; grade ii : tear of the membranous part with subcutaneous emphysema and/or pneumomediastinum, no oesophageal injury or mediastinitis ; grade iiia : full disruption of the tracheal wall, no oesophageal injury or mediastinitis ; grade iiib : any tracheal injury associated with oesophageal injury or mediastinitis. in patients with grade i - iiia injuries, cardillo. used conservative treatment including bronchoscopic application of 1 - 2 ml fibrin glue to seal the injury., in patients with grade iiib injuries surgical repair via right thoracotomy should be performed, supplemented with broad - spectrum antibiotics, anti - cough medication and parenteral nutrition. there was a strong predominance of women (85.7%), which corresponds to the structure of our group. elective intubation was the cause of injury in 65.9% of patients, emergency intubation in 27.4%, and in the remaining 12 patients no data in this regard were available. as the number of elective intubations largely exceeds the emergency ones, the results confirm very high risk of tracheal injury associated with emergency intubation. in 53.3% of cases a single - lumen tube was used, and in the remaining 46.7% a double lumen tube. again, due to the relatively small number of intubations with double - lumen tubes, the results confirm the high risk associated with their use. in 17% of patients, injury was diagnosed during the procedure and in the remaining 83% with a delay of up to 240 hours. the most common symptoms included subcutaneous emphysema (64%), followed by pneumomediastinum, pneumothorax, dyspnoea and haemoptysis. emergency intubation, advanced patient age and delay in diagnosis were associated with increased mortality. according to other authors, factors increasing mortality include delay in diagnosis > 20 hours, difficult intubation, age > 60 years, female gender and obesity [10, 19 ]. in our group, 2 patients died within the first 24 hours ; both underwent emergency extra - hospital intubation due to cardiac arrest and had severe neurological pathology. the remaining two patients in our group died due to severe sepsis and multi - organ failure after oesophageal resection. the direct cause of all deaths in our group was not related to the airway injury. in the study of 19 patients with iatrogenic airway injuries, hofmann reported mortality of 42%. regarding conservative treatment, the results presented by minambres are in concordance with other authors, who believe that it should be used in cases of small lacerations and with no progressive general symptoms [8, 9, 11, 21 ]. used a self - expanding stent for 4 cm laceration of the membranous part of the trachea ; a similar stent was used in one of our patients. self - expanding metallic stents are not widely used, because they cause damage to the airway mucosa and their removal is difficult. we also implanted a silicone y stent in 2 patients with laceration of the membranous part of the lower trachea. the treatment was successful and the stents were removed on the 7 and 10 day ; follow - up bronchoscopy confirmed healing of the laceration. a third y stent was successfully used in a patient with injury to the right main bronchus and the oesophagus. our results with airway stenting in patients with iatrogenic injuries to the trachea and main bronchi are similar to those reported by other authors [18, 23 ]. most authors agree that open surgical repair is required in case of full - thickness airway injury with pneumothorax, pneumomediastinum, progressive respiratory insufficiency or technical difficulties in ventilation [4, 5, 15, 19, 22, 24 ], whilst stenting should be used in medically inoperable patients [18, 23 ]. in order to limit surgical trauma, some authors advocate the use of minimally invasive techniques [12, 20, 24, 25 ]. welter presented endoscopic repair of lacerations of the membranous part of the trachea using a videotracheoscope and endoscopic instruments, with general anaesthesia and jet ventilation. videobron - choscope - assisted repair of tracheal laceration was presented by okada.. | introductioniatrogenic tracheobronchial injuries are rare.aimto analyse the mechanism of injury, symptoms and treatment of these patients.material and methodsretrospective analysis of hospital records of all patients treated for main airway injuries between 1990 and 2012 was performed.resultsthere were 24 patients, including 21 women and 3 men. mean time between injury and initiation of treatment was 12 hours (range : 2 - 48). in 16 patients the injury occurred during tracheal intubation, in 1 during rigid bronchoscopy, in 1 during rigid oesophagoscopy, in 1 during mediastinoscopy and in 5 during open surgery. mean length of airway tear was 3.8 cm (range : 1.5 - 8). in 1 patient there was an injury to the cervical trachea and in the remaining 23 in the thoracic part of the airway. the treatment included repair of the membranous part of the trachea performed via right thoracotomy in 10 patients (in 1 patient additionally coverage with a pedicled intercostal muscle flap was used), a self - expanding metallic stent in 1 patient, suture of the right main bronchus and the oesophagus in 1, left upper sleeve lobectomy in 1, right upper lobectomy in 1, implantation of a silicone y stent in 3, mini - tracheostomy in 1, and conservative treatment in 5 patients.conclusionsintubation is the most frequent cause of iatrogenic main airway injuries. patients with these life - threatening complications require an individualised approach and treatment in a reference centre. |
the most commonly reported etiologic microorganisms of this rare condition are citrobacter diversus proteus mirabilis, pseudomonas aeruginosa, serratia marcescens, and other enterobacteriaceae. a male preterm of approximate 34 weeks gestation, weighing 1.9 kg, appropriate for gestational age was delivered vaginally to a primigravida mother at a private nursing home. the baby had cried immediately after birth and was asymptomatic on day 1 of life. on day 2, the baby was admitted in neonatal intensive care unit of a tertiary care hospital with complaints of excessive cry and refusal to feed. there was a history of leaking per vaginum for 3 days prior to the delivery. the baby exhibited weak cry, shallow, and irregular breathing pattern with the inability to maintain oxygen saturation in room air. fever, progressively worsening respiratory distress, apnea, cyanosis, seizures, bulging anterior fontanelle, vomiting, and pallor were, however not observed. neurological examination revealed normal occipito - frontal circumference, wakefulness, reflexes and physiological hypotonia without a focal neurological deficit. total leukocyte count = 4000/l, absolute neutrophil count = 1800/l, serum c - reactive protein = 4.8 mg / dl (semiquantitative latex slide agglutination kit, vital diagnostics, thane, india, normal cut - off limit = 0.6 mg / dl) normal blood sugar (4.62 mmol / l), serum calcium (2.35 mmol / l), normal chest x - ray and an arterial blood gas. the patient was administered a combination of cefotaxime at 50 mg / kg / dose q 12 hourly and amikacin at 7.5 mg / kg / dose q 12 hourly owing to laboratory confirmation of sepsis. the cerebrospinal fluid examination was normal with sterile culture and with no micro - organisms seen in the gram - stained slide, thus, indicating the absence of meningitis. due to the persistent clinical features of sepsis, antibiotics were changed to intravenous vancomycin at 10 mg / kg / dose q 12 hourly and meropenem at 20 mg / kg / dose q 12 hourly after 48 h of initiation of therapy. the persistence of shallow and irregular breathing pattern prompted us for cranial ultrasonograms on day 5 which revealed multiple hypo - echoic lesions in bilateral parietal lobes. magnetic resonance imaging (mri) brain was subsequently performed on day 6 which showed multiple hypo - dense lesions with peripheral enhancement suggestive of intra - parenchymal abscesses in bilateral fronto - parietal and left basal ganglionic region [figure 1a and b ]. intravenous vancomycin was continued in same doses, but dose of meropenem was increased to 40 mg / kg / dose q 12 hourly to facilitate the drug availability at the site of lesions. repeat blood culture drawn at this time grew kliebsiella pneumoniae after 48 h of incubation which was sensitive to ciprofloxacin and piperacillin tazobactam. on day 10, due to minimal change in the general condition and respiratory pattern, vancomycin and meropenem were replaced by intravenous piperacillin an improvement in general condition was observed in the ensuing 72 h, following which, the neonate started feeding well and was subsequently discharged after 6 weeks of therapy. on a 3 months follow - up, a repeat mri brain scan [figure 2 ] revealed complete resolution of lesions as shown in the earlier report. the baby had a normal neurological examination and developmental milestones at 9 months of age. mri brain images prior to initiation of therapy : (a) axial t2 image shows hyper - intense areas surrounded by hypo - intense rim seen involving the left basal ganglion, and in bilateral fronto - parietal locations. (b) sagittal contrast t1 image shows thick peripheral rim enhancement of the lesions in left basal ganglion and in the left parietal lobe. both images a and b are suggestive of parenchymal brain abscesses mri brain images post - therapy : axial t1 (left) and sagittal (right) contrast mri brain images on post - therapy follow - up scan at 3 months age shows no abscess in the corresponding regions of brain parenchyma and is suggestive of their complete resolution gram - negative micro - organisms commonly citrobacter diversus, proteus and pseudomonas species have been implicated in the pathogenesis as they bear the propensity to invade nervous tissue and cause necrotizing vasculitis. although, klebsiella is the most common organism causing neonatal sepsis in developing countries,, it has been reported in few cases of brain abscess in the world literature. however, in one of the largest series of 30 neonates with brain abscess in a tertiary center, no case was attributed to klebsiella species. this could be due to its lesser tendency to cause necrotizing lesions as compared to citrobacter and proteus species. volpe has emphasized that the bacteria cause meningitis and vasculitis initially, which may further, rarely complicate and result in the formation of a brain abscess. in this case, there was history of leaking per vaginum in the mother for 3 days prior to delivery which was probably followed by neonatal sepsis and hematogenous seeding of the brain parenchyma resulting in the multiple abscesses with no initial meningeal involvement. have hypothesized that the direct hematogenous seeding of the parenchyma could be an equally important pathogenetic mechanism for an abscess to develop and a meningeal involvement may not be an essential prerequisite always. the colonization of brain parenchyma during sepsis can be explained by the physiological right - to - left shunt of the neonatal circulation. the usual clinical presentation of brain abscess in neonatal period consists of irritability, bulging fontanelle, and rapid rise in head circumference with wide separation of sutures, vomiting, seizures, and poor feeding. the presence and persistence of shallow and irregular respiratory pattern with the inability to maintain oxygen saturation in room air necessitated a cranial ultrasonography and mri brain. the management of neonatal brain abscess is influenced not only by the anatomic location, number, size, stage, and nature of the abscesses but also by the age and neurological status of the patient. medical management saves the patient from the stress and surgical complications especially in a neonate, who may present with significant surgery - related neurological deficits and hemorrhage. the duration of the antibiotic course is usually 68 weeks and longer for immunocompromised patients. however, the right dose and duration of antimicrobial agents for neonatal brain abscess is poorly studied. in this case, there was a poor response to empirical meropenem therapy despite an increase in its dose. meropenem penetrates the blood - brain barrier well when the meninges are inflamed and achieves an excellent minimum inhibitory concentration for almost all gram - negative pathogens but neonatal data about its penetrative ability in the absence of meningeal inflammation or within an abscess cavity are lacking. on the other hand, surgical intervention provides samples for accurate diagnosis, reduces the mass of an abscess, improves the efficacy of the drugs used for treatment and in some conditions allows intrathecal, intra - ventricular or intra - cavitory administration of antibiotics. response to a 6 weeks course of piperacillin tazobactam obviated the requirement for surgical intervention in this case. aspiration was not attempted in this case owing to smaller multiple lesions which were inaccessible to this procedure. various studies have highlighted the poor iq scores and subsequent poor scholastic performance. in this case, the patient exhibited appropriate developmental milestones on follow - up. to conclude, apart from the typical features, a clinician should be aware of atypical features such as shallow and irregular breathing, inability to maintain oxygen saturation in room air, and absence of the usual clinical features. a strong clinical suspicion may thus be required for diagnosis in such cases. | brain abscesses occur as an uncommon complication of bacterial meningitis in the neonatal period. a 34 weeks preterm at - risk neonate presented with abnormal breathing pattern and inability to maintain the oxygen saturation in room air. magnetic resonance imaging (mri) study revealed intra - parenchymal brain abscesses in the left basal ganglion and bilateral fronto - parietal regions. intravenous piperacillin tazobactam was commenced and continued for 6 weeks in neonatal intensive care unit. no surgical intervention was required. the patient responded to the medical management and was discharged after the documentation of radiological clearance in repeat mri study. no complications were recorded. an appropriate neuro - developmental outcome was observed on follow - up. brain abscesses may not be preceded by meningitis in all neonates. a strong clinical suspicion is required for the diagnosis especially in cases with atypical presentation. |
the gfi1 mouse line (gfi1, mgi : 2449921) was provided by dr. vaginal plugs were checked the next morning before 10:00 am, and the time - mated embryos were dissected as described elsewhere. all animal procedures and experiments were approved by the cleveland clinic animal care and use committee. tissues were fixed in 4% paraformaldehyde at 4c overnight, were washed in 1 phosphate - buffered saline (pbs), and were equilibrated in 30% sucrose before embedding in optimal cutting temperature compound, before the cryosectioning. the tissue samples were stained with the primary antibodies as detailed in supplementary table 1. for the anti - hnf6 antibodies staining, the tyramide signal amplification fluorescence system was used (perkinelmer, waltham, ma). before applying the blocking reagents, the tissue samples underwent peroxidase (3% in pbs) treatment for 7 minutes. after incubating in the primary antibody (rabbit anti - hnf6) for overnight and being washed with pbs for 3 times (5 minutes / wash), the slides were incubated in biotinylated secondary antibody for 30 minutes at room temperature. after washing 3 times in 1pbs (5 minutes / wash), the slides were further incubated in streptavidin - peroxidase conjugate for 15 minutes, which was then followed by three washes (5 minutes / wash). then the slides were incubated in fluorophore tyramide (amplification reagent) for 3 to 10 minutes, which was followed by washing. binding of the primary antibodies was detected by immunofluorescence by incubating with texas red-, cy2-, or aminomethylcoumarin - conjugated secondary antibodies (jackson immunoresearch laboratories, west grove, pa). after washing with 1pbs, tissue samples were mounted (slowfade antifade kit ; molecular probes / life technologies, eugene, or). immunohistochemical analysis for cell apoptosis was performed by using an in situ apoptosis detection kit with tunel (terminal deoxynucleotidyl transferase - mediated dutp nick end labeling) according to the manufacturer s instructions (roche, indianapolis, in). to evaluate the cell proliferation, we intraperitoneally injected mice with bromodeoxyuridine (brdu) (50 g / g of body weight). two hours after the injection, the mice were dissected, and the pancreas were fixed and processed following the same protocol as previously mentioned for immunofluorescence analysis. images were obtained using either an olympus bx51 upright epifluorescence microscope (olympus america, center valley, pa), equipped with a qimaing retiga 2000r cooled ccd camera (qimaging, surrey, british columbia, canada) or a leica sp5 confocal microscope (leica microsystems, buffalo grove, il). morphometry was done by measuring the number of positively stained cells per unit area using imagepro 4.1 software image analysis system (media cybernetics, rockville, md). the probe for the in situ hybridization analysis was generated by using the digoxigenin (dig) labeling kit from roche, according to the manufacturer s protocol. the probe was made from a linearized plasmid containing the gfi1 cdna fragment (pcr - gfi1) and t7 polymerase for antisense probes or t3 polymerase for sense probes by using the dig rna labeling mix (roche). in situ hybridization after postfixation and protease treatment, sections were prehybridized at 65c for 3 hours, followed by overnight hybridization with either antisense or sense probes at 65c. sections then were blocked with 5% goat serum and 2% hybridization blocking reagent (roche) for subsequent overnight incubation with an anti - dig antibody conjugated with horseradish peroxidase at 4c. after washing with 1pbs/0.1% tween 20, nbt (nitro - blue tetrazolium chloride)/bcip (5-bromo-4-chloro-3-indolyphosphate p - toluidine salt) (roche) age- (10-week - old) and sex - matched wild - type (wt) (n = 4) and gfi1-null mice (n = 3) were fasted for 16 hours. before the test louis, mo) was injected intraperitoneally (1 g / kg body weight), and blood was sampled at 0, 15, 30, 45, 60, 90, 120, and 150 minutes after the injection. the blood glucose level was measured with an accu - check glucometer (roche). mouse pancreas (at the age of e18.5 and 1 month) (wt, n = 5 ; gfi1-null, n = 4) were dissected, and small pieces were immediately fixed in 2% glutaraldehyde in 0.1 m sodium cacodylate buffer containing 0.1 m sucrose and 3 mm calcium chloride (cacl2) (ph 7.4) at room temperature for 4 to 8 hours, then stored at 4c until further processing. specimens were rinsed in 0.15 m sodium cacodylate buffer containing 3 mm cacl2 (ph 7.4), were postfixed in 2% osmium tetroxide in 0.07 m sodium cacodylate buffer containing 1.5 mm cacl2 (ph 7.4) at 4c for 2 hours, were gradually dehydrated in ethanol, followed by acetone, and then were embedded in epon resin. semi - thin sections were cut and stained with toluidine blue and were used for light microscopic analysis. ultrathin sections (4050 nm) were cut and contrasted with 2% uranyl acetate followed by lead citrate. the ultrathin sections were examined on a philips cs12/stem transmission electron microscope (fei company, hillsboro, or) at cleveland clinic lerner research institute imaging core. the total number of acini individually observed were 28 for wt and 20 for gfi1-null. the embryonic and adult mouse pancreas at different developmental stages was isolated in rnalater (promega, madison, wi). the total rna were isolated and extracted by using rnaeasy rna extraction kit following the manufacturer s manual (qiagen, valencia, ca). total rna extracted were reverse transcribed to cdna using the superscript first - strand synthesis system based on the manufacturer s protocols (invitrogen / life technologies, carlsbad, ca). quantitative real - time polymerase chain reaction (qrt - pcr) was performed with iq sybr green supermix (bio - rad laboratories, hercules, ca) according to the manufacturer s instructions and the protocols described elsewhere. the expression levels of genes tested were normalized to the expression levels of the housekeeping gene gapdh using the ct method. primer sets used were hnf1 (tcf2), 5-cca tcc tca aag agc tcc ag-3, 5-ctc cct ctg ggg gat att gt-3 ; hnf6 (oc1), 5-ctg tga aac tcc ccc agg ta-3, 5-ggt gat gat ggt gag gga ac ; sox9, 5-tgc agc aca aga aag acc ac-3, 5-cag cgc ctt gaa gat agc at-3 ; ptf1a, 5-cag agg acc cca gaa aac tca-3, 5-gtc aaa ggt gct tca gga aat c-3 ; and gapdh, 5-tgc gac ttc aac agc aac tc-3, 5-atg tag gcc cat gag gtc cac-3. the data are shown as mean sd. the gfi1 mouse line (gfi1, mgi : 2449921) was provided by dr. vaginal plugs were checked the next morning before 10:00 am, and the time - mated embryos were dissected as described elsewhere. all animal procedures and experiments were approved by the cleveland clinic animal care and use committee. tissues were fixed in 4% paraformaldehyde at 4c overnight, were washed in 1 phosphate - buffered saline (pbs), and were equilibrated in 30% sucrose before embedding in optimal cutting temperature compound, before the cryosectioning. the tissue samples were stained with the primary antibodies as detailed in supplementary table 1. for the anti - hnf6 antibodies staining, the tyramide signal amplification fluorescence system was used (perkinelmer, waltham, ma). before applying the blocking reagents, the tissue samples underwent peroxidase (3% in pbs) treatment for 7 minutes. after incubating in the primary antibody (rabbit anti - hnf6) for overnight and being washed with pbs for 3 times (5 minutes / wash), the slides were incubated in biotinylated secondary antibody for 30 minutes at room temperature. after washing 3 times in 1pbs (5 minutes / wash), the slides were further incubated in streptavidin - peroxidase conjugate for 15 minutes, which was then followed by three washes (5 minutes / wash). then the slides were incubated in fluorophore tyramide (amplification reagent) for 3 to 10 minutes, which was followed by washing. binding of the primary antibodies was detected by immunofluorescence by incubating with texas red-, cy2-, or aminomethylcoumarin - conjugated secondary antibodies (jackson immunoresearch laboratories, west grove, pa). after washing with 1pbs, tissue samples were mounted (slowfade antifade kit ; molecular probes / life technologies, eugene, or). immunohistochemical analysis for cell apoptosis was performed by using an in situ apoptosis detection kit with tunel (terminal deoxynucleotidyl transferase - mediated dutp nick end labeling) according to the manufacturer s instructions (roche, indianapolis, in). to evaluate the cell proliferation, we intraperitoneally injected mice with bromodeoxyuridine (brdu) (50 g / g of body weight). two hours after the injection, the mice were dissected, and the pancreas were fixed and processed following the same protocol as previously mentioned for immunofluorescence analysis. images were obtained using either an olympus bx51 upright epifluorescence microscope (olympus america, center valley, pa), equipped with a qimaing retiga 2000r cooled ccd camera (qimaging, surrey, british columbia, canada) or a leica sp5 confocal microscope (leica microsystems, buffalo grove, il). morphometry was done by measuring the number of positively stained cells per unit area using imagepro 4.1 software image analysis system (media cybernetics, rockville, md). the probe for the in situ hybridization analysis was generated by using the digoxigenin (dig) labeling kit from roche, according to the manufacturer s protocol. the probe was made from a linearized plasmid containing the gfi1 cdna fragment (pcr - gfi1) and t7 polymerase for antisense probes or t3 polymerase for sense probes by using the dig rna labeling mix (roche). in situ hybridization after postfixation and protease treatment, sections were prehybridized at 65c for 3 hours, followed by overnight hybridization with either antisense or sense probes at 65c. sections then were blocked with 5% goat serum and 2% hybridization blocking reagent (roche) for subsequent overnight incubation with an anti - dig antibody conjugated with horseradish peroxidase at 4c. after washing with 1pbs/0.1% tween 20, nbt (nitro - blue tetrazolium chloride)/bcip (5-bromo-4-chloro-3-indolyphosphate p - toluidine salt) (roche) age- (10-week - old) and sex - matched wild - type (wt) (n = 4) and gfi1-null mice (n = 3) were fasted for 16 hours. before the test, the animals were weighed, and their preinjection blood glucose levels were measured. louis, mo) was injected intraperitoneally (1 g / kg body weight), and blood was sampled at 0, 15, 30, 45, 60, 90, 120, and 150 minutes after the injection. the blood glucose level was measured with an accu - check glucometer (roche). mouse pancreas (at the age of e18.5 and 1 month) (wt, n = 5 ; gfi1-null, n = 4) were dissected, and small pieces were immediately fixed in 2% glutaraldehyde in 0.1 m sodium cacodylate buffer containing 0.1 m sucrose and 3 mm calcium chloride (cacl2) (ph 7.4) at room temperature for 4 to 8 hours, then stored at 4c until further processing. specimens were rinsed in 0.15 m sodium cacodylate buffer containing 3 mm cacl2 (ph 7.4), were postfixed in 2% osmium tetroxide in 0.07 m sodium cacodylate buffer containing 1.5 mm cacl2 (ph 7.4) at 4c for 2 hours, were gradually dehydrated in ethanol, followed by acetone, and then were embedded in epon resin. semi - thin sections were cut and stained with toluidine blue and were used for light microscopic analysis. ultrathin sections (4050 nm) were cut and contrasted with 2% uranyl acetate followed by lead citrate. the ultrathin sections were examined on a philips cs12/stem transmission electron microscope (fei company, hillsboro, or) at cleveland clinic lerner research institute imaging core. the total number of acini individually observed were 28 for wt and 20 for gfi1-null. the embryonic and adult mouse pancreas at different developmental stages was isolated in rnalater (promega, madison, wi). the total rna were isolated and extracted by using rnaeasy rna extraction kit following the manufacturer s manual (qiagen, valencia, ca). total rna extracted were reverse transcribed to cdna using the superscript first - strand synthesis system based on the manufacturer s protocols (invitrogen / life technologies, carlsbad, ca). quantitative real - time polymerase chain reaction (qrt - pcr) was performed with iq sybr green supermix (bio - rad laboratories, hercules, ca) according to the manufacturer s instructions and the protocols described elsewhere. the expression levels of genes tested were normalized to the expression levels of the housekeeping gene gapdh using the ct method. primer sets used were hnf1 (tcf2), 5-cca tcc tca aag agc tcc ag-3, 5-ctc cct ctg ggg gat att gt-3 ; hnf6 (oc1), 5-ctg tga aac tcc ccc agg ta-3, 5-ggt gat gat ggt gag gga ac ; sox9, 5-tgc agc aca aga aag acc ac-3, 5-cag cgc ctt gaa gat agc at-3 ; ptf1a, 5-cag agg acc cca gaa aac tca-3, 5-gtc aaa ggt gct tca gga aat c-3 ; and gapdh, 5-tgc gac ttc aac agc aac tc-3, 5-atg tag gcc cat gag gtc cac-3. the data are shown as mean sd. we first analyzed expression of gfi1 in the mouse pancreas at different developmental stages by performing qrt - pcr and in situ hybridization. from the mice embryonic stages of e12.5 to e16.5, gfi1 mrna were present in pancreas (figure 1a). at later stages of embryonic development (e17.5e18.5) and the postnatal stage (p0 and p6), gfi1 was continuously expressed in the adult pancreas (1 month old and 4 months old, 1 m and 4 m). to assess the cell - type specific expression of gfi1, we performed in situ hybridization on developing mouse pancreas tissues (figure 1b). at all stages analyzed, the pancreatic mesenchymal tissue was devoid of gfi1 expression, and the adjacent liver, containing hematopoietic cells, served as a useful positive control (figure 1b). gfi1 was expressed in e11.5 pancreatic epithelium. at e14.5, when the pancreas progenitor population had segregated into the trpc / tippc regions, and acinar and endocrine / ductal differentiation had commenced, gfi1 became restricted to tip regions, gradually being excluded from the central domain the trunk regions although the cells displaying intermediate levels of gfi1 mrna could still be detected (arrows in figure 1b, e14.5). at e15.5, gfi1 expression was fully excluded from trunk - located cells. at e16.5, gfi1 remained abundantly expressed in the cells at tips, and we observed a strict boundary between gfi1-expressing cells at the tip location to adjacent epithelial gfi1-negative cells (e16.5 in figure 1b). the abundance of gfi1 mrna in the postsecondary transition pancreas is suggestive of a functional role for gfi1 in pancreas development at the late developmental stages. to functionally investigate gfi1 in pancreas, we bred for gfi1 nullizygous embryos and offspring using a global deletion model of exon 23 of gfi1. null animals suffer from severe neutropenia, loss of hearing, and a general growth deficiency.16, 17, 18, 19 visual inspection of midgestational (e14.5), and late - gestational (e18.5) endodermal organs revealed a normal pancreatic size and gross appearance. analysis of hematoxylin and eosin (h&e)-stained tissue at the embryonic stage of e14.5 and e18.5 did not reveal major changes in pancreatic morphology (figure 2a d). however, in adult mice (4 months old, 4 m), morphologic changes were observed in the acinar compartment. the acinar architecture was poorly organized, marked by the presence of vacuolized structures within the acini (figure 2f), when compared to the wt littermates (figure 2e). at all stages considering that gfi1 is expressed during the secondary transition when endocrine cells form, we first investigated the development and function of pancreatic endocrine cells. the presence and organization of pancreatic endocrine cells were analyzed by immunofluorescence analysis against polypeptide hormones and chromogranin a. the presence of pancreatic endocrine cell types was confirmed by staining for insulin (), glucagon (), and ghrelin () at e18.5. the central core of insulin - positive -cells in the islets of langerhans, surrounded by glucagon - positive -cells, was observed in both the wt (figure 2i) and gfi1 pancreas (figure 2j). no difference in the expression of pdx1 was observed in gfi1 mice (figure 2h). pdx1 was confined to the nuclei of pancreatic insulin - producing cells at e18.5 (figure 2h), as observed in wt littermates (figure 2 g), and was less abundantly expressed in acinar cells (arrows, figure 2 g and h). expression of ghrelin and the panendocrine marker chromogranin a (chroma) were identified in gfi1 mice (figure 2l) as compared with wt mice (figure 2k). we found no evidence of increased proliferation in the endocrine compartment at e18.5 using the m - phase marker phh3 compared with wt (figure 2i and j). analysis of the e14.5 pancreas revealed a normal onset of pancreatic endocrine cell differentiation at the secondary transition, with no apparent delay. to assess adult -cell function, we performed a glucose tolerance test in adult (10-week - old) gfi1-null mice and wt littermates. the glucose clearing rate and fasting glycemic levels of the gfi1-nulls remained comparable to the age - matched wt mice (figure 2s). we have concluded that gfi1 is not essential for initiation of pancreatic endocrine cell differentiation, endocrine subtype fate assignment, the process of islet formation, or -cell function. the endocrine compartment remained normal, so we turned our investigation to the formation and structure of the acinar compartment. to more specifically define the onset of the acinar pancreatic phenotype, we first characterized the forming acinar compartment at e14.5. the initiation of acinar development can be visualized at e14.5 by the tip - cell expression of ptf1a and the emergence of mist1, which is expressed only in maturing acinar cells. both these proteins were expressed at e14.5 in gfi1-null mice (figure 3b and d) in a pattern similar to wt littermates (figure 3a and c). using antibodies directed against carboxypeptidase a1 (cpa1) and amylase at e18.5, we found that the acinar compartment displayed similar immunoreactivity in the acinar cytoplasm between gfi1-null and wt littermates. the acinar cells appeared to develop normally in the gfi1-null mouse pancreas prenatally, but postnatally both cpa1 and amylase immunostaining revealed abnormal acinar units at the age of 1 month (1 m) (figure 3f and h) when compared with wt (figure 3e and g). in addition, the typical pyramidal, wedgelike shape of individual acinar cells was lost in the gfi1-null pancreas (figure 3f and h). furthermore, brdu staining and tunel staining revealed increased cell proliferation (at e18.5) and cell apoptosis (at 1-month - old) in the gfi1-null pancreas (figure 2n and p) compared with wt littermates (figure 2 m and o), which was made further evident by quantitative analysis (figure 2q and r). to better visualize the changes in the acinar / centroacinar units at the ultrastructural level, we performed transmission electronic microscopy analysis. electron micrographs (em) of wt pancreas showed the regular distribution of zymogen granules within the acinar cells (figure 4a and c), a regular lumen with well - organized microvilli (figure 4d), and the highly organized rer (figure 4e). in contrast, em of gfi1-null pancreas revealed accumulated zymogen granules in the acinar units (figure 4b and f) and autolysosomes (arrows in figure 4b), a distorted lumen with disorganized microvilli (figure 4 g), and accumulation of cell secretions (arrows in figure 4 g). we noted a highly disorganized, excessively dilated rer in gfi1-null pancreas (figure 4h). furthermore, em revealed significant disturbance to the apical region of almost all acini in the gfi1-null pancreas. this was evident by the irregular apical surfaces, indentations between individual exocrine cells, and vacuoles (figure 4f). the ultrastructural analysis also revealed the presence of cac in wt (highlighted with a dotted line in figure 5a), surrounded by zymogen - rich acinar cells, with which they share a lumen. in contrast to the wt pancreas, we noted that cells of a cac morphology (small, cytoplasm sparse) and location (adjacent to the exocrine apical surface) were difficult to detect within the gfi1-null pancreas (figure 5b). to further investigate whether cacs were affected in gfi1-null mice, we performed an immunohistochemical analysis to localize and characterize these cell types, covering a larger field than provided by em imaging. we chose to stain the pancreatic tissues by using fluorescently labeled bauhinia purpurea lectin (bpl), a specific lectin that we have found only labels the acinar lumen. bpl displays little to no reactivity against pancreatic ductal cells. at e18.5, when the acinar units are maturing, bpl reactivity is highly restricted to the emerging acinar lumen (figure 5c) ; an increase in bpl reactivity is noted as the organ matures (1 m, figure 5e). the bpl reactivity was completely absent in the e18.5 gfi1-null pancreas (figure 5d). bpl reactivity was disorganized and was observed with the distorted acinar units in the 1-month - old gfi1-null pancreas (figure 5f), reflecting the em morphology. we also used antibodies directed against claudin10 and mucin1 (muc1) to detect the morphology of acinar apical membranes. claudin10 is highly specific for the cells in the cac position (figure 5 g). this pattern in the pancreas reflects the presence of claudin10 in terminal tubules in the murine submandibular gland. claudin10 immunoreactivity was lost in the gfi1-null pancreas at e18.5 (figure 5h). at the postnatal stage (6 days after birth, p6), specific defects were further observed at the level of pancreatic cacs in gfi1-null mice when compared with wt littermates (figure 5o and p). in the wt pancreas, claudin10 protein specifically localizes at the apical membranes of acinar cells (arrows in figure 5o), where the duct cells including sox9 + cacs (arrowheads, figure 5o) interconnect with acinar cells. however, in gfi1-null mice, claudin10 expression at the apical membranes of acinar cells dramatically decreased (arrows in figure 5p), and sox9 + cacs were rarely found (arrowheads in figure 5p). to examine the topology of ductal structures in the pancreatic tissues of gfi1-null mice, we performed immunofluorescent staining with antibodies directed against mucin1 (muc1), a membrane protein expressed in the epithelial cells lining glands and ducts in multiple organs. in the e18.5 wt pancreas, mucin1 antibodies labeled all cells of the ductal system, including intercalated ducts and cacs, showing the well - structured branching of the ductal system and the radiations of small luminal areas (figure 5i). gfi1-null pancreas contained intercalated ductal cells but showed a lack of the terminal, radiating, branching structures as observed in wt (figure 5j). the phenotype was more pronounced at 1 month of age (1 m) (figure 5l) compared with wt littermates (figure 5k). progression and worsening of acinar structural defects continued over time ; at 4 months old (4 m), the mucin1-positive areas outlined the extensive vacuoles observed in the gfi1-null pancreas (figure 5n) compared with wt (figure 5 m). we surmised that the ongoing deterioration of acinar function might be paralleled by a compensatory regenerative program. when compared with the wt (figure 2o), we found a prevalence of apoptotic cells (tunel - positive staining) restricted to the pancreatic acinar compartment in the gfi1-null pancreas (figure 2p). correspondingly, there was also an acinar - compartment - specific increased fraction of cells in the s - phase, as detected by short - term brdu labeling (figure 2n). such a significant regenerative response would suggest substantial adaptive responses from one or more cell types in the pancreas, which could include dedifferentiation and redifferentiation events of existing cells (facultative replenishment) as seen after chemical injury,24, 25 or alternatively could involve the activation of genetic programs normally only observed during embryogenesis, such as ngn3 reactivation. expression of ngn3 mrna was not found to be increased at e18.5 or postnatally (p6) in the gfi1-null pancreas when compared with wt littermates via qrt - pcr analysis (figure 2 t and u), arguing against the reactivation of endocrine progenitor cells. it is possible that gfi1 would be required for facultative replenishment on a continuous basis, explaining the inability of the organ to recover ; however, it is also possible that the initial effect of gfi1 loss led to a defective pancreatic structure from which regeneration was not possible, regardless of the status of gfi1. further investigations into this matter would demand an ability to perform a postnatal rescue of gfi1. the developmental origin of cacs is currently unresolved but has been speculated to be ductal, occurring from sox9-expressing cells. ductal cell differentiation is initiated at the secondary transition in mice (e13.5e14.5), resulting from bipotential trpc - type progenitors segregating into endocrine and ductal cells. the gfi1-null pancreas displayed a normal distribution and amount of hnf1- and sox9-expressing cells at e14.5 (figure 6b, d, f, and h) when compared with the wt littermates (figure 6a, c, e, g). such cells coexpressed the ductal marker dolichos biflorus agglutinin (dba) (figure 6f and h), confirming their ductal identity. the hnf1- and sox9-expressing cells comprised the entire forming ductal system and extended to the forming acinar units (figure 6b, d, f, and h), comparable to the wt pancreas (figure 6a, c, e, and g). we conclude that the gfi1-null pancreas initiated ductal development appropriately, and that the organ underwent an intact process of organ domain patterning between e11.5 and e13.5. at e16.5, after completion of the secondary transition, a normal complement of hnf1+, sox9 +, dba+ cells was observed, extending throughout the ductal system (figure 6j, l, n, and p) compared with the wt pancreas (figure 6i, k, m, and o). however, at e18.5, hnf6 +, sox9 +, and hnf1+ cells, proximal to acinar units and thus corresponding to a centroacinar location, were reduced in the gfi1-null pancreas (figure 7b, b, d, d, f, and f) but were easily identifiable in the wt pancreas based on their expression, spatial location, and the typical centroacinar morphology of being cytoplasm sparse and containing flattened nuclei (figure 7a, a, c, c, e, and e). over time, the reduction of hnf6-, hnf1-, and sox9-expressing cacs remained, and only few such cells could be detected at the age of 1 month (figure 8b, b, d, d, f, and f) when compared with the wt littermates (figure 8a, a, c, c, e, and e). the reduced expression of these transcriptional factors in gfi1-null mice was further confirmed by qrt - pcr for the mrna levels of these genes (figure 7 g). interestingly, the expression of the acinar / progenitor expressed gene ptf1a was similarly reduced (figure 7 g), yet expression of the acinar terminal marker gene bhlhb8, encoding mist1, remained expressed at normal levels and was detectable throughout the acinar cells in the gfi1-null pancreas. the mist1 protein expression pattern in the gfi1-null pancreas at 1 month old (1 m) (figure 8h, h, j, and j) did not appear to be noticeably different when compared with wt littermates (figure 8 g, g, i, and i). these data suggest that the gfi1-null pancreas fails to establish a cac population and that this process occurs between e16.5 and e18.5 in mice. this time period correlates very well with the significant increase in gfi1 gene expression between e17.5 and e18.5 (figure 1a) in mouse development. we first analyzed expression of gfi1 in the mouse pancreas at different developmental stages by performing qrt - pcr and in situ hybridization. from the mice embryonic stages of e12.5 to e16.5, gfi1 mrna were present in pancreas (figure 1a). at later stages of embryonic development (e17.5e18.5) and the postnatal stage (p0 and p6), gfi1 was continuously expressed in the adult pancreas (1 month old and 4 months old, 1 m and 4 m). to assess the cell - type specific expression of gfi1, we performed in situ hybridization on developing mouse pancreas tissues (figure 1b). at all stages analyzed, the pancreatic mesenchymal tissue was devoid of gfi1 expression, and the adjacent liver, containing hematopoietic cells, served as a useful positive control (figure 1b). gfi1 was expressed in e11.5 pancreatic epithelium. at e14.5, when the pancreas progenitor population had segregated into the trpc / tippc regions, and acinar and endocrine / ductal differentiation had commenced, gfi1 became restricted to tip regions, gradually being excluded from the central domain the trunk regions although the cells displaying intermediate levels of gfi1 mrna could still be detected (arrows in figure 1b, e14.5). at e15.5, gfi1 expression was fully excluded from trunk - located cells. at e16.5, gfi1 remained abundantly expressed in the cells at tips, and we observed a strict boundary between gfi1-expressing cells at the tip location to adjacent epithelial gfi1-negative cells (e16.5 in figure 1b). the abundance of gfi1 mrna in the postsecondary transition pancreas is suggestive of a functional role for gfi1 in pancreas development at the late developmental stages. to functionally investigate gfi1 in pancreas, we bred for gfi1 nullizygous embryos and offspring using a global deletion model of exon 23 of gfi1. null animals suffer from severe neutropenia, loss of hearing, and a general growth deficiency.16, 17, 18, 19 visual inspection of midgestational (e14.5), and late - gestational (e18.5) endodermal organs revealed a normal pancreatic size and gross appearance. analysis of hematoxylin and eosin (h&e)-stained tissue at the embryonic stage of e14.5 and e18.5 did not reveal major changes in pancreatic morphology (figure 2a d). however, in adult mice (4 months old, 4 m), morphologic changes were observed in the acinar compartment. the acinar architecture was poorly organized, marked by the presence of vacuolized structures within the acini (figure 2f), when compared to the wt littermates (figure 2e). at all stages studied, the pancreatic acinar morphology of gfi1 heterozygous mice was normal. considering that gfi1 is expressed during the secondary transition when endocrine cells form, we first investigated the development and function of pancreatic endocrine cells. the presence and organization of pancreatic endocrine cells were analyzed by immunofluorescence analysis against polypeptide hormones and chromogranin a. the presence of pancreatic endocrine cell types was confirmed by staining for insulin (), glucagon (), and ghrelin () at e18.5. the central core of insulin - positive -cells in the islets of langerhans, surrounded by glucagon - positive -cells, was observed in both the wt (figure 2i) and gfi1 pancreas (figure 2j). no difference in the expression of pdx1 was observed in gfi1 mice (figure 2h). pdx1 was confined to the nuclei of pancreatic insulin - producing cells at e18.5 (figure 2h), as observed in wt littermates (figure 2 g), and was less abundantly expressed in acinar cells (arrows, figure 2 g and h). expression of ghrelin and the panendocrine marker chromogranin a (chroma) were identified in gfi1 mice (figure 2l) as compared with wt mice (figure 2k). we found no evidence of increased proliferation in the endocrine compartment at e18.5 using the m - phase marker phh3 compared with wt (figure 2i and j). analysis of the e14.5 pancreas revealed a normal onset of pancreatic endocrine cell differentiation at the secondary transition, with no apparent delay. to assess adult -cell function, we performed a glucose tolerance test in adult (10-week - old) gfi1-null mice and wt littermates. the glucose clearing rate and fasting glycemic levels of the gfi1-nulls remained comparable to the age - matched wt mice (figure 2s). we have concluded that gfi1 is not essential for initiation of pancreatic endocrine cell differentiation, endocrine subtype fate assignment, the process of islet formation, or -cell function. the endocrine compartment remained normal, so we turned our investigation to the formation and structure of the acinar compartment. to more specifically define the onset of the acinar pancreatic phenotype, we first characterized the forming acinar compartment at e14.5. the initiation of acinar development can be visualized at e14.5 by the tip - cell expression of ptf1a and the emergence of mist1, which is expressed only in maturing acinar cells. both these proteins were expressed at e14.5 in gfi1-null mice (figure 3b and d) in a pattern similar to wt littermates (figure 3a and c). using antibodies directed against carboxypeptidase a1 (cpa1) and amylase at e18.5, we found that the acinar compartment displayed similar immunoreactivity in the acinar cytoplasm between gfi1-null and wt littermates. the acinar cells appeared to develop normally in the gfi1-null mouse pancreas prenatally, but postnatally both cpa1 and amylase immunostaining revealed abnormal acinar units at the age of 1 month (1 m) (figure 3f and h) when compared with wt (figure 3e and g). in addition, the typical pyramidal, wedgelike shape of individual acinar cells was lost in the gfi1-null pancreas (figure 3f and h). furthermore, brdu staining and tunel staining revealed increased cell proliferation (at e18.5) and cell apoptosis (at 1-month - old) in the gfi1-null pancreas (figure 2n and p) compared with wt littermates (figure 2 m and o), which was made further evident by quantitative analysis (figure 2q and r). to better visualize the changes in the acinar / centroacinar units at the ultrastructural level, we performed transmission electronic microscopy analysis. electron micrographs (em) of wt pancreas showed the regular distribution of zymogen granules within the acinar cells (figure 4a and c), a regular lumen with well - organized microvilli (figure 4d), and the highly organized rer (figure 4e). in contrast, em of gfi1-null pancreas revealed accumulated zymogen granules in the acinar units (figure 4b and f) and autolysosomes (arrows in figure 4b), a distorted lumen with disorganized microvilli (figure 4 g), and accumulation of cell secretions (arrows in figure 4 g). we noted a highly disorganized, excessively dilated rer in gfi1-null pancreas (figure 4h). furthermore, em revealed significant disturbance to the apical region of almost all acini in the gfi1-null pancreas. this was evident by the irregular apical surfaces, indentations between individual exocrine cells, and vacuoles (figure 4f). the ultrastructural analysis also revealed the presence of cac in wt (highlighted with a dotted line in figure 5a), surrounded by zymogen - rich acinar cells, with which they share a lumen. in contrast to the wt pancreas, we noted that cells of a cac morphology (small, cytoplasm sparse) and location (adjacent to the exocrine apical surface) were difficult to detect within the gfi1-null pancreas (figure 5b). to further investigate whether cacs were affected in gfi1-null mice, we performed an immunohistochemical analysis to localize and characterize these cell types, covering a larger field than provided by em imaging. we chose to stain the pancreatic tissues by using fluorescently labeled bauhinia purpurea lectin (bpl), a specific lectin that we have found only labels the acinar lumen. bpl displays little to no reactivity against pancreatic ductal cells. at e18.5, when the acinar units are maturing, bpl reactivity is highly restricted to the emerging acinar lumen (figure 5c) ; an increase in bpl reactivity is noted as the organ matures (1 m, figure 5e). the bpl reactivity was completely absent in the e18.5 gfi1-null pancreas (figure 5d). bpl reactivity was disorganized and was observed with the distorted acinar units in the 1-month - old gfi1-null pancreas (figure 5f), reflecting the em morphology. we also used antibodies directed against claudin10 and mucin1 (muc1) to detect the morphology of acinar apical membranes. claudin10 is highly specific for the cells in the cac position (figure 5 g). this pattern in the pancreas reflects the presence of claudin10 in terminal tubules in the murine submandibular gland. claudin10 immunoreactivity was lost in the gfi1-null pancreas at e18.5 (figure 5h). at the postnatal stage (6 days after birth, p6), specific defects were further observed at the level of pancreatic cacs in gfi1-null mice when compared with wt littermates (figure 5o and p). in the wt pancreas, claudin10 protein specifically localizes at the apical membranes of acinar cells (arrows in figure 5o), where the duct cells including sox9 + cacs (arrowheads, figure 5o) interconnect with acinar cells. however, in gfi1-null mice, claudin10 expression at the apical membranes of acinar cells dramatically decreased (arrows in figure 5p), and sox9 + cacs were rarely found (arrowheads in figure 5p). to examine the topology of ductal structures in the pancreatic tissues of gfi1-null mice, we performed immunofluorescent staining with antibodies directed against mucin1 (muc1), a membrane protein expressed in the epithelial cells lining glands and ducts in multiple organs. in the e18.5 wt pancreas, mucin1 antibodies labeled all cells of the ductal system, including intercalated ducts and cacs, showing the well - structured branching of the ductal system and the radiations of small luminal areas (figure 5i). gfi1-null pancreas contained intercalated ductal cells but showed a lack of the terminal, radiating, branching structures as observed in wt (figure 5j). the phenotype was more pronounced at 1 month of age (1 m) (figure 5l) compared with wt littermates (figure 5k). progression and worsening of acinar structural defects continued over time ; at 4 months old (4 m), the mucin1-positive areas outlined the extensive vacuoles observed in the gfi1-null pancreas (figure 5n) compared with wt (figure 5 m). we surmised that the ongoing deterioration of acinar function might be paralleled by a compensatory regenerative program. when compared with the wt (figure 2o), we found a prevalence of apoptotic cells (tunel - positive staining) restricted to the pancreatic acinar compartment in the gfi1-null pancreas (figure 2p). correspondingly, there was also an acinar - compartment - specific increased fraction of cells in the s - phase, as detected by short - term brdu labeling (figure 2n). such a significant regenerative response would suggest substantial adaptive responses from one or more cell types in the pancreas, which could include dedifferentiation and redifferentiation events of existing cells (facultative replenishment) as seen after chemical injury,24, 25 or alternatively could involve the activation of genetic programs normally only observed during embryogenesis, such as ngn3 reactivation. expression of ngn3 mrna was not found to be increased at e18.5 or postnatally (p6) in the gfi1-null pancreas when compared with wt littermates via qrt - pcr analysis (figure 2 t and u), arguing against the reactivation of endocrine progenitor cells. it is possible that gfi1 would be required for facultative replenishment on a continuous basis, explaining the inability of the organ to recover ; however, it is also possible that the initial effect of gfi1 loss led to a defective pancreatic structure from which regeneration was not possible, regardless of the status of gfi1. further investigations into this matter would demand an ability to perform a postnatal rescue of gfi1. the developmental origin of cacs is currently unresolved but has been speculated to be ductal, occurring from sox9-expressing cells. ductal cell differentiation is initiated at the secondary transition in mice (e13.5e14.5), resulting from bipotential trpc - type progenitors segregating into endocrine and ductal cells. the gfi1-null pancreas displayed a normal distribution and amount of hnf1- and sox9-expressing cells at e14.5 (figure 6b, d, f, and h) when compared with the wt littermates (figure 6a, c, e, g). such cells coexpressed the ductal marker dolichos biflorus agglutinin (dba) (figure 6f and h), confirming their ductal identity. the hnf1- and sox9-expressing cells comprised the entire forming ductal system and extended to the forming acinar units (figure 6b, d, f, and h), comparable to the wt pancreas (figure 6a, c, e, and g). we conclude that the gfi1-null pancreas initiated ductal development appropriately, and that the organ underwent an intact process of organ domain patterning between e11.5 and e13.5. at e16.5, after completion of the secondary transition, a normal complement of hnf1+, sox9 +, dba+ cells was observed, extending throughout the ductal system (figure 6j, l, n, and p) compared with the wt pancreas (figure 6i, k, m, and o). however, at e18.5, hnf6 +, sox9 +, and hnf1+ cells, proximal to acinar units and thus corresponding to a centroacinar location, were reduced in the gfi1-null pancreas (figure 7b, b, d, d, f, and f) but were easily identifiable in the wt pancreas based on their expression, spatial location, and the typical centroacinar morphology of being cytoplasm sparse and containing flattened nuclei (figure 7a, a, c, c, e, and e). over time, the reduction of hnf6-, hnf1-, and sox9-expressing cacs remained, and only few such cells could be detected at the age of 1 month (figure 8b, b, d, d, f, and f) when compared with the wt littermates (figure 8a, a, c, c, e, and e). the reduced expression of these transcriptional factors in gfi1-null mice was further confirmed by qrt - pcr for the mrna levels of these genes (figure 7 g). interestingly, the expression of the acinar / progenitor expressed gene ptf1a was similarly reduced (figure 7 g), yet expression of the acinar terminal marker gene bhlhb8, encoding mist1, remained expressed at normal levels and was detectable throughout the acinar cells in the gfi1-null pancreas. the mist1 protein expression pattern in the gfi1-null pancreas at 1 month old (1 m) (figure 8h, h, j, and j) did not appear to be noticeably different when compared with wt littermates (figure 8 g, g, i, and i). these data suggest that the gfi1-null pancreas fails to establish a cac population and that this process occurs between e16.5 and e18.5 in mice. this time period correlates very well with the significant increase in gfi1 gene expression between e17.5 and e18.5 (figure 1a) in mouse development. here, we report that gfi1 plays a role in late gestational development of the mouse pancreas. the phenotype manifests between e16.5 and e18.5 and is correlated with a significant activation of gfi1 mrna abundance. the main hallmark of the pancreatic response to a lack of gfi1 is the apparent lack of the distal - most ductal cell type in the organ, typically referred to as the cac. our data reveal a general disturbance to the organization of the acinar unit, impacting acinar cell polarity and resulting in a specific deficiency reflected by ultrastructural changes including internal vacuolization, expanded rer, and a particular deficiency in the organization of the apical cellular region. the apical side of the acinar cell is normally connected to the intercalated ductal network, allowing for effective drainage of zymogens. we suggest that retention of zymogens in the acinar cells, the dilation of the acinar rer, and the loss of claudin10 apical expression are secondary to the structural defects resulting from a lack of cacs, although an intrinsic role of gfi1 in the acinar cell can not be ruled out. the apical deficiency of the acinar unit is significant as it appears to involve a gradual loss of hnf1-, hnf6-, and sox9-expressing cells at the very tip of the ductal network. however, the majority of the pancreatic ducts appear unaffected by a lack of gfi1, and importantly both the development of the ductal lineage and the formation of a ductal tree structure, which occurs well before e18.5, are normal. how gfi1 functions in the proper development of the interface between the ductal tree and the acinar cells of the pancreas is still unclear. expression of gfi1 ranges from the hematopoietic and lymphoid system to the sensory epithelia such as inner ear hair cells as well as in purkinje cells, lung and intestinal epithelial cells, and parts of the central nervous system.17, 18, 19, 20 gene knockout studies have shown that loss of gfi1 affects pre - t - cell differentiation, the development of granulocytes, the development and function of dendritic cells, the development pulmonary of neuroendocrine cells, the integrity of inner ear hair cells, and the proliferation, differentiation, specification, and self - renewal of hematopoietic stem cells.17, 18, 19, 20 in the gut, gfi1 serves as a prodifferentiation factor that regulates the secretory versus endocrine fate switch. except for the demonstrated role of gfi1 in the maintenance of hematopoietic stem cells, most studies implicate a specific role for gfi1 in cell fate determination. considering that the pancreatic regulatory program shares multiple determinants with that of the gut, it is tempting to speculate that gfi1 may operate also in the pancreas for the determination of cell fate, here specifically related to the generation of cacs. the pancreas is, similar to the gut, composed of secretory and endocrine components. considering that gfi1 in the gut plays a specific role opposed to neurog3 (ngn3) in facilitating the development of goblet and paneth cells, a possibility is that in the pancreas gfi1 is involved in differentiating a unique secretory cell type, the cacs, from the ductal compartment at a late stage (e17.5e18.5). at this point, it is currently unclear whether the lineage origin of the cacs is ductal or acinar, and experimental clarification would require lineage tracing from either compartment into the cacs, preferably under conditions in the absence or presence of the gfi1 gene function. because of the known plasticity of the acinar cell compartment in adult pancreatic regeneration, cacs might descend from acinar cells. although we used multiple commercial and published antibody reagents to gfi1, these attempts failed to provide convincing data on the cell - type or spatial expression of gfi1 protein at the time of cac differentiation. of note, gfi1 mrna expression data determined by in situ hybridization revealed a tip - cell (ie, acinar - fated) expression of gfi1 in early pancreatic development (figure 1b). it is thus possible that a subpopulation of already specified acinar cells commit to a cac fate via the expression of gfi1. because multiple markers, such as hnf6, hnf1, and sox9, are commonly expressed between ductal cells and cacs, we hypothesize that gfi1 expression operates to segregate the distal - most ductal cells into specific cacs, and that in absence of gfi1 such specification fails. however, it is also possible that existing acinar cells begin to express the complement of these genes upon a cac conversion. it is intriguing to speculate that gfi1 may be related to the maintenance of notch signaling in the cacs, as it was previously shown that conditional deletion of rbpj in the pancreas led to conversion of cacs into acinar cells. importantly, gfi1 has been shown to be required for the ability of immature hematopoietic cells to competently integrate notch signaling. regardless of the outstanding question on cacs origin, it is significant that the gfi1 model provides evidence that there is a specific genetic function that helps create the terminal ductal structures of the organ and that this event happens well after the initial specification of the ductal lineage. the acinar / centroacinar unit has been referred to as a highly specialized compartment, having a fenestrated structure apical to the acinar cell that allows for effective draining of acinar zymogens. regulation of ph appears to be important to immediately neutralize immature zymogen granule content should they enter this very restricted and limited space. therefore, it perhaps is not unexpected that genetic mechanisms operate to secure proper development at this level, including the specification of a uniquely functioning cell cac at the distal - most intercalated ductal network. the gfi1 gene encodes a 55-kda nuclear repressor, recruiting histone deacetylases to target promoters. at its c - terminus, it bears six typical c2h2-type zinc - finger domains that mediate sequence - specific dna binding and the interaction with other proteins. at its n - terminus, gfi1 has a 20-amino acid stretch that was named the snag (snail / gfi-1) domain because it is also found in the proteins snail and slug, which similarly have repressor functions. it is unclear whether these also represent pancreatic partners for gfi1, so it remains unknown which genomic regions are targeted by gfi1 in the pancreas as well. among the possible gfi1-repressed genes in the pancreas is ngn3, as gfi1 and ngn3 are antagonistic in the gut ; however, we did not observe an increase in endocrine cell formation at e14.5 in the gfi1-null pancreas or obtain any evidence of endocrine cell differentiation at later stages or postnatally. we believe that due to the late - gestational phenotype, gfi1 repressor functions are more likely to be related to suppression of the gene - activation programs in the duct or acinar cells, allowing for their further specification. mechanistic studies focused on gfi1 may provide new insight to the biology of the cacs. it should be recognized that we can not determine whether the lack of gfi1 leads to a complete block in the differentiation of cacs. it remains possible that such cells differentiate but lack expression of particular factors such as possibly hnf6, hnf1, sox9, which could be necessary for their structural program. in the absence of a specific intercalated ductal / cac marker that would signify the presence of such a partially specified subpopulation, it is difficult to conclude that the cells are completely absent or present in another differentiated form however, transmission electron microscopy imaging leads us to conclude that there are fewer cellular structures present in gfi1-nulls at the apical exocrine position that are not provided by the exocrine cells themselves. we believe that the presence of structures provided by such dysfunctional intercalated ductal cells would be detected as a membrane lipid bilayer contribution proximal to the acinar cell if partially differentiated cells were present. evidently, the acinar units in gfi1-null mice were abnormal, and the loss of claudin10 supported the notion that tight junctions at the apical position of the exocrine cells are defective in gfi1-null pancreas. in conclusion, we have reported on a novel gene in the pancreas, gfi1, which serves a late role in organ development. these studies also invite further exploration of the possible role of gfi1 in pancreatic disease and suggest a potential avenue for therapeutic intervention. louis, moa8273077h90441:500anti - ghrelingoatsanta cruz biotechnology, santa cruz, casc10368f01051:200anti - chrom arabbitabcam, cambridge, maab17064gr192521 - 11:250anti - glucagonmousesigma - aldrichab10988g26541:100anti - insulinguinea pignovo - nordisk, bagsvaerd, denmarka0564100578501:500anti - pdx1goatdr. wright, nashville, tn1:2000anti - sox9rabbitmillipore, billerica, maab553521671531:2000anti - hnf6rabbitsanta cruz biotechnologysc-13050c03101:50 (tsa)anti - phh3rabbitmillipore06 - 57020660521:200anti - mucin 1hamsterneomarkers, fremont, cahm-1630-p1630p805a1:200anti - brdumousebd biosciences, san jose, ca347580560651:100anti - cpa1mouseabcamab84999gr107104 - 11:200anti - claudin10rabbitabcamab24792 - 1004557761:200anti - hnf1rabbitsanta cruz biotechnologysc-22840i02051:100anti - e - cadherinratinvitrogen, frederick, md131900736501551:200fluorescein - bauheniapurpurea lectinvector labs, burlingame, cafl-1281q09101:100fluorescein - dolichosbiflorus agglutininvector labsfl-1031w11301:100tsa, tyramide - stimulated amplification. | background & aimsthe genetic specification of the compartmentalized pancreatic acinar / centroacinar unit is poorly understood. growth factor independence-1 (gfi1) is a zinc finger transcriptional repressor that regulates hematopoietic stem cell maintenance, pre - t - cell differentiation, formation of granulocytes, inner ear hair cells, and the development of secretory cell types in the intestine. as gfi1/gfi1 is expressed in human and rodent pancreas, we characterized the potential function of gfi1 in mouse pancreatic development.methodsgfi1 knockout mice were analyzed at histological and molecular levels, including qrt - pcr, in situ hybridization, immunohistochemistry, and electron microscopy.resultsloss of gfi1 impacted formation and structure of the pancreatic acinar / centroacinar unit. histologic and ultrastructural analysis of gfi1-null pancreas revealed specific defects at the level of pancreatic acinar cells as well as the centroacinar cells (cacs) in gfi1/ mice when compared with wild - type littermates. pancreatic endocrine differentiation, islet architecture, and function were unaffected. organ domain patterning and the formation of ductal cells occurred normally during the murine secondary transition (e13.5e14.5) in the gfi1/ pancreas. however, at later gestational time points (e18.5), expression of cellular markers for cacs was substantially reduced in gfi1/ mice, corroborated by electron microscopy imaging of the acinar / centroacinar unit. the reduction in cacs was correlated with an exocrine organ defect. postnatally, gfi1 deficiency resulted in severe pancreatic acinar dysplasia, including loss of granulation, autolytic vacuolation, and a proliferative and apoptotic response.conclusionsgfi1 plays an important role in regulating the development of pancreatic cacs and the function of pancreatic acinar cells. |
lung cancer is considered the most common cancer worldwide, with estimates of 1.6 million new cancers diagnosed each year. it is also the leading cause of cancer - related deaths, accounting for an estimated 1.4 million deaths per year.1 some studies estimate that 30 to 70% of the patients who die from cancer have spinal metastases at autopsy, and roughly 14% of these patients will have a symptomatic lesion over their disease course.2 3 moreover, in the united states it is estimated that there are more than 20,000 cases of metastatic epidural spinal cord compression diagnosed per year.2 3 the mean survival for patients with breast, renal, or prostate cancer that has spread to distant organs is estimated to average from 1 to 2 years, whereas the mean survival time for patients diagnosed with lung cancer that has spread to distant organs is only 6 months.4 lung cancer has a propensity to metastasize to bone resulting in skeletal complications that include bony destruction causing instability, pathologic fractures, pain, and spinal cord compression.5 6 7 each of these skeletal complications can decrease the quality of life, can cause significant morbidity, and is associated with increased mortality for patients.7 8 for the patients with non small cell lung cancer (nsclc), the spinal column is the most common site of bone metastasis. some studies demonstrate that 30% of patients with nsclc develop skeletal metastasis over their clinical course, and 50% of these metastases are in the spinal column.9 the surgical treatment of spinal metastases is often dictated by the systemic burden and life expectancy. some studies advocate a life expectancy greater than 3 months as the cutoff for surgical intervention.2 4 10 for patients with a life expectancy less than 3 months, less invasive interventions such as kyphoplasty, vertebroplasty, and/or radiotherapy are advocated irrespective of whether the patient has a neurologic deficit.2 most commonly, the opinion of the clinicians managing the primary lesion is given priority in determining the life expectancy of patients diagnosed with spinal metastases. nonetheless, this opinion can be inaccurate even when the estimations of clinicians from other departments are included in preoperative determination of the overall survival.11 the majority of studies to date have attempted to identify the prognostic factors that predict postoperative surgical outcomes through the assessment of preoperative demographic, radiologic, and functional status and/or adjuvant therapy variables. despite these assessments, the majority of the studies contain a heterogeneous composition of patients with multiple different tumor subtypes. studies that assess prognostic factors associated with specific tumor subtypes are needed to make better predictions of the optimal surgical candidate and moreover the specific interventions that will be ideal for a given patient. we performed a retrospective analysis of patients diagnosed with nsclc metastasis to the spine stratified by survival either greater than or less than 3 months to determine variables that were associated with prolonged survival. our objective was to identify preoperative prognostic factors associated with survival greater than or less than 3 months in patients with spinal metastasis from lung carcinoma. we performed a retrospective analysis of 26 patients diagnosed with lung carcinoma metastatic to the spinal column at a single institution from the years 2002 to 2011. only patients who had an estimated life expectancy greater than 3 months, as determined by the clinical assessment of their medical oncologist, were deemed potential candidates for surgical intervention. using the clinical cutoff for whether surgical intervention should be employed of 3 months, patients were stratified into two groups for analysis : patients who survived less than 3 months and patients who survived more than 3 months. we collected patient information including demographics, preoperative neurologic condition, functional status, primary disease location, systemic disease burden, other treatments, intraoperative and postoperative data on neurologic status, number of vertebral bodies removed, estimated blood loss, perioperative blood transfusions, crystalloid replacement, and complications stratified by neurologic, hematologic, respiratory, gastrointestinal, infectious, wound dehiscence, and hardware failure. magnetic resonance imaging was also used to determine extension of vertebral lesion into the ventral, lateral or paraspinal area. karnofsky performance score (kps) and modified rankin score were determined for each patient preoperatively and postoperatively based on the patient records. the modified rankin score is calculated based on the following criteria : 0 = no symptoms ; 1 = no significant disability, able to carry out all usual activities, despite some symptoms ; 2 = slight disability, requires some help, but walks unassisted ; 3 = moderate disability, requires some help, but able to walk unassisted ; 4 = moderately severe disability, unable to attend to own bodily needs without assistance, and unable to walk unassisted ; 5 = severe disability, requires constant nursing care and attention, bedridden, incontinent ; 6 = dead. statistical analysis was performed using graphpad prism (la jolla, california, united states) software. the university 's institutional review board (irb protocol # na_00067508) approved this study. using the clinical cutoff of 3 months ' survival for whether surgical intervention should be employed, patients with a diagnosis of lung adenocarcinoma spinal metastasis were stratified into those who survived less than 3 months and those who survived more than 3 months. the difference in survival between patients who survived less than 3 months compared with those who survived more than 3 months was statistically significant according to the log rank (mantel cox) test (p 70 and 7 (58%) patients with a baseline kps 70 and 6 (43%) patients with a baseline kps 70 and 12 (83%) patients with a baseline kps 70 in the group that survived more than 3 months, which approached statistical significance (p = 0.1032 ; fig. 3). for patients with a postoperative kps 70 (p = 0.10) that approached significance and no significant difference in the proportion of patients with a baseline kps > 70 when the group that survived less than 3 months was compared with the group that survived more than 3 months. there was a statistically significant difference in the postoperative modified rankin scores (p = 0.0236) and no significant difference in the baseline modified rankin scores when the group that survived less than 3 months was compared with the group that survived more than 3 months. p < 0.05 indicates statistical significance. for postoperative adjuvant therapies, 3 (25%) patients underwent postoperative radiotherapy in the group that survived less than 3 months compared with 6 (43%) in the group that survived more than 3 months, which was not significant. no patients underwent postoperative chemotherapy in the group that survived less than 3 months compared with 10 (71%) patients in the group that survived more than 3 months, which was statistically significant (p < 0.001) and most likely reflects the subset of patients who survived long enough to receive chemotherapy. there were 13 complications in the group that survived less than 3 months compared with 7 complications in the group that survived more than 3 months, which was not statistically significant (p = 0.2002) ; complications included deep vein thromboses, pulmonary embolism, pneumonias, wound infections, wound dehiscence, and cerebrospinal fluid fistulae. there was one hardware revision in each group : in the group that survived greater than 3 months, a t4 pedicle screw that broke out laterally, close to the aortic arch. in the group that survived less than 3 months, one patient with recurrence of tumor and severe cord compression lost the ability to walk for 2 to 3 days prior to surgery (table 3). recent advances in molecular biology, genomics, and surgical resection have demonstrated that nsclc is comprised of multiple tumor subtypes with specific genetic alterations that determine the growth characteristics, treatment paradigms, and prognosis.12 13 the armamentarium of pharmacologic inhibitors, which include epidermal growth factor inhibitors and anaplastic lymphoma kinase fusion oncogene inhibitors, combined with an increased understanding of the genetic background of nsclc, has resulted in more patients living with lung cancer, and this number is expected to increase in the upcoming years. with the increased prevalence of lung cancer and better treatments to control systemic disease and local recurrence, the incidence of patients developing spinal metastases is also expected to increase.3 with the increased prevalence of patients diagnosed with lung cancer spinal metastasis, surgeons will be confronted with the challenge of determining which patients will benefit from surgical intervention to improve functional status, reverse neurologic deficit, alleviate pain, or improve quality of life.4 our objective in this study is to identify factors that are associated with survival loner than 3 months in patients with spinal metastasis from lung carcinoma. we identified the presence of extrathoracic metastasis, visceral metastasis, and ventral extension of the tumor as factors associated with patients living less than 3 months. a preoperative and postoperative kps less than 40 was associated with survival less than 3 months. other scores of functional status, such as modified rankin score, demonstrated that higher scores were associated with less than 3-month survival as well. a variety of studies have identified factors that predict postoperative surgical outcomes and/or prolonged or shortened survival utilizing a heterogeneous group of tumor subtypes. studies have cited several favorable prognostic factors associated with metastatic spine disease including 0 to 2 ecog (eastern cooperative oncology group) scale of performance status, higher kps, female gender, primary histologic diagnosis of adenocarcinoma, absence of appendicular metastases, lack of pathologic fracture, use of adjuvant chemoradiation, preoperative ambulation, and slower preoperative primary tumor growth rate.14 15 16 17 18 19 in contrast, previously discovered negative prognostic factors include pathologic fracture in extraspinal metastases, complete pathologic vertebral fracture, primary histologic diagnosis of small cell lung carcinoma, absence of adjuvant chemoradiation, visceral metastases, and greater number of spinal segments affected by metastatic lesions.17 20 21 22 in 2005, tokuhashi proposed a scoring system to evaluate prognosis and potential treatment strategies, relegating lung cancer to the poor prognosis group and thus better suited to palliative or conservative treatments.23 weigel also performed a retrospective analysis of 76 patients and concluded that survival was worse in the patients with lung cancer in comparison with other tumor subtypes.24 some authors contend that these studies may indicate that patients with lung cancer should be discussed separately from other cancer subtypes.25 with this consideration in mind, fukuhara and colleagues performed a retrospective analysis of patients with metastatic lung cancer to the spine and identified 13 patients with a median postoperative survival of 5 months (range : 1 to 25 months). the authors also demonstrated that good postoperative performance was associated with better median postoperative survival.25 in our study, patients showed no significant difference in baseline kps or modified rankin scores preoperatively ; however, there was a significant difference, with better scores (i.e., higher kps and lower modified rankin scores) in the group with survival greater than 3 months. our findings support the claim that improvement and/or maintenance of functional status is associated with improved survival. improvements in adjuvant treatment modalities, including chemotherapy, radiotherapy, small molecule inhibitors, and immunotherapies, have resulted in increased control of systemic disease and longer life expectancies for patients diagnosed with lung cancer spinal metastases.4 although there was no significant difference with respect to brain metastases, we found that the group that survived less than 3 months had a higher proportion of patients with visceral metastasis compared with the group that survived more than 3 months ; the difference was statistically significant (p = 0.0447). similar to other studies, control of the systemic disease was a good prognostic factor.23 in our series, 10 patients underwent postoperative chemotherapy in the group that survived more than 3 months in comparison with no patients who survived less than 3 months. these findings are likely associated with the better overall functional outcomes in the patients who survived more than 3 months and their ability to undergo postoperative chemotherapy (better nutritional status and higher kps, among other factors). although postoperative chemotherapy may also play an important role in the overall survival of patients with spinal metastasis secondary to lung adenocarcinoma, the lack of postoperative chemotherapy in patients who lived less than 3 months is not likely to have played a significant role in our study. this observation is based on the fact that these patients had a median survival of only 1.5 months after surgery, and they did survive long enough to be considered appropriate candidates for adjuvant chemotherapy. cetin evaluated the incidence of bone metastasis and skeletal - related events (spinal cord compression, fracture, bone surgery, radiation) in patients with lung cancer, demonstrating a 1-year survival of 37.4% for patients with no bone metastasis, 12.1% for patients with bone metastasis and no skeletal - related events, and 5.1% for patients with both bone metastasis and skeletal - related events.7 we found a significant difference between both groups with 6 (50%) patients who survived less than 3 months having extrathoracic metastasis compared with 1 (7%) who survived more than 3 months (p = 0.0261). the presence of extrathoracic metastases may indicate the presence of more disseminated disease ; however, larger studies determining the natural history of the disease progression would be needed to determine whether this observation is in fact the case. the complication rates associated with different surgical approaches must be weighed against the benefits of providing better local recurrence control, increased quality of life, and ultimately increased overall survival. the determination of the ideal surgical intervention should avoid shortening the patients ' life or lowering their quality of life relative to the natural history of their associated disease.26 27 lee assessed surgical outcomes, complications, and mortality in patients with spinal metastases from multiple tumor subtypes who underwent either en bloc resection, tumor debulking, or palliative surgery. twenty - one percent of their cases were lung cancer (42 cases) and 15 of these patients underwent en bloc resection. they demonstrated that patients who underwent en bloc resection had the longest mean survival postoperatively, but they did not analyze the subgroup of patients with lung cancer.27 similarly, ratasvuori demonstrated that en bloc resection for solitary bone metastases (any skeletal metastasis) resulted in a significant improvement in overall postoperative survival rate for all tumor subtypes when compared with other surgical strategies.28 interestingly, we found that the three longest survivors in our series underwent en bloc resection with an average survival of 25.8 months (range 9.9 to 51.8 months). weighing the increased survival seen in the patients undergoing en bloc resection against the complications in this group of three patients, one patient had three complications, whereas the remaining two patients had none. these findings suggest that in appropriate circumstances en bloc resection could be considered an appropriate treatment option ; however, larger studies will be needed to substantiate this claim. our study was limited by the relatively small number of patients and lack of a control group of nonsurgically treated patients with lung cancer spinal metastasis and/or patients treated with radiotherapy. furthermore, we are only able to make associations as a retrospective study. larger multicenter prospective randomized trials focused on individual tumor subtypes will be needed to make more formidable conclusions that will influence the current treatment paradigms for this disease. the determination of which patients with lung cancer spinal metastases will benefit from surgical intervention is a multifactorial process that is often dictated by the patient 's predicted life expectancy. several factors associated with poorer prognosis include age, functional status, visceral metastases, and extrathoracic metastases. although the prognosis for patients with lung cancer spinal metastases is poor, some patients may benefit long term from surgical intervention. | study design retrospective study. objective our objective was to identify preoperative prognostic factors associated with survival in patients with spinal metastasis from lung carcinoma. methods a retrospective analysis of 26 patients diagnosed with lung carcinoma metastatic to the spinal column was performed to determine factors associated with survival. we used 3 months survival as the clinical cutoff for whether surgical intervention should be performed. we analyzed patients who survived less than 3 months compared with those who survived more than 3 months. demographic, preoperative, operative, and postoperative factors including functional scores were collected for analysis. results the median survival for all patients in our study was 3.5 months. we found a statistically significant difference between the group that survived less than 3 months and the group that survived greater than 3 months in terms of extrathoracic metastasis, visceral metastasis, and average postoperative modified rankin score. conclusion determining which patients with lung cancer spinal metastases will benefit from surgical intervention is often dictated by the patient 's predicted life expectancy. factors associated with poorer prognosis include age, functional status, visceral metastases, and extrathoracic metastases. although the prognosis for patients with lung cancer spinal metastases is poor, some patients may experience long - term benefit from surgical intervention. |
multiple sclerosis (ms) is a complex and clinically heterogeneous inflammatory demyelinating disease of the central nervous system that often results in marked physical and cognitive impairment. during the course of the disease, the tissue of the entire central nervous system is affected by demyelination, axonal damage, gliosis, inflammation and, sometimes, remyelination, which can not be clearly distinguished in vivo by conventional mri (schmierer., 2007). the corpus callosum (cc) is the largest white matter bundle in the human brain and has been found affected by lesions in up to 53% of ms patients (barnard and triggs, 1974 ; gean - marton., 1991 ; lumsden, 1971 ; simon., cc is one of the few white matter tracts that can be identified with low uncertainty by quantitative mri and diffusion tensor imaging (dti), which allow the study of its macro- and microstructures respectively. although it is well known that cc damage progresses along the course of ms (pelletier., 2001), studies on the correlation between bundle abnormalities and disability have led to inconsistent results (hasan., 2005 ; sigal., 2012 ; rimkus., 2013 ; the majority of these studies adopted metrics averaged on a priori subdivisions of the cc to investigate its involvement in ms. given the limited correlation previously found between mri measures of the cc and physical and cognitive disability, we hypothesized that a stronger relationship could be uncovered by analyzing region - specific callosal changes, detected by multimodal mri. our assumption was that, without any a priori constraint upon the topography of the bundle, macro- and micro - structural abnormalities might yield complementary information about disability in a cohort of patients with a relapsing remitting course of ms (rrms). forty - seven consecutive patients with a diagnosis of rrms according to the 2010 revised mcdonald criteria (polman., 2011) were involved in this study. inclusion criteria were absence of clinical relapses in the month before the mri scan and assumption of a first - line disease modifying therapy (interferon - beta or glatiramer acetate). since mechanisms by which disease - modifying agents may act on the brain are not fully understood (zivadinov., 2008), we chose to homogenize our sample in terms of immuno - modulating therapy, to exclude confounding effects on mri measures that could be triggered by medications and not only by ms pathology. other exclusion criteria were : evidence of major depression or other psychiatric disorders ; past or current history of traumatic brain injury or other coexisting medical disorders ; consumption of antidepressant, anxiolytic, antipsychotic or antiepileptic drugs ; and consumption of steroids in the month before the neuropsychological testing, since these substances may influence cognitive performance. on the same day of the mri acquisition, in all participants physical disability was assessed by the expanded disability status scale (edss) score while cognitive and executive functions were explored through the brief repeatable battery of neuropsychological tests and the stroop test, administered by an expert (c.c.) with more than 10 years of experience in ms. the following domains were explored : a) verbal memory : long term storage, consistent long term recall, and selective reminding test delayed ; b) language : word list generation ; c) visuo - spatial memory : spatial recall test (immediate and delayed) ; and d) executive functions : symbol digit modalities test and stroop test. cut - off values for each test were extracted by previously published normative data on the italian population (amato., 2006). in order to define the degree of neuropsychological impairment, an overall cognitive score was determined for each patient as the number of failed tests. all participants provided written informed consent and the study was approved by the institutional review board. patients were examined using a 3-tesla ge mr750 scanner (ge healthcare, rahway, nj). the mri protocol included whole - brain, three - dimensional, t1-weighted (bravo), spoiled gradient recall echo (te / tr = 3.7/9.2 ms, flip angle 12, voxel size = 1 1 1 mm) ; dti (b = 0, 1000 ; diffusion weighting along 27 non - collinear gradient directions ; matrix size 128 128 ; 80 axial slice ; number of b0 images = 4 ; nex = 2 ; voxel size = 2 2 2 mm) ; and fast fluid - attenuated inversion - recovery (flair) axial images (tr = 9500 ms, te = 100 ms ; matrix size 512 512, fov : 24 cm ; 36 slices, 4 mm slices, 0 mm gap). image processing was performed using the functional magnetic resonance imaging of the brain (fmrib) analysis group software library (fsl) (oxford university, united kingdom). image distortions induced by eddy currents and head motion in the dti data were corrected by applying a 3d full affine (mutual information cost function) alignment of each image to the mean no diffusion weighting (b0) image. after distortion corrections, dti data were averaged and concatenated into twenty - eight (1 mean b0 + 27 b1000) volumes. a diffusion tensor model was fit at each voxel, generating fractional anisotropy (fa) and mean diffusivity (md) maps. the fa maps created were then registered to brain - extracted whole - brain volumes from t1-weighted images using a full affine (correlation ratio cost function) alignment with nearest - neighbor resampling. the calculated transformation matrix was then applied to the md maps with identical resampling options. normal appearing cc was manually segmented by two experts (p.p. and a.g.) on the midsagittal plane of t1-weighted images, and subsequently averaged on a consensus binary mask. to accurately and automatically extract the bundle thickness profile we used an approach recently proposed by adamson. briefly, the thickness model was derived by computing a solution to laplace 's equation evaluated on callosal voxels. the streamlines from this solution formed non - overlapping, cross - sectional contours, the lengths of which were modeled as the callosal thickness. a smooth center line, obtained with cubic spline interpolation between measurement points, was divided into 51 regions of interest (rois) of equal lengths by 50 nodes. at each node, the distance of the line extending orthogonally to each boundary of the cc represented its thickness. we then extracted the values of md and fa from the same 50 rois by overlaying the thickness profile onto the t1-coregistered dti maps. at each roi, we computed the md / fa value as a weighted 2d gaussian average with radius 2 mm. manual delineation of t1 and flair lesions was performed by an expert radiologist (p.p.), who traced the lesion outline with a mouse - controlled cursor by using mricron software (http://www.mccauslandcenter.sc.edu/crnl). lesion volume across whole brain, whole cc and midsagittal cc was assessed by using fsl. a graphical description of the processing workflow is shown in fig. 1. spearman 's correlation was tested between each of the clinical / neuropsychological variables and, in turn, each of the three imaging features from the entire cc (i.e., thickness, fa, md). correlation significance level was set at p = 0.05 after correcting for multiple comparisons according to the false discovery rate method (benjamini and hochberg, 1995). in order to understand which of the three imaging variables had a greater role in explaining clinical and cognitive status in rrms, we built three different linear regression models in which thickness, md and fa of the roi were explanatory variables and edss, disease duration and cognitive performance, in turn, represented the dependent variable. also, gender was added to the model as a covariate, since it has been shown to have an effect on disease progression (voskuhl and gold, 2012). in each model (i.e., on each roi of the bundle) we identified the explanatory variable that better described the response variable by comparing the regression coefficients. to take into account the influence of ms lesions, regression analyses were repeated adding lesion load (either total or regional) in each model as the fourth explanatory variable. since we analyzed the entire bundle, we used the witelson scheme as a guide to interpret results (witelson, 1989). the topography of the cc is described by five sections, defined as arithmetic fractions of the maximum anterior posterior extent. section i, including the rostrum, genu, and rostral body, is crossed by fibers from the prefrontal, premotor, and supplementary motor cortical areas. motor fibers are assumed to cross the cc through the anterior midbody (section ii), whereas the somaesthetic and posterior parietal fiber bundles should cross the cc through the posterior midbody (section iii). the isthmus (section iv) is assigned to the posterior parietal and superior temporal cortical areas, and the splenium (section v), is assigned to the inferior temporal, parietal, and occipital cortical regions. thirteen patients (27.7%) failed at least three out of nine neuropsychological tests and were considered cognitively impaired. as shown in fig. 2, correlation between edss and thickness was largely significant in the anterior midbody (section ii, r values ranging from 0.50 to 0.63, p < 0.05). the significance pattern for md and fa showed significant correlation in the splenium (section v ; md only : r values ranging from 0.42 to 0.44, p < 0.05), in section ii (fa : r values ranging from 0.46 to 0.49, p < 0.05 ; md : r values ranging from 0.49 to 0.59, p < 0.05) and in section i (fa : r values ranging from 0.38 to 0.45, p < 0.05 ; md : r values ranging from 0.52 to 0.59, p < 0.05). disease duration showed a differential pattern of correlation along the bundle thickness profile : we found that correlation was most significant across section iii (r values ranging from 0.39 to 0.49, p < 0.05) and sections ivv (r values ranging from 0.38 to 0.46, p < 0.05), while barely significant in section i (r values ranging from 0.38 to 0.39, p < 0.05), according to witelson 's classification. correlation with fa was significant in section i (r values ranging from 0.36 to 0.44, p < 0.05) and section v (r = 0.38, p < 0.05). the cognitive score correlated with all the imaging variables along the whole bundle, revealing peaks of significant correlation with thickness in section i (r values ranging from 0.46 to 0.59, p values < 0.05), with fa in sections i and iv (i : r values ranging from 0.46 to 0.55, p < 0.05 ; iv : r values ranging from 0.44 to 0.55, p < 0.05) and with md across sections iii and iv (iii : r values ranging from 0.46 to 0.51, p < 0.05 ; iv : r values ranging from 0.42 to 0.52, p < 0.05) and in section i (r values ranging from 0.43 to 0.50, p < 0.05). as shown in fig. 3, multivariate linear regression analysis confirmed the spatially heterogeneous relationship between key variables of rrms disability and multimodal imaging characteristics of the entire cc. in particular, when considering only thickness, md, and fa as independent variables, relationship with edss in sections ii and iv was mainly guided by thickness, while md gave significant contributions to model variance in sections i and iii. fa contributions were barely present only in region i. relationship between the bundle and disease duration highlighted a significant contribution from thickness in sections ii and iv, from md in the splenium and from fa in the anterior third. when modeling cognitive score, instead, fa contributed consistently in section i and to a less extent in region iv, combined with md in sections iii and v, and with thickness in the rostrum. we used lesion volume both in whole white matter and in a callosal roi centered on the midsagittal cc section : since results were very similar, we reported only those relative to the whole - brain lesion load. by including lesion volume in the analysis, the relative importance of the independent variables in explaining the variance of each model remained roughly unchanged and, as expected, the insertion of lesion load increased the overall variance explained by the models. in the present study, we have analyzed microstructural abnormalities across the entire cc in rrms patients. to the best of our knowledge, this is the first study correlating callosal fiber thickness, md and fa in different cc rois with disability. in contrast to the majority of studies, we carried out the analysis without imposing any a priori subdivision on the bundle. astonishingly, the rois significantly involved in explaining disability were naturally matched to the functional sections described by witelson (1989) and to the fiber composition of the bundle observed by aboitiz. (1992), which were used only as a guide during the post - hoc interpretation of results. in particular, associations between physical and cognitive disability and structural abnormalities were spatially segregated. previous studies had discovered progressive damage of the cc in ms patients compared to controls (pelletier., 2001), but findings about the relationship between the bundle and disease variables have been rather inconsistent, ranging from no relationship at all (hasan., 2005) 2013) mainly with interhemispheric dysfunction measures rather than clinical disability variables (pelletier., 2001 ; ozturk., 2010 ; yaldizli., 2014), or among different disease courses (pelletier., 2001 ; hasan., 2005 ; sigal., 2012 ; rimkus., 2013 ; furthermore, to our knowledge, none had yet modeled clinical variables through continuous imaging features within rrms only. we believe that, in this framework, it is possible to study the specific disease course without the influence of those mechanisms that promote progression to other courses. disease duration was associated with bundle thickness, which is in line with different other studies confirming that atrophy progresses along the course of the disease (pelletier., 2001). as expected, edss score, which measures physical disability, correlated with regions of the bundle crossed by motor and premotor fibers. the cognitive score, on the other hand, correlated with cc properties of those rois crossed by fibers from the prefrontal, temporal and parietal cortical areas, which are all interconnected in order to exploit higher - level brain functions. we believe that the highly significant correlations, discrepant with respect to previous research (hasan., 2005 ; sigal., 2012 ; rimkus., 2013), might be largely explained by the fact that we assessed cc damage in a continuous fashion, without recurring to a priori subdivisions, and consequently to metrics averaged over pre - determined sections, which might have led to loss of space - variant information. multiple regression analyses uncovered the differential pattern of contributions from the imaging characteristics of the entire cc to the global measures of disease severity. disease duration was mainly explained by reductions in bundle thickness over the sections crossed by sensorimotor and temporo - parietal fibers, by md increases in regions corresponding to occipital fibers and by fa decreases in prefrontal fibers. 1992) that small fibers are densely distributed in the genu and to some extent in the splenium, while large myelinated fibers are mainly found in the body. in the light of this, the significant contribution to the model of disease duration carried by md and fa rather than thickness of prefrontal and occipital fibers could be interpreted as an early marker of fiber damage that has not yet resulted in macroscopic atrophy, due to the large number of fibers with a small diameter that populate those regions of the cc. on the contrary, atrophy was more evident where there was loss of large, heavily myelinated axons (motor fibers). regression models of edss showed that thickness reductions in the splenium and in motor areas, together with md increases in the prefrontal and sensory areas, gave the most significant contributions to the model of physical disability. as shown in fig. 3, by comparing the models of duration and physical disability, it can be seen that the beta coefficients relative to disease duration assume smaller values. this is in line with the evidence that there is no direct correspondence between disease duration and disease severity (fisniku., 2008). finally, when modeling cognitive impairment, fa gave the most consistent contribution to the analysis in the regions crossed by prefrontal and temporal fibers, thus suggesting damage of fibers involved in executive function and memory circuits, while md contributed in sections related to somatosensory and occipital fibers. since trials of potential neuroreparative agents are increasingly important in the spectrum of ms research, the need for appropriate imaging markers has been recently pointed out (mallik., 2014). these measures should be computationally lean to obtain from mr images, should be sensitive and specific to myelin and, of course, should be reproducible and clinically meaningful. in this context, we believe that the present analysis method could be a convenient mri marker in a clinical trial, from several points of view. first, it is computationally lean and reproducible : in fact, although in this testing phase it has been applied on manual segmentation of the cc, the entire analysis can be easily automated (adamson., 2014). third, dti metrics extracted continuously from the cc might indirectly bring information on demyelination / remyelination. despite the well - known limitations of diffusion - tensor modeling, these affect the dti metrics mainly when fiber orientation per voxel can not be correctly estimated, which should not be frequent when dealing with highly directional structures like the cc. even taking into account dti pitfalls, novel methods for a better reconstruction of fiber orientation are currently being implemented and could be easily integrated in our analysis (for a comparison of most recently developed methods see daducci., 2014). last, but not least, we believe that the cc might represent a crucial structure for monitoring the effects of neuroreparative drugs because of the varied nature of the fibers that form it. damage or repair to its different regions, as measured with this convenient marker, might bring further insights not only on the integrity of callosal fibers, but also, indirectly, on the status of cortical regions that these fibers connect. for all these reasons and from a wider perspective, continuous measures of callosal macro- and micro - structural integrity could be adopted to monitor drug efficacy in clinical trials focusing on neurorepair in multiple sclerosis, thus improving the treatment of disability and, as a consequence, the quality of life of patients suffering from this disease. although the present study displays coherent relationships it has some limitations : first, the sample size is relatively small, especially given the heterogeneity of the disease. for this reason future studies should foresee a follow - up stage on a larger cohort of patients. our efforts will thus focus on the progression of damage along the individual disease course, which is especially relevant when designing new treatment strategies. in this work, we demonstrated the ability of multimodal mri metrics to uncover spatially coherent patterns of relationship between cc damage and rrms disability. the lack of a priori subdivisions on the bundle did not preclude the discovery of associations between variables ; instead, it might have strengthened our results, leading us to the conclusion that the macro- and micro - structural cc damage might be a very sensitive marker of disease progression. | significant corpus callosum (cc) involvement has been found in relapsing remitting multiple sclerosis (rrms), even if conventional magnetic resonance imaging measures have shown poor correlation with clinical disability measures. in this work, we tested the potential of multimodal imaging of the entire cc to explain physical and cognitive disability in 47 patients with rrms. values of thickness, fractional anisotropy (fa) and mean diffusivity (md) were extracted from 50 regions of interest (rois) sampled along the bundle. the relationships between clinical, neuropsychological and imaging variables were assessed by using spearman 's correlation. multiple linear regression analysis was employed in order to identify the relative importance of imaging metrics in modeling different clinical variables. regional fiber composition of the cc differentially explained the response variables (expanded disability status scale [edss ], cognitive impairment). increases in edss were explained by reductions in cc thickness and md. cognitive impairment was mainly explained by fa reductions in the genu and splenium. regional cc imaging properties differentially explained disability within rrms patients revealing strong, distinct patterns of correlation with clinical and cognitive status of patients affected by this specific clinical phenotype. |
histologically it can be categorized as one of five histologic subtypes : capillary, cavernous, venous, arteriovenous or mixed. case reports describe hemangiomas of the chest wall arising from intercostal muscles (intramuscular, intercostal) or from the medullary canal of the rib (bone hemangioma). we describe a patient with an intercostal venous hemangioma presenting as a chest wall tumor, for which resection of the chest wall was successfully accomplished. a 36-year - old male was admitted to the clinic of thoracic surgery for a slowly progressing chest mass and six - month pain of the thoracic spine and left chest. there was no medical history of trauma of the chest wall and no oncological disease. during clinical examination a slowly growing soft resistance located dorso - laterally in the range of the seventh to ninth rib on the left chest chest magnetic resonance (mr) imaging showed a well - bordered tumor of the left chest wall, with a size of 9.5 x 9 x 3 cm with calcifications, overgrowing to the seventh and eighth intercostal space (fig. the patient underwent surgery. a complete resection of the tumor with the left seventh, eighth and ninth ribs and their intercostal muscles was successfully accomplished (fig. microscopically, structures of benign vascular lesions corresponding to intramuscular venous hemangioma with numerous phleboliths were found in adipose tissue and striped muscle in the resected chest wall. the patient was discharged on the seventh postoperative day, and presently, ten months after the operation, he is doing well, without any evidence of local recurrence. coronal t2-weighted mr images of the intercostal hemangioma of the chest wall (a) and enhancement by contrast media (b) reconstruction of the chest wall with used polypropylene mesh (a). intramuscular hemangiomas occur most often in people under 30 years of age and show no sex predilection. generally they are considered to be congenital, but factors such as chest trauma or repetitive bruises may play a role in development of intramuscular hemangioma [2, 3 ]. patients with intercostal hemangioma clinically may present with palpable mass of the thoracic wall, soft - tissue bulging, pain or a combination of these signs and symptoms. intercostal hemangioma should be included in the differential diagnosis of primary soft tissue chest wall tumors, such as fibromas, lipomas, giant cell tumors, neurogenic tumors, desmoids, soft tissue sarcomas, connective tissue tumors and metastatic tumors. conventional radiography hemangiomas may be seen as an ill - defined soft - tissue mass. regional ribs may show periostitis, trabecular coarsening and cortical changes such as thickening, thinning or erosion. computed tomography (ct) of the chest localizes and characterizes chest wall hemangioma, its effect on adjacent structures and it can also reveal phleboliths. a preliminary diagnosis may be established by mr examination, when classic mr findings are present : tumor mass of low or intermediate signal intensity in t1-weighted images and a high signal intensity in t2-weighted images (vascularity is manifested as a high signal intensity). invasive diagnostic techniques, such as open or percutaneous (ct - guided or echo - guided) needle biopsy are also available, but needle biopsy of hemangioma may cause bleeding. in the case of big hemangiomas, a preoperative embolization of hemangiomas with the aim to minimize the risk of intraoperative complications like excessive bleeding, intercostal hemangiomas are managed by complete surgical chest wall resection with clean surgical margins. the next decision about skeletal reconstruction posterior defects that lie above the fourth rib and are covered by the scapula do not need to be reconstructed. the choice of synthetic prosthetic material depends on the location of the defect, its size and surgeon 's preference and experience. in conclusion presumptive diagnosis was based on mr, resection was performed for definitive diagnosis and treatment. | the authors describe a case of a 36-year - old patient who had six months pain of the thoracic spine and left chest. a soft slowly growing resistance was present on the dorso - lateral side of the left chest wall, in the range of the seventh to ninth rib. according to the medical history, the patient did not have any prior trauma and malignancy. a well - defined tumor of the left chest wall with calcifications, which grew to the seventh and eighth intercostal space, was present on computed tomography (ct) and magnetic resonance (mr) scans. the patient underwent resection of the tumor with the chest wall and reconstruction with polypropylene mesh. histologically, it was a venous hemangioma, one of very rare tumors of the chest wall. |
the development of multidrug resistance in pathogenic bacteria is a significant public health risk across the world. methicillin - resistant staphylococcus aureus (mrsa) has become one of the pathogens of greatest concern due to its ability to cause a wide range of infections ranging from localized skin conditions to life threatening pneumonia and sepsis and its high prevalence in hospital- and community - associated settings.1 skin and soft tissue infections (sstis) are the most common manifestation of mrsa infection in the community setting.2 recent studies found that mrsa now accounts for 59% of sstis presenting to emergency departments in the us,3 and the national cost associated with community - acquired mrsa sstis ranges from $ 108 to $ 343 million annually.4 the emergence of community - acquired mrsa infections over the last decade has been notable because those affected are typically young, healthy individuals without any apparent risk factors,2 the infecting strains tend to be more virulent than nosocomial isolates,5 and it correlates with a coincident increase in the total number of hospitalizations in the affected patients.6 current trends indicate the expanding reservoir of mrsa in the community is likely to become a source for recurrent transmission into hospitals where it would put many more patients at risk for developing highly virulent and multidrug resistant infections.7 due to the steady decrease in the rate of new antibiotics reaching the market, an urgent need exists for the development of alternate therapeutic approaches. one potential strategy for circumventing multidrug resistance mechanisms that has gained interest in recent years is the use of light - based therapies to induce chemical or physical damage to the bacteria.813 while photodynamic therapy involving use of photosensitizing dyes has been widely shown to be effective at killing bacteria via generation of reactive oxygen species,13 use of metallic nanomaterials as photoabsorbers offers many advantages over this approach. gold nanoparticles (gnps) in particular are viewed as a promising platform for light - based therapies because they are predicted to have 45 orders of magnitude higher energy absorption and greater photostability than conventional photosensitizing dyes.14 additional advantages of gnps include ease of synthesis, straightforward conjugation to a variety of targeting molecules, ability to tune the optical properties to absorb at specific wavelengths, and utility in multimodal applications such as simultaneous imaging and treatment.14,15 importantly, gold nanomaterials are also considered to exhibit relatively good biocompatibility, and therapies involving use of gnps are currently undergoing testing in clinical trials.16 previous studies have shown that use of gnps with continuous wave or pulsed laser irradiation can significantly decrease the viability of several types of bacteria via photothermal cell lysis.912,17,18 zharov proposed that the precision of microbial killing could be maximized and collateral host tissue damage minimized by combining nanomaterials functionalized with antibodies against specific bacterial cell wall components and nanosecond pulsed laser exposure. the antibody increases the specificity of nanoparticle binding, thereby targeting the thermally induced damage to the vicinity of the bacterial surface. similarly, use of short laser pulses, compared to continuous wave irradiation, allows less time for heat diffusion to surrounding host tissue during exposure for more localized bacterial damage and reduced nonspecific damage to normal tissue ; this may also allow integration of detection through photoacoustic or other mechanisms into the treatment platform.8,10,19 using this approach, one research group reported a 95% killing of a methicillin - sensitive strain of s. aureus, in vitro, using laser pulses at 532 nm and a two - step nanoparticle targeting strategy involving binding of primary antibodies to the bacteria followed by treatment with secondary antibodies conjugated with gold nanospheres.10 the purpose of the current study was to test the antibacterial effect of this laser - induced photothermal technique against both methicillin - sensitive and methicillin - resistant s. aureus using a simplified, one - step method for targeting of the gnps to the bacteria. methicillin - sensitive staphylococcus aureus (mssa ; atcc 29213) and methicillin - resistant s. aureus (mrsa ; atcc 33591) strains of s. aureus were obtained from the american type culture collection (manassas, va, usa). most of the experiments were conducted using only the mssa strain to simplify sample handling and for experimental convenience. mssa and mrsa cultures were grown aerobically in tryptic soy broth or nutrient broth (bd, franklin lakes, nj, usa), respectively, on a shaking incubator at 250 rpm and 37c to an od600 of 0.50.7. cultures were centrifuged at 5,000 g for 5 minutes at room temperature and the bacterial pellets were washed and resuspended in phosphate buffered saline (pbs ; gibco, grand island, ny, usa) with 0.1% bovine serum albumin (bsa ; sigma - aldrich, st louis, mo, usa). biotinylated monoclonal antibodies specific to s. aureus peptidoglycan (1.3 mg / ml, clone 702, catalog number bm3066b ; acris antibodies, san diego, ca, usa) were added to the samples at a 1:100 volumetric ratio. control samples were prepared for both bacterial strains as described above, except no antibody was added. all samples were incubated for 1 hour at room temperature on an orbital mixer, then pelleted via centrifugation. bacteria were washed twice using pbs with 0.1% bsa and resuspended in the original volume in pbs containing 0.1% bsa, 1% goat serum (jackson immunoresearch laboratories, inc., west grove, pa, usa), and alexa fluor 488-affinipure goat anti - mouse igg (1.5 mg / ml, catalog number 115 - 545 - 146 ; jackson immunoresearch laboratories, inc.) at a 1:400 dilution. after incubation for 1 hour at room temperature on an orbital mixer, samples were centrifuged, washed three times with pbs, and resuspended in pbs. six aliquots (100 l) of each sample were placed into individual wells of a 96-well microtiter plate. a synergy ht microplate reader (biotek, winooski, vt, usa) was used to read od630 for the amount of bacteria and fluorescence at excitation / emission settings of 485/528 nm for the amount of bound antibody. the fluorescent signal was divided by the od630 reading to standardize for the amount of bacteria in each sample. standardized values for mssa and mrsa from three independent experiments were compared using a student s two - tailed t - test (statistica, v10 ; statsoft, inc., tulsa, ok, usa), with p<0.05 considered significant. sterile - filtered 40 nm gold nanospheres coated with streptavidin were obtained commercially (15 od ; bioassay works, ijamsville, md, usa). aliquots containing approximately 6.510 nanoparticles were diluted with 1 ml of pbs containing 0.1% bsa and 10% glycerol (sigma - aldrich ; vehicle containing pbs, bsa, and glycerol hereafter referred to as pbg) and centrifuged at 7,400 g for 10 minutes at room temperature to remove the original vehicle. the supernatant was removed, the pellet was suspended in 0.5 ml of pbg, and biotinylated monoclonal antibodies specific to s. aureus peptidoglycan (acris antibodies) were added at a 1:100 volumetric ratio. pilot testing using different volumetric ratios from 1:50 to 1:50,000 indicated 1:100 was the lowest antibody concentration required to provide maximal bacterial killing (data not shown). nonfunctionalized gold nanospheres were prepared as described above for functionalized particles except antibody was not added. bacterial cultures were centrifuged at 5,000 g for 5 minutes at room temperature, the supernatant was removed, and the bacterial pellets were suspended in half the original culture volume in pbg. tubes containing either the functionalized or nonfunctionalized gnps were removed from the orbital shaker and 0.5 ml of the bacterial suspension was added to each tube. for vehicle control samples, 0.5 ml of the bacterial suspension in pbg was added to a tube containing 0.5 ml of pbg without any nanoparticles. all tubes were then returned to the orbital mixer for 90 minutes of incubation at room temperature. pilot experiments indicated antibody alone had no significant effect on mssa colony forming ability (data not shown), and therefore this experimental group was not included in subsequent testing. laser exposures were performed using an nd : yag q - switched laser (model crf400 ; big sky / quantel, bozeman, mt, usa) with a wavelength of 532 nm, an 8 ns pulse duration, and a pulse repetition rate of 1 hz. the optical system included a 250 mm focal length lens and a variable aperture arranged to provide a 2 mm diameter beam with a maximum energy density of approximately 5 j / cm / pulse.10 the approximate size and alignment of the beam was confirmed using zap - it paper (zap - it corporation, salisbury, nh, usa). neutral density filters were placed in the beam path to reduce the pulse energy in the experiments conducted to test the effect of laser fluence on bacterial killing. a nova ii laser energy meter with a pyroelectric energy sensor (model pe25bf - dif - c) and starlab 2.0 software (all from newport, irvine, ca, usa) were used to measure and record the pulse energies for each experiment. laser dosimetry was performed immediately pre- and postexposure by recording the energy of ten successive pulses. these pre- and postexposure values were combined to calculate the mean exposure energy, which was used to calculate laser fluence (energy per unit area) assuming a 2 mm beam diameter. triplicate or quadruplicate 75 l aliquots from each bacterial sample were exposed in quartz cuvettes with a 2 mm wide window and 10 mm light path (precision cells, farmingdale, ny, usa). during exposures, cuvettes were placed in a holder with black side walls to minimize light scatter while allowing the laser beam to pass through the front and back windows. vehicle controls and bacteria treated with gnps alone were transferred to cuvettes and subjected to sham exposure for the approximate duration of laser treatment without laser activation. after irradiation with 100 pulses or sham exposure, the aliquots were gently mixed by pipetting, transferred to fresh tubes, serially diluted in pbs, and plated in triplicate on tryptic soy agar. data in graphs are expressed as percent survival compared to the vehicle controls and represent the mean and standard deviation (sd) of three to six independent experiments. statistical analysis of results was conducted using repeated measures analysis of variance (anova) tests followed by post hoc tukey or dunnett tests (statistica ; statsoft, inc.), with p<0.05 considered significant.20 linear regression was performed using sigmaplot (v10.0 ; systat software, inc., san jose, ca, usa). selected samples of mssa were collected on 0.22 m filters immediately after sham or laser exposure. the bacteria on the filters were washed once in pbs and twice in double - distilled h2o, fixed in 2.5% glutaraldehyde in pbs, and then dehydrated through a series of increasing alcohol concentrations. after mounting the filters on specimen stubs, samples were coated using a hummer 6.2 sputter coater (anatech usa, union city, ca, usa) with a gold palladium (50%50%) target and imaged using a sigma - vp40 field emission scanning electron microscope (carl zeiss inc., methicillin - sensitive staphylococcus aureus (mssa ; atcc 29213) and methicillin - resistant s. aureus (mrsa ; atcc 33591) strains of s. aureus were obtained from the american type culture collection (manassas, va, usa). most of the experiments were conducted using only the mssa strain to simplify sample handling and for experimental convenience. mssa and mrsa cultures were grown aerobically in tryptic soy broth or nutrient broth (bd, franklin lakes, nj, usa), respectively, on a shaking incubator at 250 rpm and 37c to an od600 of 0.50.7. cultures were centrifuged at 5,000 g for 5 minutes at room temperature and the bacterial pellets were washed and resuspended in phosphate buffered saline (pbs ; gibco, grand island, ny, usa) with 0.1% bovine serum albumin (bsa ; sigma - aldrich, st louis, mo, usa). biotinylated monoclonal antibodies specific to s. aureus peptidoglycan (1.3 mg / ml, clone 702, catalog number bm3066b ; acris antibodies, san diego, ca, usa) were added to the samples at a 1:100 volumetric ratio. control samples were prepared for both bacterial strains as described above, except no antibody was added. all samples were incubated for 1 hour at room temperature on an orbital mixer, then pelleted via centrifugation. bacteria were washed twice using pbs with 0.1% bsa and resuspended in the original volume in pbs containing 0.1% bsa, 1% goat serum (jackson immunoresearch laboratories, inc., west grove, pa, usa), and alexa fluor 488-affinipure goat anti - mouse igg (1.5 mg / ml, catalog number 115 - 545 - 146 ; jackson immunoresearch laboratories, inc.) at a 1:400 dilution. after incubation for 1 hour at room temperature on an orbital mixer, samples were centrifuged, washed three times with pbs, and resuspended in pbs. six aliquots (100 l) of each sample were placed into individual wells of a 96-well microtiter plate. a synergy ht microplate reader (biotek, winooski, vt, usa) was used to read od630 for the amount of bacteria and fluorescence at excitation / emission settings of 485/528 nm for the amount of bound antibody. the fluorescent signal was divided by the od630 reading to standardize for the amount of bacteria in each sample. standardized values for mssa and mrsa from three independent experiments were compared using a student s two - tailed t - test (statistica, v10 ; statsoft, inc., tulsa, ok, usa), with p<0.05 considered significant. sterile - filtered 40 nm gold nanospheres coated with streptavidin were obtained commercially (15 od ; bioassay works, ijamsville, md, usa). aliquots containing approximately 6.510 nanoparticles were diluted with 1 ml of pbs containing 0.1% bsa and 10% glycerol (sigma - aldrich ; vehicle containing pbs, bsa, and glycerol hereafter referred to as pbg) and centrifuged at 7,400 g for 10 minutes at room temperature to remove the original vehicle. the supernatant was removed, the pellet was suspended in 0.5 ml of pbg, and biotinylated monoclonal antibodies specific to s. aureus peptidoglycan (acris antibodies) were added at a 1:100 volumetric ratio. pilot testing using different volumetric ratios from 1:50 to 1:50,000 indicated 1:100 was the lowest antibody concentration required to provide maximal bacterial killing (data not shown). nonfunctionalized gold nanospheres were prepared as described above for functionalized particles except antibody was not added. bacterial cultures were centrifuged at 5,000 g for 5 minutes at room temperature, the supernatant was removed, and the bacterial pellets were suspended in half the original culture volume in pbg. tubes containing either the functionalized or nonfunctionalized gnps were removed from the orbital shaker and 0.5 ml of the bacterial suspension was added to each tube. for vehicle control samples, 0.5 ml of the bacterial suspension in pbg was added to a tube containing 0.5 ml of pbg without any nanoparticles. all tubes were then returned to the orbital mixer for 90 minutes of incubation at room temperature. pilot experiments indicated antibody alone had no significant effect on mssa colony forming ability (data not shown), and therefore this experimental group was not included in subsequent testing. laser exposures were performed using an nd : yag q - switched laser (model crf400 ; big sky / quantel, bozeman, mt, usa) with a wavelength of 532 nm, an 8 ns pulse duration, and a pulse repetition rate of 1 hz. the optical system included a 250 mm focal length lens and a variable aperture arranged to provide a 2 mm diameter beam with a maximum energy density of approximately 5 j / cm / pulse.10 the approximate size and alignment of the beam was confirmed using zap - it paper (zap - it corporation, salisbury, nh, usa). neutral density filters were placed in the beam path to reduce the pulse energy in the experiments conducted to test the effect of laser fluence on bacterial killing. a nova ii laser energy meter with a pyroelectric energy sensor (model pe25bf - dif - c) and starlab 2.0 software (all from newport, irvine, ca, usa) were used to measure and record the pulse energies for each experiment. laser dosimetry was performed immediately pre- and postexposure by recording the energy of ten successive pulses. these pre- and postexposure values were combined to calculate the mean exposure energy, which was used to calculate laser fluence (energy per unit area) assuming a 2 mm beam diameter. triplicate or quadruplicate 75 l aliquots from each bacterial sample were exposed in quartz cuvettes with a 2 mm wide window and 10 mm light path (precision cells, farmingdale, ny, usa). during exposures, cuvettes were placed in a holder with black side walls to minimize light scatter while allowing the laser beam to pass through the front and back windows. vehicle controls and bacteria treated with gnps alone were transferred to cuvettes and subjected to sham exposure for the approximate duration of laser treatment without laser activation. after irradiation with 100 pulses or sham exposure, the aliquots were gently mixed by pipetting, transferred to fresh tubes, serially diluted in pbs, and plated in triplicate on tryptic soy agar. data in graphs are expressed as percent survival compared to the vehicle controls and represent the mean and standard deviation (sd) of three to six independent experiments. statistical analysis of results was conducted using repeated measures analysis of variance (anova) tests followed by post hoc tukey or dunnett tests (statistica ; statsoft, inc.), with p<0.05 considered significant.20 linear regression was performed using sigmaplot (v10.0 ; systat software, inc., san jose, ca, usa). selected samples of mssa were collected on 0.22 m filters immediately after sham or laser exposure. the bacteria on the filters were washed once in pbs and twice in double - distilled h2o, fixed in 2.5% glutaraldehyde in pbs, and then dehydrated through a series of increasing alcohol concentrations. after mounting the filters on specimen stubs, samples were coated using a hummer 6.2 sputter coater (anatech usa, union city, ca, usa) with a gold palladium (50%50%) target and imaged using a sigma - vp40 field emission scanning electron microscope (carl zeiss inc., figure 1 shows the results of testing the antibacterial effect of nonfunctionalized or antibody - functionalized gnps either alone or in combination with 532 nm pulsed laser irradiation against mssa. exposure of bacteria to nonfunctionalized or functionalized gnps without laser treatment resulted in 88%8% and 79%5% (mean sd, n=4) survival, respectively, relative to the vehicle control group. when laser irradiation was combined with nonfunctionalized gnps, viability remained at 75%7% of the controls and was not significantly different from survival observed in the two experimental groups treated with nanoparticles alone. in contrast, use of gnps coated with anti - s. aureus antibodies with pulsed laser exposure decreased viability to 36%7% of controls, and this was significantly less than survival in the controls and other three treatment groups (p=0.0002). though the combination of nonfunctionalized gnps and laser exposure caused a slight but significant reduction in viability compared to the control group, the results illustrate the positive influence of the targeting antibody on bacterial killing and are in agreement with prior reports showing use of a targeting moiety was necessary to induce pulsed laser - induced photothermal killing in bacteria and mammalian cells.10,15 selected mssa samples were collected on filters for scanning electron microscopy (sem) imaging to visually confirm attachment of antibody - functionalized gnps to the bacteria and determine the presence of morphological changes indicative of laser - induced photothermal damage to the cells. backscattered electron images of mssa incubated with vehicle alone as the control, nonfunctionalized gnps, or antibody - functionalized gnps are shown in figure 2a. in mssa samples treated with nonfunctionalized gnps, almost all of the visible particles, which show up as bright white dots, appear on the collection filter rather than in close contact with the bacteria. in contrast, a much greater number of gnps appear attached to the bacteria when functionalized with the anti - s. these findings are in line with the results reported in figure 1 and further support the hypothesis of a highly localized photothermal effect generated by the pulsed laser around the nanoparticles, necessitating close contact between the gnps and bacteria for optimal killing. secondary electron images of mssa incubated with functionalized gnps followed by sham or pulsed laser exposure are shown in figure 2b. sham exposed samples appear as typical clumps of cocci with gnps attached individually or as clusters, whereas laser exposed samples indicate the presence of damaged bacteria that appear as shrunken or flattened cells. mechanisms of pulsed laser - induced photothermal damage to bacteria may involve generation of acoustic and shock waves, bubble formation, thermal injury, and melting or fragmentation of the nanoparticles.10,18,19 not all of the bacteria showed overt signs of damage after laser exposure, including some that had nanoparticles attached to the surface. it is possible that the typical growth pattern of s. aureus as clumps may shield some of the bacteria from the incident laser energy or binding of a sufficient number of gnps to induce complete killing. the antibacterial effect of targeted gnps and pulsed laser irradiation was tested at varying laser fluences from 0 to 5 j / cm to characterize the dose survival in the 0 j / cm (sham exposed) group was 86%20% (mean sd, n=6) compared to the vehicle controls, and increasing the laser dose from 1 to 5 j / cm resulted in decreasing levels of viability from 75%11% to 31%8%. bacteria exposed to 2, 3, or 5 j / cm had significantly lower survival rates compared to the 0 j / cm group, whereas viability in the bacteria exposed to 1 j / cm did not differ significantly from the sham exposed group. regression analysis of the data revealed a linear relationship between photothermal killing and laser fluence with an r value of 0.97. overall, the data show a similar pattern of decreasing viability as that previously published for mssa exposed to targeted gnps and nanosecond pulsed laser irradiation at 532 nm from 0 to 5 j / cm.10 the prior report showed 10% bacterial survival for laser fluences at or above 3 j / cm, which is somewhat lower than that achieved here. this disparity may be due to use of different strains of s. aureus in the two investigations or use of a one - step approach for targeting the gnps to the bacteria in the present study instead of the two - step method utilized previously. in addition, antibodies specific to peptidoglycan were employed in the current investigation, rather than anti - protein a antibodies as used in the earlier study, because growth phase- and s. aureus strain - dependent variations in protein a expression may affect efficacy of antibody - targeted antimicrobial therapies.21 the efficacy of the pulsed laser - induced photothermal treatment was tested against an mrsa strain in addition to the mssa strain used in the above experiments. as shown in figure 4, laser exposure alone did not induce a significant reduction in viability in either strain compared to the vehicle control group. incubation with functionalized gnps alone did not significantly affect viability of the mrsa strain (figure 4b) but decreased survival to 59%11% (mean sd, n=3) in the mssa strain, though this was of borderline statistical significance with p=0.04. the combination of functionalized gnps and pulsed laser exposure, however, caused a significant reduction in survival to 31%8% and 58%10%, respectively, in the mssa and mrsa strains. though comparison of results from the two strains indicate a potential difference in sensitivity, the data from mssa and mrsa were not compared statistically because testing for mssa and mrsa was conducted in separate experimental runs. differential sensitivity of these two bacterial strains could be due to multiple factors including variations in the epitope to which the antibody binds and other structural characteristics affecting the inherent sensitivity of the cells to photothermal effects. increased cell wall thickness, for example, has been associated with antibiotic resistance in s. aureus22,23 and could decrease susceptibility of mrsa to photoacoustic damage. in addition, variations in surface antigenicity of mssa and mrsa24,25 could influence the affinity of the targeting antibody to the individual strains leading to differences in sensitivity. with this possibility in mind, binding of the biotinylated anti - s. aureus primary antibody to the mssa and mrsa strains was tested using a fluorescently labeled anti - mouse secondary antibody in a microtiter plate assay. values for fluorescence intensity standardized to od630 were 249,36637,294 and 276,92216,189 (mean sd, n=3) for mrsa and mssa, respectively, and were not significantly different (p=0.31). these results indicate that differences in the level of binding of the antibody to the bacteria do not account for the variation in killing between strains observed in this study. however, further testing using a larger panel of bacterial strains and antibodies against other surface antigens is required to determine if true variations exist in the efficacy of the laser - induced photothermal treatment against different strains of s. aureus. figure 1 shows the results of testing the antibacterial effect of nonfunctionalized or antibody - functionalized gnps either alone or in combination with 532 nm pulsed laser irradiation against mssa. exposure of bacteria to nonfunctionalized or functionalized gnps without laser treatment resulted in 88%8% and 79%5% (mean sd, n=4) survival, respectively, relative to the vehicle control group. when laser irradiation was combined with nonfunctionalized gnps, viability remained at 75%7% of the controls and was not significantly different from survival observed in the two experimental groups treated with nanoparticles alone. in contrast, use of gnps coated with anti - s. aureus antibodies with pulsed laser exposure decreased viability to 36%7% of controls, and this was significantly less than survival in the controls and other three treatment groups (p=0.0002). though the combination of nonfunctionalized gnps and laser exposure caused a slight but significant reduction in viability compared to the control group, the results illustrate the positive influence of the targeting antibody on bacterial killing and are in agreement with prior reports showing use of a targeting moiety was necessary to induce pulsed laser - induced photothermal killing in bacteria and mammalian cells.10,15 selected mssa samples were collected on filters for scanning electron microscopy (sem) imaging to visually confirm attachment of antibody - functionalized gnps to the bacteria and determine the presence of morphological changes indicative of laser - induced photothermal damage to the cells. backscattered electron images of mssa incubated with vehicle alone as the control, nonfunctionalized gnps, or antibody - functionalized gnps are shown in figure 2a. in mssa samples treated with nonfunctionalized gnps, almost all of the visible particles, which show up as bright white dots, appear on the collection filter rather than in close contact with the bacteria. in contrast, a much greater number of gnps appear attached to the bacteria when functionalized with the anti - s. these findings are in line with the results reported in figure 1 and further support the hypothesis of a highly localized photothermal effect generated by the pulsed laser around the nanoparticles, necessitating close contact between the gnps and bacteria for optimal killing. secondary electron images of mssa incubated with functionalized gnps followed by sham or pulsed laser exposure are shown in figure 2b. sham exposed samples appear as typical clumps of cocci with gnps attached individually or as clusters, whereas laser exposed samples indicate the presence of damaged bacteria that appear as shrunken or flattened cells. mechanisms of pulsed laser - induced photothermal damage to bacteria may involve generation of acoustic and shock waves, bubble formation, thermal injury, and melting or fragmentation of the nanoparticles.10,18,19 not all of the bacteria showed overt signs of damage after laser exposure, including some that had nanoparticles attached to the surface. it is possible that the typical growth pattern of s. aureus as clumps may shield some of the bacteria from the incident laser energy or binding of a sufficient number of gnps to induce complete killing. the antibacterial effect of targeted gnps and pulsed laser irradiation was tested at varying laser fluences from 0 to 5 j / cm to characterize the dose response relationship (figure 3). survival in the 0 j / cm (sham exposed) group was 86%20% (mean sd, n=6) compared to the vehicle controls, and increasing the laser dose from 1 to 5 j / cm resulted in decreasing levels of viability from 75%11% to 31%8%. bacteria exposed to 2, 3, or 5 j / cm had significantly lower survival rates compared to the 0 j / cm group, whereas viability in the bacteria exposed to 1 j / cm did not differ significantly from the sham exposed group. regression analysis of the data revealed a linear relationship between photothermal killing and laser fluence with an r value of 0.97. overall, the data show a similar pattern of decreasing viability as that previously published for mssa exposed to targeted gnps and nanosecond pulsed laser irradiation at 532 nm from 0 to 5 j / cm.10 the prior report showed 10% bacterial survival for laser fluences at or above 3 j / cm, which is somewhat lower than that achieved here. this disparity may be due to use of different strains of s. aureus in the two investigations or use of a one - step approach for targeting the gnps to the bacteria in the present study instead of the two - step method utilized previously. in addition, antibodies specific to peptidoglycan were employed in the current investigation, rather than anti - protein a antibodies as used in the earlier study, because growth phase- and s. aureus strain - dependent variations in protein a expression may affect efficacy of antibody - targeted antimicrobial therapies.21 the efficacy of the pulsed laser - induced photothermal treatment was tested against an mrsa strain in addition to the mssa strain used in the above experiments. as shown in figure 4, laser exposure alone did not induce a significant reduction in viability in either strain compared to the vehicle control group. incubation with functionalized gnps alone did not significantly affect viability of the mrsa strain (figure 4b) but decreased survival to 59%11% (mean sd, n=3) in the mssa strain, though this was of borderline statistical significance with p=0.04. the combination of functionalized gnps and pulsed laser exposure, however, caused a significant reduction in survival to 31%8% and 58%10%, respectively, in the mssa and mrsa strains. though comparison of results from the two strains indicate a potential difference in sensitivity, the data from mssa and mrsa were not compared statistically because testing for mssa and mrsa was conducted in separate experimental runs. differential sensitivity of these two bacterial strains could be due to multiple factors including variations in the epitope to which the antibody binds and other structural characteristics affecting the inherent sensitivity of the cells to photothermal effects. increased cell wall thickness, for example, has been associated with antibiotic resistance in s. aureus22,23 and could decrease susceptibility of mrsa to photoacoustic damage. in addition, variations in surface antigenicity of mssa and mrsa24,25 could influence the affinity of the targeting antibody to the individual strains leading to differences in sensitivity. with this possibility in mind, binding of the biotinylated anti - s. aureus primary antibody to the mssa and mrsa strains was tested using a fluorescently labeled anti - mouse secondary antibody in a microtiter plate assay. values for fluorescence intensity standardized to od630 were 249,36637,294 and 276,92216,189 (mean sd, n=3) for mrsa and mssa, respectively, and were not significantly different (p=0.31). these results indicate that differences in the level of binding of the antibody to the bacteria do not account for the variation in killing between strains observed in this study. however, further testing using a larger panel of bacterial strains and antibodies against other surface antigens is required to determine if true variations exist in the efficacy of the laser - induced photothermal treatment against different strains of s. aureus. in conclusion, our findings support the use of laser - activated, functionalized gnps as an adjunct therapy for s. aureus infections. the treatment can be targeted to specific organisms, and the bactericidal effect is linearly related to the laser fluence. while not completely eliminating all viable bacteria, at least in the treatment s present form, this novel therapy has a potential application as an adjunct to conventional therapy for reducing the infectious load in a lesion, thus facilitating the complete eradication with the concomitant administration of a suitable antibiotic. because the systemic elimination of nanoparticles is incompletely understood at present,26 this therapy is most suited for treatment of cutaneous infections, in which the subsequent removal of the particles following the treatment is not a serious concern. finally, the efficacy of the nanoparticle therapy may likely be optimized in future studies by adjusting the size and shape of the nanoparticles and the laser parameters to regulate the nature of the induced shock waves, the extent of thermal diffusion from the metal particle, and laser tissue interactions. it may also be possible to improve the therapy by modifying the particular bacterial antigen targeted by the functionalizing ligand on the nanoparticle. | purposethe continued emergence of multidrug resistant bacterial infections and the decline in discovery of new antibiotics are major challenges for health care throughout the world. this situation has heightened the need for novel antimicrobial therapies as alternatives to traditional antibiotics. the combination of metallic nanoparticles and laser exposure has been proposed as a strategy to induce physical damage to bacteria, regardless of antibiotic sensitivity. the purpose of this study was to test the antibacterial effect of antibody - targeted gold nanoparticles combined with pulsed laser irradiation.methodsgold nanoparticles conjugated to antibodies specific to staphylococcus aureus peptidoglycan were incubated with suspensions of methicillin - resistant and methicillin - sensitive s. aureus (mrsa and mssa). bacterial suspensions were then exposed to 8 ns pulsed laser irradiation at a wavelength of 532 nm and fluences ranging from 1 to 5 j / cm2. viability of the bacteria following laser exposure was determined using colony forming unit assays. scanning electron microscopy was used to confirm the binding of nanoparticles to bacteria and the presence of cellular damage.resultsthe laser - activated nanoparticle treatment reduced the surviving population to 31% of control in the mssa population, while the survival in the mrsa population was reduced to 58% of control. significant decreases in bacterial viability occurred when the laser fluence exceeded 1 j / cm2, and this effect was linear from 0 to 5 j / cm2 (r2=0.97). significantly less bactericidal effect was observed for nonfunctionalized nanoparticles or functionalized nanoparticles without laser activation.conclusionlaser-activated nanoparticles targeted to s. aureus surface antigens significantly reduced the percentage of viable organisms and represents a promising new treatment modality that could be used either alone or as an adjunct to existing, conventional antibiotic therapy. |
diabetic nephropathy is a complex disease affecting approximately 30% of patients with type 2 diabetes mellitus (t2 dm) and leading to increased morbidity and mortality. the pathogenesis of diabetic nephropathy is multifactorial ; however, the mechanisms that drive its development remain largely undetermined [2, 3 ]. during the course of diabetic nephropathy, the functional impairment and structural remodeling of the kidney, induced by hyperglycemic injury, adiponectin - knockout (ad) mice exhibited increased albuminuria and fusion of podocyte foot processes, whereas infusion of adiponectin reduced oxidative stress and reversed the albuminuria. however, the beneficial roles of adiponectin may not be completely applied to the entire course of diabetic nephropathy. in early diabetic nephropathy nevertheless, in advanced diabetic nephropathy with macroalbuminuria or renal insufficiency, increased serum adiponectin levels were observed [68 ], predicting coexisting vascular endothelial dysfunction. in addition, high serum adiponectin levels predict mortality and progression to end stage renal disease in type i diabetic patients. the discrepancy between beneficial effects of adiponectin, observed mainly in early diabetic nephropathy, and association of adiponectin with negative prognosis in diabetic and nondiabetic patients with more advanced chronic kidney disease (ckd) is generally explained by the confounding effect of inflammation of ckd and dialysis which at the same time impacts on prognosis and triggers increased adiponectin synthesis [10, 11 ]. moreover, the expected inverse relationship between adiponectin and clinical outcomes may be modified by differential retention of high - molecular weight forms of adiponectin in renal failure, the nutritional and inflammatory status, adiponectin gene polymorphisms, and combination of these factors. the role of lipid abnormalities in the pathogenesis of glomerular injury has been demonstrated in animal models in addition to a link between hypercholesterolemia and diabetic nephropathy in humans. localized tissue oxidative stress plays a key role in the development of diabetic nephropathy and oxidized low - density lipoprotein (ox - ldl) is induced in the onset of the disease. elevated plasma ox - ldl levels were observed in t2 dm patients with macroalbuminuria, whereas significantly lower levels of ox - ldl were observed in ckd patients undergoing hemodialysis compared with controls. decreased adiponectin levels are an indicator of increased oxidative state in the arterial wall, whereas adiponectin treatment markedly suppresses foam cell formation in ox - ldl - treated macrophages from diabetic subjects. an inverse association between adiponectin and ox - ldl levels was found in t2 dm patients with coronary artery disease as well as in hemodialysis patients. however, the interplay between adiponectin and ox - ldl levels has not been studied in patients at different stages of diabetic nephropathy. although circulating adiponectin has been suggested as a possible marker of cardiovascular disease in the general population, it is a poor predictor of cardiovascular risk in both the ckd and renal transplant population. noninvasive techniques are widely used to estimate the stage of atherosclerotic changes and carotid intima - media thickness (cimt) is a useful marker of diabetic macroangiopathy as well as the extent of coronary artery disease in patients with impaired renal function. findings on the association between adiponectin levels and cimt in kidney disease are contradictory. in predialysis ckd patients, an inverse association or no association was shown [22, 23 ]. similarly, in end stage renal disease, adiponectin was shown either to correlate inversely with cimt or not to be associated with cimt. on the contrary, a positive correlation between cimt and adiponectin has been reported in patients with diabetic nephropathy. in the present study, we sought to explore (a) the association between circulating levels of adiponectin and ox - ldl in patients with diabetic nephropathy and (b) their association with cimt in the studied subjects. the criteria for determining subjects with t2 dm for inclusion in the study have been described before. all eligible patients who consented to participate in the study had to have a documented history of type 2 diabetes for at least 10 years. established diabetic nephropathy was defined by microalbuminuria (30300 mg / g creatinine) or persistent albuminuria (> 300 mg / g creatinine) in two out of three consecutive measurements in sterile spot urine sample during a 6-month period, presence of diabetic retinopathy, and absence of other kidney or urinary tract disease [27, 28 ]. diabetic retinopathy (dr) was included in the eligibility criteria for this study, since it is frequent in the presence of diabetic nephropathy and is a clue for its diagnosis. alternatively, the patients might have a history of retinal laser surgery (photocoagulation) for dr. the diagnosis and classification of ckd stages were established according to the criteria from the clinical practice guidelines for chronic kidney disease from the national kidney foundation absence of diabetic nephropathy (controls) was defined as absence of diabetic retinopathy, egfr above 60 ml / min, and persistent normoalbuminuria (030 mg / g creatinine) after at least 10 years of type 2 diabetes. patients with clinical or laboratory evidence of nondiabetic nephropathy or urinary tract disease were excluded from the study. all stage 5 ckd patients had been under regular hemodialysis, for at least 6 months, and were dialyzed thrice weekly for 4 hours per session (ckd-5d). all diabetic patients were regular patients at the diabetes clinic and nephrology clinic of academic general hospital of alexandroupolis (greece) and gave written informed consent. the study was approved by the ethics committee of the scientific council of the university general hospital of alexandroupolis and was in accordance with helsinki declaration of human rights. fasting blood was obtained from all patients and plasma was stored at 20c until analysis. blood samples were collected from hemodialysis patients after an overnight fasting of 8 h, immediately before the start of a routine 4 h hemodialysis session, as described in previous studies [17, 30 ]. blood was drawn from all patients into edta - containing tubes and into tubes without anticoagulant in order to obtain plasma, whole blood, and serum. samples for fasting blood glucose levels, hba1c, total cholesterol, low - density lipoprotein cholesterol (ldl - cholesterol), high - density lipoprotein cholesterol (hdl - cholesterol), triglycerides, and creatinine were transferred to the laboratory and assayed immediately. for adiponectin and ox - ldl, the samples were centrifuged immediately and plasma was stored at 20c until analysis. according to guidelines of american clinical practice (k / doqi), the five stages of renal insufficiency based on egfr are as follows : i 90, ii = 6089, iii = 3059, iv = 1529, and v < 15 [9, 31 ]. the glomerular filtration rate (gfr) was estimated (egfr) using the chronic kidney disease epidemiology collaboration (ckd - epi) equation, which is more accurate and less biased than the mdrd study equation, especially in patients with higher gfr, resulting in reduced misclassification of ckd. plasma concentrations of total adiponectin and ox - ldl were quantitated by enzyme - linked immunosorbent assay (elisa) according to the manufacturer 's instructions (human adiponectin elisa kit, buhlmann, switzerland ; human ox - ldl elisa kit, mercodia, sweden). according to the manufacturers, the detection limits for adiponectin and ox - ldl assays were 0.08 ng / ml and 0.3 u / l, respectively. intra- and interassay coefficients of variation were < 15% and < 10% for adiponectin and ox - ldl, respectively. measurements of cimt were obtained from 117 out of 124 patients with diabetic nephropathy recruited in this study. cimt was considered the distance from the leading edge of the lumen - intima interface to the leading edge of the intima - adventitia interface. measurements of cimt were performed online by a single trained sonographer, using a high resolution, real - time ultrasonograph with a 7,5 mhz transducer (atl ultrasound hdi 1300, philips, bothell, wa, usa). three measurements were performed for each of the common carotid arteries at 0.5, 1, and 1.5 cm distance from carotid bulb and mean cimt was calculated by averaging the six measurements. maximum cimt was defined as the greatest value of those measurement points of both sides. statistical analyses were performed using the statistical package for social sciences (spss 18.0 for windows). data for patients with diabetic nephropathy at stages 3 and 4 were merged for analysis due to the small number of subjects at stage 4 of the disease. data were tested for normality by kolmogorov - smirnov test. normally distributed continuous variables are presented as mean (s.d.), whereas nonnormally distributed continuous variables are presented as median (range). comparisons for normally distributed continuous variables among stages of diabetic nephropathy were performed with one - way anova. nonnormally distributed continuous variables were compared by mann - whitney test or kruskal - wallis test as appropriate, whereas categorical variables were compared by test. correlation analysis of cimt with various factors (age, sex, body mass index (bmi), systolic blood pressure, smoking, egfr, ldl - cholesterol, hdl - cholesterol, duration of t2 dm, and statin treatment) was conducted on an exploratory basis to determine possible predictors of cimt. stepwise multiple regression analysis was performed to study the relationship between ox - ldl and adiponectin, adjusting for confounding variables (ldl - cholesterol, triglycerides, and egfr). the same type of analysis was used to assess the significance of possible predictors of cimt (log - transformed) as determined by correlation analysis. cluster analysis methods lead to the definition of groups with similar values of certain attributes such as laboratory variables (qualitative or quantitative analytes) or other variables (e.g., anthropometric). using a matrix of distance measurements, cluster analysis finds groups of subjects more similar to each other than to those in other groups. the spss twostep cluster analysis is an algorithm that finds the optimal number of clusters using both continuous and categorical variables. in the present model, age, fasting glucose, adiponectin, and ox - ldl were included as continuous variables, whereas stage of diabetic nephropathy was included as categorical variable. anthropometric, biochemical, and clinical data of patients with diabetic nephropathy in total and according to stages of disease are presented in table 1. there was no significant difference in age and the proportion of sexes across categories of diabetic nephropathy severity. the duration of t2 dm was significantly different across groups and increased in late stages of diabetic nephropathy (p = 0.01). although total cholesterol and ldl - cholesterol did not differ significantly among groups, hdl - cholesterol decreased whereas triglycerides increased with disease progression (p < 0.001 and p = 0.006, resp.). post hoc analysis showed significant differences between control subjects and subjects with stage 5 diabetic nephropathy (p = 0.002). moreover, significant differences were observed between subjects with (a) stages 1/2 and 3/4 (p = 0.027), (b) stages 1/2 and 5 (p = 0.001), and (c) stages 3/4 and 5 (p = 0.048). conversely, circulating ox - ldl decreased significantly with disease severity (p < 0.001). post hoc analysis showed that this was due to a difference between control subjects and subjects with stage 5 diabetic nephropathy (p = 0.002) as well as between subjects with stage 3/4 and stage 5 (p = 0.001). in addition, the ratio of ox - ldl / ldl - cholesterol (u / mg) did not differ among groups (results not shown). we also investigated the effect of angiotensin - converting enzyme inhibitors, angiotensin receptor blockers, metformin, and statins on adiponectin and ox - ldl levels. the only difference identified for adiponectin was that patients on metformin (n = 35) had significantly lower levels of adiponectin compared with those not taking the drug (6.1 versus 9.2 g / ml, p = 0.001). moreover, there was an effect of statins on ox - ldl levels, as these were significantly lower in patients (n = 95) treated with statins compared with levels in untreated patients (59.1 versus 68.9 u / l, p = 0.04). finally, cimt was significantly higher at late stages of diabetic nephropathy compared with control subjects (p = 0.022). the latter had significantly lower cimt values compared with patients at stage 3/4 or 5 (p = 0.009 and p = 0.017, resp.). table 2 shows the correlation matrix between adiponectin and ox - ldl with several characteristics of the patients studied. there was a significant inverse association between adiponectin and ox - ldl in the total sample population (r = 0.29, p < 0.01) as well as in different stages of diabetic nephropathy (results not shown). moreover, adiponectin positively correlated with age, whereas it inversely correlated with egfr and bmi. after adjustment for age and bmi, plasma adiponectin positively correlated with stages of diabetic nephropathy (r = 0.23, p = 0.014). also, after adjustment for ldl - cholesterol and triglycerides, ox - ldl was inversely correlated with stages of diabetic nephropathy (r = 0.33, p < 0.001). in multiple regression analysis (table 3) including the variables which correlated with ox - ldl (adiponectin, egfr, ldl - cholesterol, and triglycerides), adiponectin was a significant negative predictor of ox - ldl levels (= 5.02, p = 0.049), independently of ldl - cholesterol, triglycerides, and egfr. there was no significant correlation between cimt and adiponectin or ox - ldl either in the total sample population (r = 0.14, p = 0.13 and r = 0.003, p = 0.97, resp.) or in any stage of diabetic nephropathy (results not shown). cimt significantly correlated with age (r = 0.24, p = 0.008), sex (r = 0.32, p = 0.001), egfr (r = 0.23, p = 0.004), and hdl - cholesterol (r = 0.18, p = 0.049). also, cimt was significantly lower in patients treated with statins compared with untreated patients (0.92 mm versus 1.00 mm, p = 0.04). in multiple regression analysis including the above variables (table 3), the only independent predictors of cimt were egfr (= 0.002, p = 0.003) and sex (= 0.102, p = 0.017). cluster analysis was performed to further explore the interrelationship of adiponectin, ox - ldl, and cimt regarding the stage of diabetic nephropathy (table 4). based on four variables entered in the model (adiponectin, ox - ldl, cimt, and age), four clusters were formed, each one distinctively characterizing each stage of diabetic nephropathy. patients with the highest cimt values, highest levels of adiponectin, and lowest levels of ox - ldl were included in cluster 3 and all assigned to stage 5 of diabetic nephropathy. conversely, patients with the lowest cimt values and low levels of adiponectin were included in cluster 4 and all assigned to the control group. figure 1 shows within cluster percent of patients according to the stage of diabetic nephropathy. in patients with diabetes mellitus as well as in those with ckd, cardiovascular disease is the leading cause of mortality. the increased cardiovascular disease risk can not solely be explained by traditional risk factors and, therefore, identification of novel modifiable risk factors is important in preventing cardiovascular disease in diabetic nephropathy. in the present study, we assessed circulating adiponectin and ox - ldl levels in patients with diabetic nephropathy as well as cimt as a noninvasive surrogate of atherosclerotic status. plasma adiponectin levels increased significantly with disease severity, in agreement with previous studies [7, 8 ]. moreover, adiponectin positively correlated with age and stages of diabetic nephropathy, whereas it inversely correlated with egfr and bmi, as previously observed. several features of uremia, such as inflammation, oxidative stress, and sympathetic overactivity, may decrease adiponectin expression, whereas other features, such as decreased renal function, proteinuria, and protein energy wasting, may increase circulating adiponectin levels. it is controversial whether adiponectin levels are increased in chronic kidney disease as a result of declining gfr and, therefore, decreased catabolism or in response to other uremic factors, since patients with advanced diabetic nephropathy have both increased adiponectin serum concentrations and increased urinary excretion. it has been suggested that increased adiponectin levels in overt diabetic nephropathy might be a physiological response to counteract renal tubular injury preventing the further progression of diabetic nephropathy through its anti - inflammatory and antiatherogenic effects. it was recently shown that renal tubular cells secrete adiponectin which increases upon inflammatory stimulus and, therefore, may contribute to the increase in circulating adiponectin levels observed in diabetic nephropathy. however, this is probably not a real effect of metformin on adiponectin levels but can be explained by the fact that this oral hypoglycemic agent is contraindicated in patients with advanced kidney disease due to the risk for lactic acidosis. the canadian diabetes association practice guidelines recommend that metformin should be stopped when the patient has progressed to stage 4 ckd (gfr < 30 ml / min/1.73 m) and should be used with extreme caution in stage 3 ckd patients (gfr between 30 and 59 ml / min/1.73 m) [38, 39 ]. in the present study, the groups of patients under metformin therapy were control patients without nephropathy, patients in stages 1 and 2, and only the minority of patients (4 out of 35) in stage 3 ckd. therefore, the difference in adiponectin levels according to metformin treatment seems to be due to confounding by its restricted administration in late stages of ckd. plasma ox - ldl levels were significantly decreased in patients with end stage diabetic nephropathy compared with early stages. despite the fact that some studies have reported higher ox - ldl levels in patients undergoing hemodialysis, contradicting results some authors found no difference between hemodialysis patients and controls [30, 40 ], whereas others described low ox - ldl levels in these patients [17, 4143 ]. possible causes of these contradictory results may be differences among study populations in age, serum lipids, or disease since most of the studies that reported elevated levels of ox - ldl in hemodialysis patients compared ox - ldl levels in nondiabetic end stage renal disease patients under hemodialysis versus healthy controls. in the present study, we assessed ox - ldl levels in diabetic patients in different stages of ckd (stages 14), diabetic patients under hemodialysis (stage 5), and diabetic controls without kidney failure. although there are reports with contradictory results on several markers of oxidative stress in patients with end stage renal disease, our results are in agreement with previous studies using the same quantitation method for ox - ldl [17, 41, 42 ]. since the ratio of ox - ldl / ldl - cholesterol did not differ among groups of patients, the proportion of ldl that is oxidized was similar across different stages of diabetic nephropathy. the reduction in ox - ldl levels in patients with end stage diabetic nephropathy does not necessarily imply a reduced risk of atherosclerosis. this could be due to a greater capture of ox - ldl by macrophage scavenger receptors which are increased in hemodialysis patients. it is also likely that decreased ox - ldl levels in hemodialysis patients are due to a reduction in ldl particles. finally, sevinc ok. reported that ox - ldl is not associated with the progression of atherosclerosis or cardiovascular / overall mortality in hemodialysis patients. it has been shown that low serum adiponectin is associated with high circulating oxidized low - density lipoprotein in patients with type 2 diabetes mellitus and coronary artery disease. similarly, we have shown an inverse association between plasma levels of adiponectin and ox - ldl in the total sample population as well as in different stages of diabetic nephropathy. in the study by lautamki., adiponectin was an independent negative predictor of ox - ldl but not lipids, whereas in the study by ribeiro. independent associations of ox - ldl were found with lipids but not adiponectin in patients under hemodialysis. nevertheless, in the present study, both lipids and adiponectin were independent predictors of ox - ldl. it has been recently shown that higher levels of adiponectin are associated with a more beneficial oxidative stress profile and lower levels of lipid peroxidation. in patients with t2 dm, however, in patients with diabetic nephropathy, adiponectin was shown to be associated with several indices of vascular dysfunction, including a positive correlation with cimt, while increased plasma adiponectin was assumed to be compensatory for early vascular endothelial damage. we did not detect a significant association between cimt and adiponectin either in the total sample population or according to disease staging, probably due to differences in patient characteristics between our study and the study by ran.. in the latter, no patients with diabetic nephropathy on hemodialysis were included, whereas all patients were free of cardiovascular complications. where circulating adiponectin was not a predictor of cimt in subjects with early stage diabetic nephropathy. similarly with adiponectin, ox - ldl was not associated with cimt in the studied population, although cimt increased with disease staging and ox - ldl decreased. since there was an effect of statins on cimt, we included statin treatment in multiple regression analysis for cimt along with age, sex, egfr, and hdl - cholesterol. clustering techniques are used when the data are expected to group physically in various categories. cluster analysis was used to describe the patterns of diabetic nephropathy based on the clinical, pathogenetic, and physiological features. according to this analysis, patients with the highest cimt values, highest levels of adiponectin, and lowest levels of ox - ldl were included in one cluster and all assigned to stage 5 of diabetic nephropathy. definitely, the repeatability of the cluster solutions should be validated in different samples and the clinical benefit must be proven. although cluster analysis is inadequate in identifying disease causes, its results may give insights into the structure of a data set and lead to hypotheses for further investigation. in patients with end stage diabetic nephropathy, the increased atherosclerotic burden is peculiarly accompanied by a favorable profile of both adiponectin and ox - ldl. therefore, apart from physiological and traditional factors associated with cimt, other pathological / protective factors affecting this surrogate marker of atherosclerosis should be explored in diabetic nephropathy. for example, other modified forms of ldl as well as circulating markers of antioxidant capacity should be examined since the latter have been shown to be negative determinants of cimt in diabetic patients. to our knowledge, this is the first study assessing the interrelationship of adiponectin and ox - ldl as well as their effect on cimt in diabetic nephropathy. however, there are certain limitations in the present study. the number of patients with diabetic nephropathy according to disease staging is small to draw definite conclusions on the associations examined. finally, only total adiponectin was measured and not its various isoforms which could show different associations with the variables examined. an inverse association between circulating adiponectin and ox - ldl was observed in diabetic nephropathy, whereas adiponectin was an independent negative predictor of ox - ldl in these patients. there was no significant association between cimt and adiponectin or ox - ldl either in the total sample population or according to disease staging. other factors affecting this surrogate marker of cardiovascular disease in diabetic nephropathy should be sought in prospective studies since the early detection of atherosclerotic changes using biomarkers could reduce adverse outcomes. | aims. we sought to determine the association between levels of adiponectin and oxidized low - density lipoprotein (ox - ldl) in patients with diabetic nephropathy as well as their effect on carotid intima - media thickness (cimt). methods. adiponectin and ox - ldl were determined in 25 diabetic patients without nephropathy and 94 patients at different stages of diabetic nephropathy including subjects on hemodialysis. cimt was measured using real - time b - mode ultrasonography. results. plasma adiponectin levels increased significantly with severity of diabetic nephropathy (p = 0.002), on the contrary to ox - ldl which decreased with disease severity (p < 0.001). cimt was significantly higher at late stages of diabetic nephropathy compared with early stages (p = 0.022). adiponectin was a significant negative predictor of ox - ldl levels (= 5.45, p = 0.023), independently of confounding factors. there was no significant correlation between cimt and adiponectin or ox - ldl either in the total sample population or according to disease staging. cluster analysis showed that patients with the highest cimt values, highest levels of adiponectin, and lowest levels of ox - ldl were included in one cluster and all assigned to stage 5 of diabetic nephropathy. conclusions. there was no significant association between adiponectin or ox - ldl and cimt and, therefore, other factors affecting this surrogate marker of cardiovascular disease in diabetic nephropathy should be sought. |
n - myristoylation is a common form of co - translational protein fatty acylation resulting from the attachment of myristate to a required n - terminal glycine residue.1,2 we show that aberrantly acquired n - myristoylation of shoc2, a leucine - rich repeat - containing protein that positively modulates ras - mapk signal flow,36 underlies a clinically distinctive condition of the neuro - cardio - facial - cutaneous disorders family. twenty - five subjects with a relatively consistent phenotype previously termed noonan - like syndrome with loose anagen hair [omim 607721]7 shared the 4a > g missense change (ser2gly) in shoc2 that introduces an n - myristoylation site, resulting in aberrant targeting of shoc2 to the plasma membrane and impaired translocation to the nucleus upon growth factor stimulation. expression of shoc2s2 g in vitro enhanced mapk activation in a cell type - specific fashion. induction of shoc2s2 g in caenorhabditis elegans engendered protruding vulva, a neomorphic phenotype previously associated with aberrant signaling. these results document the first example of an acquired n - terminal lipid modification of a protein causing human disease. |
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in korea, the incidence and mortality rate of invasive cervical cancer have decreased since the introduction of a national cervical cancer screening program. however, it has been suggested that the incidence of cervical cancer among young korean women has increased during the last two decades. furthermore, changes in sexual behavior and the prevalence of human papillomavirus (hpv) infection among korean women have also been reported. accordingly, age - period - cohort analysis could help separate the independent effects of age, period and cohort from trends in cancer rates. moreover, period effects reflect the factors that influence all age groups simultaneously, such as implementation of a screening program, while cohort effects reflect changes in lifestyle or external environmental exposures. to the best of our knowledge, only one korean study has examined the trends and age - period - cohort effects on cervical cancer - related mortality, and that study did not evaluate incidence data and used mortality data that are now > 10 years old. therefore, the present study was conducted to describe secular trends in the incidence and mortality rate of cervical cancer in korea, and to use age - period - cohort analysis to evaluate these factors independent effects. data regarding the incidence of cervical cancer between 1993 and 2012 were derived from the korea national cancer incidence database (kncidb) of the korea central cancer registry (kccr). the kncidb was launched by the korean ministry of health and welfare in 1980, and is a nationwide hospital - based cancer registry. the kccr collects data regarding approximately 80%-90% of cancer cases each year from > 180 training hospitals throughout the country. since 1999, the kccr has covered the entire population under the population - based cancer registry program. the kncidb includes information regarding age, sex, diagnosis date, geographical region, histological type, primary site, and first treatment modality. data regarding cervical cancer - related mortality between 1993 and 2012 were obtained from statistics korea (http://kosis.kr). ethical approval for the research protocol was provided by the institutional review board of the national cancer center, goyang, korea (ncc2015 - 0185). although the incidence data has high quality and completeness, the majority of mortality data for cervical cancer has been recorded as unspecified uterine cancer deaths. therefore, it is necessary to redistribute unspecified uterine cancer deaths to cervical cancer deaths using a redistribution algorithm estimation of more accurate cervical cancer mortality trends. to accomplish this, we linked the incidence database with the mortality database, then extracted registered cases of malignant neoplasms of the cervix uteri (c53) among deaths from unspecified malignant neoplasms of the uterus (c55) based on the tenth edition of the international classification of diseases. for the present study, we adjusted the numbers of cervical cancer - related deaths (c53) using the proportion of registered cases of cervical cancer (i.e., incidence, c53) to the number of deaths due to unspecified uterine cancer (c55) in each age group. we excluded the incidence and mortality data from 180 training hospitals throughout the country. since 1999, the kccr has covered the entire population under the population - based cancer registry program. the kncidb includes information regarding age, sex, diagnosis date, geographical region, histological type, primary site, and first treatment modality. data regarding cervical cancer - related mortality between 1993 and 2012 were obtained from statistics korea (http://kosis.kr). ethical approval for the research protocol was provided by the institutional review board of the national cancer center, goyang, korea (ncc2015 - 0185). although the incidence data has high quality and completeness, the majority of mortality data for cervical cancer has been recorded as unspecified uterine cancer deaths. therefore, it is necessary to redistribute unspecified uterine cancer deaths to cervical cancer deaths using a redistribution algorithm estimation of more accurate cervical cancer mortality trends. to accomplish this, we linked the incidence database with the mortality database, then extracted registered cases of malignant neoplasms of the cervix uteri (c53) among deaths from unspecified malignant neoplasms of the uterus (c55) based on the tenth edition of the international classification of diseases. for the present study, we adjusted the numbers of cervical cancer - related deaths (c53) using the proportion of registered cases of cervical cancer (i.e., incidence, c53) to the number of deaths due to unspecified uterine cancer (c55) in each age group. we excluded the incidence and mortality data from < 20-year - old women because the numbers of cases and cancer - related deaths were very low. the incidence and mortality rates of cervical cancer were grouped into thirteen age groups (most age groups covered 5 years ; 20 - 24 years old,, 80 years old). the incidence and mortality data were also arranged in four 5-year periods from 1993 - 1997 to 2008 - 2012. the age - standardized incidences and mortality rates were calculated using segi s world standard population. the joinpoint regression analysis program was employed to estimate trends in the age - standardized incidences and mortality rates, and the results were expressed as the annual percent changes (apcs) and their 95% confidence intervals (cis) for each period. age - period - cohort effects were assessed using a log - linear model to describe trends in the incidence and mortality rate using the intrinsic estimator method. the goodness of fit for each model was estimated using the likelihood ratio test and the akaike information criterion (aic). all statistical analyses were performed using the stata software ver. 12.0 (statacorp lp, college station, tx) and the age - standardized incidence of cervical cancer among 20-year - old women decreased from 32.8 per 100,000 in 1993 to 15.9 per 100,000 in 2012 (apc, 3.9% ; 95% ci, 4.2% to 3.6%) (table 1). the peak age for the incidence of cervical cancer steadily increased from 60 - 64 years during 1993 - 1997 to 75 - 79 years during 2008 - 2012 (fig. 1a). the age - specific incidence of cervical cancer exhibited a decreasing trend for all age groups, with the exception of women who were < 30 years old and 75 years old (fig. the incidence of cervical cancer among < 30-year - old women exhibited an increasing trend, and the apc among 20 - 24-year - old women (apc, 5.6% ; 95% ci, 2.9% to 8.3%) was greater than that among 25 - 29-year - old women (apc, 2.5% ; 95% ci, 1.4% to 2.5%) (data not shown). the age - standardized mortality rate for cervical cancer among 20-year - old women decreased from 5.2 per 100,000 in 1993 to 2.1 per 100,000 in 2012 (apc, 4.8% ; 95% ci, 5.1% to 4.4%) (table 1). 2a, the mortality rate for cervical cancer increased with age, except among elderly people (70 years old) during 1993 - 1997. the age - specific mortality rate for cervical cancer exhibited a decreasing trend for all age groups, with the exception of women who were 20 - 24 years old and 75 years old (fig. 2b). the age - period - cohort model of incidence had the lowest aic value from the goodness of fit tests (table 2). the age - period - cohort model of mortality had the lowest aic value, and was selected as the model with the best fit based on the goodness of fit test results (table 2). in the age - period - cohort models, the period effects for the incidence and mortality rate of cervical cancer both decreased from 1993 to 2008 (fig. the risk ratio for cervical cancer incidence and mortality decreased among women who were born between 1928 and 1973 after we adjusted for age and period effects. however, the risk ratio for cervical cancer incidence and mortality increased among women who were born after 1973. the age - standardized incidence of cervical cancer among 20-year - old women decreased from 32.8 per 100,000 in 1993 to 15.9 per 100,000 in 2012 (apc, 3.9% ; 95% ci, 4.2% to 3.6%) (table 1). the peak age for the incidence of cervical cancer steadily increased from 60 - 64 years during 1993 - 1997 to 75 - 79 years during 2008 - 2012 (fig. 1a). the age - specific incidence of cervical cancer exhibited a decreasing trend for all age groups, with the exception of women who were < 30 years old and 75 years old (fig. the incidence of cervical cancer among < 30-year - old women exhibited an increasing trend, and the apc among 20 - 24-year - old women (apc, 5.6% ; 95% ci, 2.9% to 8.3%) was greater than that among 25 - 29-year - old women (apc, 2.5% ; 95% ci, 1.4% to 2.5%) (data not shown). the age - standardized mortality rate for cervical cancer among 20-year - old women decreased from 5.2 per 100,000 in 1993 to 2.1 per 100,000 in 2012 (apc, 4.8% ; 95% ci, 5.1% to 4.4%) (table 1). 2a, the mortality rate for cervical cancer increased with age, except among elderly people (70 years old) during 1993 - 1997. the age - specific mortality rate for cervical cancer exhibited a decreasing trend for all age groups, with the exception of women who were 20 - 24 years old and 75 years old (fig. the age - period - cohort model of incidence had the lowest aic value from the goodness of fit tests (table 2). the age - period - cohort model of mortality had the lowest aic value, and was selected as the model with the best fit based on the goodness of fit test results (table 2). in the age - period - cohort models, the period effects for the incidence and mortality rate of cervical cancer both decreased from 1993 to 2008 (fig. the risk ratio for cervical cancer incidence and mortality decreased among women who were born between 1928 and 1973 after we adjusted for age and period effects. however, the risk ratio for cervical cancer incidence and mortality increased among women who were born after 1973. our findings revealed that the incidence and mortality rate of cervical cancer decreased from 1993 to 2012. the decreasing trends in the korean incidence and mortality rate are consistent with a pattern of decreasing trends throughout developed countries. our age - period - cohort models also suggest that the decrease in the period and cohort effects significantly contributed to decreases in the incidence and mortality rate of cervical cancer. however, the incidence and mortality rate of cervical cancer gradually increased in the younger birth cohorts, and the recent birth cohort effects (after the 1973 birth year) also exhibited an increasing trend. the decreasing cohort effects on the incidence and mortality rate among women who were born from 1928 - 1973 could be explained by improvements in their socioeconomic status, access to hospital - based treatment, and cervical cancer screening program. furthermore, the cohort effect that we observed was similar to that reported in mumbai. in this context, lower socioeconomic status is highly associated with the risk of hpv infection, which is a major risk factor for cervical cancer. thus, the economic growth in korea may have resulted in improved access to hospital - based treatments and greater coverage for the cervical cancer screening program. however, the risk ratio for cervical cancer incidence and mortality exhibited an increasing trend among younger cohorts. there are some possible explanations for the increased cervical cancer incidence and mortality among young women in korea. specifically, sexual behavior in younger cohorts has changed compared with older cohorts and the age at first intercourse has become earlier. these changes in sexual behavior may have led to the increased prevalence of hpv infection in more recent korean birth cohorts. additionally, there have been changes in the nature of cervical cancer in young women. for example, in situ adenocarcinoma or adenocarcinoma has increased among young women in western countries [18 - 20 ]. in korea, adenocarcinoma increased from 1993 to 2001 among young women aged 20 - 29 years old. adenocarcinoma is usually located in the cervical canal ; therefore, it was relatively hard to detect in cervical cancer screening compared to squamous cell carcinoma. indeed, it was reported that the proportion of the lymph node metastasis and tumor size have increased among young women aged < 35 years old in korea. finally, young women have a relatively low screening rate for cervical cancer and this rate showed slight decreasing trends among women aged 30 - 39 years old. taken together, these factors have likely contributed to the rise in incidence and mortality of cervical cancer among young women in korea. cervical cancer screening programs are effective and powerful tools that can reduce the risk of cervical cancer and prevent cervical cancer - related deaths. for example, the implementation of cervical cancer screening programs has decreased the incidence and mortality rate of cervical cancer in europe and north america, as well as in asia. based on the age - period - cohort analysis results, period effects imply that this screening has beneficial effects. however, using period effects as a surrogate marker for the screening effects assumes that the cervical cancer screening was implemented at the same time in all age groups. although the korea screening program was implemented in 1996, not all age groups received free access to the program at the same time. thus, differences in the participation rates may have resulted in the cohorts affecting the cervical cancer incidence and mortality rather than the periods, which were considered a surrogate marker for the screening effects. in addition, our study population represents the entire population of korea and our results are biologically plausible based on the results from other studies. first, the completeness of the incidence data from before 1999 may be lower than that from after 1999, and this discrepancy may have introduced bias into our estimated trends and age - period - cohort effects. however, the incidence data from after 1993 were collected by the gynecologic oncology committee of korea, which launched a gynecological cancer registry in 1991, and it is likely that there were very few discrepancies between the data from 1993 - 1998 and the data from 1999 - 2012. second, the early mortality data are less reliable, and the validity of the cervical cancer - related mortality data would be expected to increase over time. therefore, we adjusted these data by matching the mortality data with the cancer registry data using a redistribution algorithm. nevertheless, it is possible that residual errors were included in the cervical cancer - related mortality data, despite the use of the redistribution algorithm. third, age - period - cohort models always have an internal limitation that is related to identifiability. however, the intrinsic estimator method is a variant of the principal components regression method that provides an unbiased estimation and has a smaller variance than constrained models. the decreasing cohort effects on the incidence and mortality rate among women who were born from 1928 - 1973 could be explained by improvements in their socioeconomic status, access to hospital - based treatment, and cervical cancer screening program. furthermore, the cohort effect that we observed was similar to that reported in mumbai. in this context, lower socioeconomic status is highly associated with the risk of hpv infection, which is a major risk factor for cervical cancer. thus, the economic growth in korea may have resulted in improved access to hospital - based treatments and greater coverage for the cervical cancer screening program. however, the risk ratio for cervical cancer incidence and mortality exhibited an increasing trend among younger cohorts. there are some possible explanations for the increased cervical cancer incidence and mortality among young women in korea. specifically, sexual behavior in younger cohorts has changed compared with older cohorts and the age at first intercourse has become earlier. these changes in sexual behavior may have led to the increased prevalence of hpv infection in more recent korean birth cohorts. additionally, there have been changes in the nature of cervical cancer in young women. for example, in situ adenocarcinoma or adenocarcinoma has increased among young women in western countries [18 - 20 ]. in korea, adenocarcinoma increased from 1993 to 2001 among young women aged 20 - 29 years old. adenocarcinoma is usually located in the cervical canal ; therefore, it was relatively hard to detect in cervical cancer screening compared to squamous cell carcinoma. indeed, it was reported that the proportion of the lymph node metastasis and tumor size have increased among young women aged < 35 years old in korea. finally, young women have a relatively low screening rate for cervical cancer and this rate showed slight decreasing trends among women aged 30 - 39 years old. taken together, these factors have likely contributed to the rise in incidence and mortality of cervical cancer among young women in korea. cervical cancer screening programs are effective and powerful tools that can reduce the risk of cervical cancer and prevent cervical cancer - related deaths. for example, the implementation of cervical cancer screening programs has decreased the incidence and mortality rate of cervical cancer in europe and north america, as well as in asia. based on the age - period - cohort analysis results, however, using period effects as a surrogate marker for the screening effects assumes that the cervical cancer screening was implemented at the same time in all age groups. although the korea screening program was implemented in 1996, not all age groups received free access to the program at the same time. thus, differences in the participation rates may have resulted in the cohorts affecting the cervical cancer incidence and mortality rather than the periods, which were considered a surrogate marker for the screening effects. our results were internally consistent with the incidence and mortality rate of cervical cancer. in addition, our study population represents the entire population of korea and our results are biologically plausible based on the results from other studies. first, the completeness of the incidence data from before 1999 may be lower than that from after 1999, and this discrepancy may have introduced bias into our estimated trends and age - period - cohort effects. however, the incidence data from after 1993 were collected by the gynecologic oncology committee of korea, which launched a gynecological cancer registry in 1991, and it is likely that there were very few discrepancies between the data from 1993 - 1998 and the data from 1999 - 2012. second, the early mortality data are less reliable, and the validity of the cervical cancer - related mortality data would be expected to increase over time. therefore, we adjusted these data by matching the mortality data with the cancer registry data using a redistribution algorithm. nevertheless, it is possible that residual errors were included in the cervical cancer - related mortality data, despite the use of the redistribution algorithm. third, age - period - cohort models always have an internal limitation that is related to identifiability. however, the intrinsic estimator method is a variant of the principal components regression method that provides an unbiased estimation and has a smaller variance than constrained models. the present study revealed that the incidence and mortality rate of cervical cancer in korea have decreased since 1993. this recent decrease in the incidence and mortality rate of cervical cancer may be due to decreases in the period and cohort effects, which likely reflect the implementation of a korean cancer screening program and changes in lifestyle. however, the risks of cervical cancer incidence and mortality tended to increase in the younger cohorts. these findings suggest that a more effective cervical cancer prevention program is required for younger korean women, such as hpv vaccination, which may be a useful tool for preventing cervical cancer among this population. | purposethis study was conducted to describe the trends and age - period - cohort effects on the incidence and mortality rate of cervical cancer in korea.materials and methodsthe incidence and mortality rate of cervical cancer among 20-year - old women from 1993 to 2012 were obtained from the korea central cancer registry and the korean statistical information service. age - standardized rates were calculated and joinpoint regression was used to evaluate the trends in the incidence and mortality rate. age - period - cohort analysis was performed to investigate the independent effects of age, period and cohort.resultsthe incidence of cervical cancer decreased from 32.8 per 100,000 in 1993 to 15.9 per 100,000 in 2012 (annual percent change [apc ], 3.9% ; 95% confidence interval [ci ], 4.2% to 3.6%). the mortality rate decreased from 5.2 per 100,000 in 1993 to 2.1 per 100,000 in 2012 (apc, 4.8% ; 95% ci, 5.1% to 4.4%) ; however, the incidence and mortality rates among young women (< 30 years old) increased. an age - period - cohort model of the incidence and mortality rate showed decreasing period effects between 1993 and 2008 and decreasing cohort effects between 1928 and 1973, while birth cohorts after 1973 exhibited slight increases in the incidence and mortality rate of cervical cancer.conclusionrecent decreases in the incidence and mortality rate of cervical cancer were due to decreases in the period and cohort effects, which reflect the implementation of a cancer screening program and changes in lifestyle. however, our findings also highlighted an increase in cohort effects on the incidence and mortality rate among young women born after 1973. |
nosocomial infections are responsible for death of 4 - 56% of hospital - born neonates in various countries. thus, control of infectious agents is of much importance, particularly in the hospital environments. nosocomial infections lead a prolonged hospital stay, increased hospital costs, and anxiety of parents and families. intravascular catheterization is a risk factor for nosocomial infections and insertion of catheters puts the patients at the risk of localized and systemic infections. however, using catheters are necessary for ill neonates to take medications, water, electrolytes, and nutrients. there are plenty of antiseptic agents available, such as 70% alcohol, chlorhexidine, and povidone - iodine. the most common antiseptics in the world are povidone - iodine and chlorhexidine, both of which are available in the forms of aqueous as well as alcoholic solutions. many studies have suggested that the incidence of infections caused by inserted catheters was less in the patients whose catheter placement region was disinfected by chlorhexidine rather than povidone - iodine solution. in a meta - analysis study conducted by chaiyakunapruk. (2002), 50% reduction rate in septicemia was found when 2% chlorhexidine solution or 0.5% alcoholic chlorhexidine were used in compare to 10% povidone - iodine. yet, in a study conducted by garland. in 2009 on infants, no significant difference was found for catheter 's bacterial colonization when disinfection was done either with povidone - iodine or with chlorhexidine. with regard to the inconsistencies of the findings of different studies nevertheless, according to the existing information aqueous povidone - iodine solution is the most common antiseptic used for skin disinfection in iran. given that bacterial resistance changes over time and places, and because no studies have yet compared the efficacy of disinfectant solutions on bacterial skin flora in iranian infants, and as the efficacy of antiseptics should be evaluated periodically, this study aims to compare the effects of povidone - iodine solution and chlorhexidine solution on skin bacterial flora among iranian hospitalized infants. this is a pre - post - test and single - blinded clinical trial with within - subjects design. the within - subjects design is a powerful design which provides the most control over subject variables because the same subjects are used in each group and undergo both interventions. the sample size computed based on confidence of 0.95, power of 0.8, and mean differences equal to 0.4 sd (standard deviation). subjects were 98 bedridden infants who were hospitalized in a neonatal ward and neonatal intensive - care units of two teaching hospitals affiliated to isfahan university of medical sciences, that are the main referral centers for ill newborns in isfahan province, from 06.08.2011 until 06.11.2011. the neonates were hospitalized for a minimum duration of 24 h, their birth weights were > 1000 g, and their gestational age was more than 28 weeks. they were not suffering from any skin lesion or infectious diseases, and were selected by a continuous convenient sampling method. subjects were excluded and replaced by another infant if one of the sampling sites of their skin was used for therapeutic procedures or disinfected by any solution, or if the infant died before taking all required culture samples. before sampling, cleaning packages which contained six pieces of cotton were placed in a graft paper, sealed by an operator and sterilized in an autoclave. for sampling, the regions at the back of hand, anterior medial elbow and ankle, were coded as 1, 2, 3 respectively, and via drawing lots one of the codes was selected. through coin flipping (heads or tails), it was decided that which side of the body was supposed to get disinfected with 10% povidone - iodine solution and which side was to be disinfected by 2% chlorhexidine solution. then, by using a drawing template, the researcher selected a 2 2 cm region on the skin of the randomly selected region of each hand. before disinfecting, a skin culture sample was taken from the specified region. another two sets of culture samples were taken from the same region again, one immediately after the disinfection and the other one, 2 h after disinfection. the same procedure has been done for the other hand except for the applied disinfecting solution. therefore, the sampling ended - up with 294 (98 3) skin cultures for each group of chlorhexidine or povidone - iodine. all the skin cultures were taken from a previously specified region by means of sterilized swabs moistened in sterile normal saline solution, rubbed five times horizontally and two times vertically on the skin area based on the method utilized by darmstadt. all samples were immediately immersed in an amies transport medium (amies transport medium with charcoal, for staphylococcus aureus atcc 25923, and for escherichia coli atcc 25922) and were immediately sent to the hospital laboratory within 2 h. in the laboratory, the swabs were cultured on agar eosin - methylene blue plates (atcc 25922, ptcc 1609) and sheep blood agar (atcc 25922, atcc 33400, and atcc 8668) by a microbiologist who was unaware of the utilized disinfection solutions and also the tie of sampling. to identify the kind and count of the bacteria, the culture media were kept in the temperature of 37c for 48 h using an identical standard method by one laboratory technician. after 48 hours, efforts were made to identify the nine prevalent pathogens which are among the major causes of neonatal sepsis in developing countries including klebsiella pneumoniae, staphylococcus aureus, acinetobacter spp., escherichia coli, coagulase - negative staphylococcus in the culture media and count the colonies. those skin - cultured samples that contained one or more of the aforementioned pathogens were considered positive. the data were recorded in a data gathering sheet in two parts : demographic characteristics and the laboratory data about culture results. c - test was used to compare the frequency distribution of microorganism types before and after disinfection with povidone - iodine or with chlorhexidine, and to compare the frequency distribution of the kinds of the microorganism types at different intervals between the two disinfection methods. at the end, it is worth mentioning that the researchers in this study did not receive any monetary benefits from the companies which produced the solutions that were used in the current research, and the researchers were not biased toward any of the solutions. scientific and ethical content of this study have been approved by isfahan university of medical sciences and all the parents agreed to enter their infants in this study by signing an informed written consent. out of the 98 infants who were enrolled in the study, 59 infants (60.2%) were males and 39 infants (39.8%) were females. their gestational age was between 28 and 41 weeks and 95% confidence interval of their mean gestational age was 33 (3.5) weeks. confidence interval of the subjects average postnatal age was recorded at 9.94 (8.66) days ranging between 1 and 28 days. for average birth weight of the infants, it was 2005 (8.33) g ranging between 1010 and 4430 g. five out of the total number of the infants were excluded (in two cases due to using the sampling site for insertion of peripheral intravenous line (piv), in one case because the infant was referred to the neonatal surgery ward and in two other cases the losses were resulted from the contamination of the sampling site by the personnel). a total of 588 skin cultures were taken as the samples for the study, of which 294 cultures were taken from the skin site which were disinfected by povidone - iodine, and 294 cultures were taken from the skin sites that chlorhexidine was used for disinfection. the results of the skin cultures before, immediately after, and 2 hours after disinfection are given in table 1, which shows a significant difference between povidone - iodine and chlorhexidine groups only immediately after disinfection in terms of frequency of positive skin cultures which is 3.1% in the povidone - iodine group compared to 17.3% in the chlorhexidine group (p = 0.001). frequencies of positive and negative cultures at three different times in two groups the frequency distribution of the microorganisms in positive skin cultures before, immediately, and 2 h after disinfection are shown in table 2. in the povidone - iodine group, before disinfection, the most frequency pertained to staphylococcus epidermidis, acinetobacter, and enterobacter with 37.1% (n = 36), 4.1% (n = 4), and 4.1% (n two hours after disinfection, staphylococcus epidermidis shrank to 3.1% (n = 3), enterobacter decreased to 1% (n = 1), and acinetobacter reduced to 0% (n = 0). chi - squared tests indicated that the frequency distribution of the types of the microorganisms in the povidone - iodine group has a significant difference at three aforementioned intervals (p < 0.001 and = 132.99) [table 2 ]. comparative distribution frequency of microorganisms species, three different times within each group, and between groups in the chlorhexidine group before disinfection, the highest frequency was related to staphylococcus epidermidis, klebsiella, acinetobacter, and enterobacter with 36.1% (n = 35), 11.3% (n = 11), 7.2% (n = 7), and 3.1% (n = 3), respectively. in this group, staphylococcus epidermidis and klebsiella shrank to 3.1% (n = 3), acinetobacter reduced to 1% (n = 1), and enterobacter to 0% 2 h after disinfection. -tests indicated that the frequency distribution of the types of the microorganisms in the chlorhexidine group has a significant difference at three aforementioned different intervals (p < 0.001 and = 79.82) [table 2 ]. comparison between the frequency distribution of microorganism types in both groups showed that there were no significant differences between the frequency distribution of the kinds of microorganisms before disinfection (p = 0.84 and = 4.16) and also 2 h after disinfection (p = 0.13 and = 7.01). while the difference was significant immediately after disinfection (p = 0.002 and = 19.18) with higher frequency of negative skin cultures in the povidone - iodine group and higher frequencies of positive cultures with growth of staphylococcus epidermis, klebsiella pneumonia, and acinetobacter in the chlorhexidine group [table 2 ]. the results also showed that there were no significant differences between the frequency distribution of the kinds of microorganisms immediately and 2 h after disinfection in the povidone - iodine group (p = 0.48 and = 1.43). this difference was not significant in the chlorhexidine group too (p = 0.14 and = 6.58). this study indicated that the effectiveness of 2% chlorhexidine solution in the reduction of pathogens on the skin, immediately after disinfection, was significantly less than that of 10% povidone - iodine solution. meanwhile, there was no significant difference in effectiveness of the solutions 2 h after disinfection. garland and colleagues (2009) showed that there was no significant difference between the bacterial colonization on catheter 's tip in those infants whose catheter insertion site got disinfected by chlorhexidine solution (13%), and those infants whose insertion site got disinfected by povidone - iodine solution (4%). the results of skin cultures 2 h after intervention of our study are in line with those of garland 's study ; since, although seemingly the overall rate of positive skin cultures in the chlorhexidine group was higher than that of the povidone - iodine group, there was no significant statistical difference between those groups in the rate of bacterial colonization on the skin 2 h after disinfection. (2011), showed that the effect of povidone - iodine solution on the reduction of the number of those pathogens which cause hospital infection was stronger than that of chlorhexidine solution. valles and colleagues (2008), who compared the effect of three disinfecting solutions of 10% povidone - iodine, 2% chlorhexidine, and 0.5% alcoholic chlorhexidine on catheter 's bacterial colonization, showed that the rate of catheter 's bacterial colonization in the chlorhexidine group and the alcoholic chlorhexidine group was significantly higher than that of the povidone - iodine group (p = 0.03). the results of our study from those culture samples that got immediately after disinfection are similar to these results, since in this study, the percentage of positive skin cultures, immediately after disinfection, in the chlorhexidine group was higher than the povidone - iodine group. suwanpimolkul and colleagues (2008) indicated that the infection rate in blood cultures was 3.2% after disinfecting with 2% chlorhexidine and alcoholic chlorhexidine while it was 6.9% after disinfecting with povidone - iodine and this difference was significant (p < 0.001). this difference in the results can be because of using alcoholic chlorhexidine for disinfection in suwanpimolkul 's study rather than aqueous chlorhexidine in our study and also may be related to difference in sample sizes or in environmental factors such as bacterial resistance. chaiyakunapruk meta - analysis study (2002) indicated that only in those studies that alcoholic chlorhexidine solutions were used, there has been a significant decrease in the rate of catheter colonization compared to the studies which used povidone - iodine solution. other studies have shown that using alcoholic antiseptics has been more effective on the reduction of infection in blood cultures as well as catheter 's infection compared with other antiseptics. in our study, because of lack of knowledge about the probable side effects of using chlorhexidine - ethanol on infants and the prohibition of its use in infants, alcoholic mixtures were not used. kasuda and colleagues (2002) indicated that in the two groups, staphylococcus epidermis was the most frequent bacterial species which was separated from the skin cultures at the catheter 's insertion sites as well as the cultures of catheters tip and there was no significant difference between the two groups in terms of bacteria types. suwanpimolkul and colleagues (2008) also showed that 80.6% of the blood cultures infection after disinfecting the skin with povidone - iodine and chlorhexidine solutions pertained to coagulase - negative staphylococci, and the frequency distribution of the bacteria between the povidone iodine group and the chlorhexidine group was the same. our study supports the results of kasuda and suwanpimolkul, because in our study, the highest percent of bacteria species pertained to staphylococcus epidermis before and 2 h after disinfection too, and there was no significant difference between the two groups in terms of bacteria species. however, frequency of bacterial species was significantly higher in the chlorhexidine group immediately after disinfection than in the povidone - iodine group that shows greater impact of povidone - iodine on skin bacterial flora at the time frame in which insertion of iv devices usually takes place. the results of the studies by kasuda and suwanpimolkulas also have a double emphasis on the fact that bacterial skin flora enters internal body tissues through invasive methods including catheterization, venous puncture, and piv insertion ; causing nosocomial infections. darmstadt and colleagues (2007) showed that the rate of positive skin cultures 2 h after skin disinfection with chlorhexidine solution was decreased by 35 - 55%. however, in our study, positive skin cultures in the chlorhexidine group, immediately and 2 h after disinfection, dropped by 70% and 88%, respectively. this difference could be due to the difference in the density of chlorhexidine solutions which were used in darmstadt 's study (0.25%) and the present study (2%). it can also be related to the difference in the sites which were used for sampling skin culture in the two studies. in darmstadt 's study, after a primary bathing and cleansing the skin, cultures were collected from the three sites of axillary, periumbilical regions, and inguinal regions. while in our study, the cultures were collected from the regions at the back of the hand, anterior medial elbow, and ankle. therefore, it can be concluded that there is a higher probability of bacterial colonization in those regions where the skin is more wrinkled. thus, the regions at the back of the hands, anterior medial elbow and ankle can be preferably used for the venous puncture, blood sampling and inserting central venous catheter or peripherally inserted central catheter (picc) in newborns. this could be a limitation as some researchers suggested potential absorption of cholorhexidine solution despite no observed irritation or systemic side effects. the results of this study showed that 10% povidone - iodine solution is more effective than 2% chlorhexidine solution in reducing the skin bacterial flora immediately after disinfection. thus, it is recommended to use 10% povidone - iodine solution to disinfect the skin before conducting procedures such as catheterization, venous puncture, and obtaining piv and inserting picc in neonatal sectors. the probable side effects of the used solutions including occurrence of allergic reactions, probable thyroid complications related to unwanted absorption of iodine, and neurologic responses to probable absorption of chlorhexidine were not examined and investigated in this study. however, mothers were asked to inform the researcher in case of any symptom of skin allergic reaction detected. however, no one referred to the researcher for this side effect. future studies should consider the measurement of the side effects of using these solutions for infants as antiseptic solutions. | background : infection control is an essential part of caring for hospitalized infants. with regard to the change of bacterial resistance over time and places, as well as the need for periodic studies on the effectiveness of antiseptics, this study aims to compare the effects of both solutions of povidone - iodine and chlorhexidine on skin bacterial flora among hospitalized infants.materials and methods : this clinical trial recruited 98 hospitalized infants and each of the above - mentioned solutions has been applied to a small area in the left or right side of the infants bodies. skin cultures were taken before, immediately after and 2 h after the randomly chosen infants skin areas that were disinfected by each solution (588 skin cultures in total). colony count and determination of microorganism types were done by only one person in a single laboratory. the study has been conducted in two teaching hospitals in isfahan, iran.results:staphylococcus epidermidis was the most common microorganism prior to skin disinfection by either solution. two hours after disinfection, staphylococcus epidermidis and staphylococcus epidermidis and kelebsila had the highest frequencies of 3.1% and 3.1%, respectively. before and 2 h after disinfection, distribution of different types of microorganisms had no significant difference between the two groups (p = 0.84 and 0.13, respectively) ; however, the difference was significant immediately after disinfection, p < 0.01.conclusion:the present study demonstrated that 10% povidone - iodine solution has more significant effect on reduction of skin pathogens promptly after application compared to 2% chlorhexidine. therefore, prior to any catheterization procedures, it is imperative to use 10% povidone - iodine solutions for skin disinfection. |
dna is a highly polar molecule, evolved to be stable in high - dielectric environments, such as aqueous solution, which is the main environment considered in most experimental and theoretical studies. however, understanding the properties of dna in low - polarity environments is crucial for many nanotechnological applications of dna and is instrumental in understanding how nucleic acids acting as antigene or antisense drugs pass through the highly hydrophobic cellular membrane, and how dna can be transported in the body by means of liposome carriers. water and counterions are crucial to screen the electrostatic repulsion among charged phosphates and also favor the apolar stacking of bases. accordingly, the large impact of solvent modification on the properties of dna is not surprising. for example, a subtle change in the neutralizing cation can lead to a dramatic conformational change or even to a complete alteration in the sequence - stability rules of the duplex. addition of ethanol to an aqueous solution induces drastic changes in the duplex structure, forcing a b a transition. some organic solvents, such as methanol, formamide, pyridine, or dimethyl sulfoxide, induce unfolding and strand separation, or formation of toroid - like conformations, while others, such as glycerol or room - temperature ionic liquids, maintain the duplex structure. more surprisingly, a dna duplex does not lose its structure completely, and the two strands remain bound when dna is transferred from aqueous solution to the gas phase. it is clear that changes in the balance between stabilizing and destabilizing terms controlling dna structure due to alterations in the solvent are complex and still not well understood. experimental studies in apolar solvents are challenging because of the difficulty of transferring dna from water to such media. this problem has motivated several theoretical studies, most of them using molecular dynamics (md) simulations. have examined the structure and stability of dna in octane, correlating the observed changes with those required for helicase activity. lin. explored the stability of decorated dnas in interfaces between dna and chloroform and have previously considered the reaction of dna to high concentrations of pyridine. finally, khalid. used coarse - grained models to study the insertion of duplex dna into a lipid bilayer, finding a significant associated free energy barrier, even for small duplexes. here we use large - scale atomistic md simulations and discrete path sampling to investigate the conformations sampled by a short dna duplex in carbon tetrachloride (ccl4), a medium comparable to the interior of a biological membrane. we have also explored the (free) energy and structural differences associated with the change of phase of a duplex dna from water to ccl4. finally we analyzed for the first time the possibility that dna acts as a proton donor / acceptor when it crosses from a polar to an apolar medium. our results suggest that dna, at least a short fragment, can maintain its duplex structure for significant periods of time in an apolar solvent and, surprisingly, appears more rigid in such media than when immersed in aqueous solution. transfer from aqueous to apolar solvents is, obviously, strongly disfavored, especially if the dna remains fully charged. typically, the transferred dna is not fully dehydrated but maintains (irrespective of its formal charge) a significant amount of water around it. calculations suggest that a neutral dna state with a compact structure (not too different from that in aqueous solution) is the most prevalent form of dna in apolar solvent. we considered a small oligonucleotide (d(gcgaagc)) as a model system for all our calculations. in aqueous solution, this small dna forms a stable hairpin with a gaa triloop and a two - d(cg)-pair stem, organized in a canonical b - type duplex, as confirmed by nmr. experiments and theory have shown that this oligonucleotide folds on a (multi)microsecond time scale in water, and should unfold on a similar time scale in a denaturant solvent such as pyridine. the small size of the system accelerates calculations, favors sampling, and reduces memory effects, which might be very important for longer oligonucleotides. previous studies have demonstrated the ability of current force field and md protocols to simulate this structure properly in a variety of environments. the 7-mer dna hairpin was first simulated in aqueous solution as a reference for the native trajectory. the nmr structure was neutralized by adding six na ions (placed at optimal classical molecular interaction potential (cmip) positions) and solvated by 7200 water molecules, defining an octahedral box. the system was optimized, thermalized, and preequilibrated using standard procedures with triplicated simulation windows, and equilibrated for 100 ns prior to 1 ms of production trajectory at constant temperature and pressure (t = 300 k ; p = 1 atm). long - range electrostatic corrections were represented by means of periodic boundary conditions (pbc) and the particle mesh ewald (pme) correction with default parameters. starting from the aqueous solution equilibrium structure, we performed unbiased md simulations in ccl4 considering the canonical dna protonation state (q = 6) as well as a fully neutralized system, in which all of the phosphates were protonated (q = 0) following the same procedure used previously for the study of gas - phase structures. the simulation cell contained one dna molecule and around 4288 ccl4 molecules. the optimization, thermalization, and equilibration procedures were the same as those used in the aqueous simulations. trajectories were also collected for 1 ms at constant temperature and pressure (t = 300 k ; p = 1 atm) using pbc pme to account for long - range effects. note that the ccl4-simulation systems do not contain counterions, which means that ewald s plasma is responsible for charge balance in these simulations. to discard possible ewald artifacts in this case, we repeated the calculations for the q = 6 system using a reaction field correction with a 1.1 nm cutoff to define the boundary between the atomistic system and the exterior dielectric continuum. to explore the conformational space of the hairpin in ccl4, we performed replica exchange replica exchange molecular dynamics (rexmd) simulations for the two charge states (q = 6 and q = 0) starting from (i) a fully extended conformation and (ii) the aqueous - equilibrated structure. as an additional test, we extended the rexmd simulations to a minimum - hydration hairpin (50 water molecules) obtained from our simulations of the transfer of the hairpin from water to ccl4 (see later discussion). a total of 25 temperatures was selected to span the range of 300440 k. the distribution of temperatures was chosen to guarantee an average exchange probability around 25%. each of the temperature replicas was explored for 150 ns (for a total simulation time of 2 2 35 0.150 = 15 ms), with exchanges attempted every 1,000 md steps. disconnectivity graphs provide a simple and powerful way to visualize the dna potential or free energy landscape in terms of local minima and transition states connecting them. this approach groups minima that are mutually accessible at a given energy threshold into disjoint sets, which are connected by vertical branches. the shorter the branches linking the minima, the smaller the activation barrier needed to move from one group to the other. the stationary points (minima and transition states) required to construct the disconnectivity graph were obtained with the discrete path sampling (dps) method, implemented in the programs pathsample and optim. dps runs were started from an initial connected path of minima and intervening transition states between two specified end points. to identify suitable end points, we used 400,000 snapshots obtained from md trajectories at low (298 k) and high (450 k) temperatures for the q = 0 system in explicit ccl4 (that for which rexmd simulations were not conclusive ; see later discussion). the lowest energy structure was chosen as one representative of the native form, while an extended structure without any native contacts was designated as the unfolded form. the connections between stationary points were calculated by approximate steepest - descent paths obtained by energy minimization following infinitesimal displacements parallel and antiparallel to the eigenvector corresponding to the unique negative eigenvalue for each transition state. we define minima and transition states as stationary points on the potential energy surface with zero and one negative hessian eigenvalues, respectively. local minimization of minima and transition states was carried out using a modified limited - memory broyden the doubly nudged elastic band (dneb) algorithm was used to identify transition - state candidates, which were accurately refined using a hybrid eigenvector - following approach. once an initial connected path was found, the database was enlarged by adding all of the minima and transition states found during successive connection - making attempts for existing pairs of minima. finally, to construct disconnectivity graphs on the basis of free energy, we estimated the relative free energies of minima and transition states from their potential energy and normal - mode frequencies using the harmonic superposition approximation for the density of states. the energy and analytical first derivatives of the energy, required for geometry optimization, were computed through an interface for the amber9 package available for optim using the parm99+parmbsc0 force field. for disconnectivity graph calculations we represented the ccl4 solvent with the generalized born model implemented in amber and without distance cutoffs for the nonbonded interactions. to obtain an atomistic representation of the transfer of the hairpin from aqueous solution to ccl4, we performed a steered md simulation, where we moved the hairpin across the major axis of an orthorhombic box (size 5.85 5.85 19.4 nm) containing 9,460 ccl4 molecules (placed in the center of the long axis) and 34,935 water molecules (placed at both sides of the ccl4 phase ; see supporting information figure s1). simulations for the q = 6 dna included six na counterions (see previous discussion for neutralization protocol), while no explicit counterions were included for the q = 0 simulations. the steered md simulations either move the hairpin from one water box to the other passing through the ccl4 phase or from the center of the ccl4 phase to one of the neighboring water boxes. a slow steering velocity [0.05 nm / ns ; with force 1,000 kj/(mol nm) ] was used to reduce hysteresis effects. the steered md trajectory was then used to extract 75 starting configurations (spaced 0.2 nm along the major axis of the simulation system ; see supporting information figure s1) from which umbrella sampling (us) calculations were performed. prior to the us calculations all configurations selected from the steered md simulations were relaxed for 20 ns. for the us procedure, we used the position of the dna hairpin in the transfer direction (z ; see supporting information figure s1) as the reaction coordinate, and a harmonic umbrella potential (k = 1,000 kj/(mol nm)) with 75 overlapping windows, centered at consecutive values of the reaction coordinate that span the complete waterccl4water transfer path. we define the central window as that centered at the center of mass (com) of the ccl4 box (z = 0 nm) and centered the first and last windows at points located at z = 7.3 and z = + 7.3 nm with respect to this com. individual trajectories for each of the 75 us windows extend for 100 ns (for a total simulation time of 9 s system). the potential of mean force (pmf) was calculated using the cyclic implementation of the weighted histogram analysis method (wham) in gromacs 4.5.5. because iact is subject to uncertainties due to limited sampling, we have smoothed the iact along the reaction coordinate using a gaussian filter of width of 0.5 nm. boltzmann (pb) equation with a very fine grid resolution as implemented in our cmip code, to determine the change in solvation free energy when the dna moves from regions of low (ccl4) and high (h2o) dielectrics. dielectric boundaries were defined from the placement of the interfaces between phases in the us calculations. for each of the 75 windows considered in the us calculations we selected 50 individual dna conformations (for q = 6 and q = 0 states) performing then a total of 15,300 pb calculations from which an average free energy of solvation for each point of the transfer process was determined. to solve the pb equation we used a regular grid (spacing 0.05 nm) with two solvent regions (ccl4 = 2 and h2o = 80). the d(gcgaagc) oligonucleotide forms a very stable dna hairpin in aqueous solution, with a well - organized and stable triloop and a short, but stable, b - gpc dna stem. the native nmr conformation is stabilized by eight base - pairing hydrogen bonds (h - bonds)six d(gc) bonds plus two d(ga) bonds that we term canonical h - bonds. despite backbone fluctuations, around 98% of these canonical h - bonds our long md simulations in aqueous solvent suggest that the dna native hairpin conformation is stable at room temperature (supporting information figure s1), in agreement with previous experimental (root mean square deviation (rmsd) = 0.08 0.01 nm with respect to the nmr average structure) and md studies. unbiased microsecond md simulations for the dna hairpin in ccl4 reveal that for both q = 6 and q = 0 charge states the aqueous conformation can survive in ccl4 for a significant amount of time, undergoing only small structural changes (supporting information figure s1). in fact, unbiased md simulations show rmsd fluctuations smaller for trajectories in ccl4 than for equivalent trajectories (at the same temperature) in water (see supporting information figure s2). clearly, the ability of water to establish h - bond interactions with the dna favors flexibility, since in aqueous solution temporarily lost intra - dna interactions (due to thermal fluctuations) are compensated with dna water contacts, while this is impossible in ccl4, or any other apolar environments. to verify that the, perhaps surprising, microsecond - scale stability of the q = 6 hairpin was not related to a pme artifact, we repeated the simulation using a reaction field to correct for distant interactions, instead of the default pbc as shown in supporting information figure s3 and figure s1, reaction field and ewald simulations give (as expected) nearly equivalent results, confirming that aqueous solution structure is indeed a microsecond - stable structure of the hairpin in ccl4, even when no neutralization of the phosphates occurs. it is, however, unclear whether or not the solution structure corresponds to the real free energy minima in ccl4 or it is simply a metastable conformation that becomes trapped due to the stiffness of the structure. to clarify this point, we performed large - scale rexmd simulations with a wide range of temperatures and an aggregated simulation time of more than 15 s (see methods and figure 1a d). as previously described, to check for memory artifacts, we started simulations from both an extended and a native - like irrespective of the starting conformation, both rexmd simulations of the q = 6 hairpin lead to quite extended configurations at the end of the simulations (figure 1b). summary of rexmd results for the dry hairpin in ccl4 solution (q = 6 and q = 0) started from the native conformation (the folded state in water) and a fully extended structure. the results correspond to the last 50 ns of the replicas at room temperature (extracted from an aggregated simulation time of 3.75 s ; see methods). the structures sampled have a significant number of h - bonds, but few of them are native (figure 1a, b, d). the loop and general hairpin structure is also destroyed, and it is difficult to find any signal of the original hairpin structure in the sampled structures at room temperature (figure 1). from these rexmd and unbiased md simulations, we conclude that the native structure is a metastable (on the microsecond time scale) conformation for a hypothetical fully charged hairpin, but it is not the most favorable conformation for this oligonucleotide in ccl4. the results of the rexmd simulations are less clear for the q = 0 state. by comparing the independent simulations, we conclude that the conformational space is dominated by compact structures (see figure 1), but it is not clear whether or not these compact structures are close to the native one. thus, simulations starting from the native state remain very close to the aqueous structure, maintaining the pattern of native h - bonds (figure 1), while simulations starting from the extended state collapse to a variety of different compact, but nonnative, structures. clearly, replica exchange seems to be nonoptimal to explore low - lying minima in this complex conformational equilibrium. thus, for the hairpin (q = 0) we decided to complement rexmd simulations with dps calculations to build disconnectivity graphs (see methods). the results in figure 2 show that the lowest free energy structures correspond to folded hairpins with a high content of native base - pairing (native in blue and near - native in green in this figure). the vertical axis represents the free energy difference with respect to the global minimum, and the spacing of the branches on the horizontal axis was chosen to reveal the structure as clearly as possible. the branches terminate at the energies defined by the individual free energy minima and are joined together at energy thresholds where the barriers separating different sets can be overcome. branches are colored according to the fraction of native base - pairing h - bond contacts (from no contacts in red to all native contacts in blue) and highlight the diversity of conformations adopted at different energies (unfolded, misfolded, near - native, and native). the graph reveals that the lowest free energy funnel corresponds to folded structures with a high content of native contacts (blue), and the existence of a funnel of misfolded structures with no native contacts (red) separated from the global minimal by a high free energy barrier. the long branches (> 30 kj / mol on the vertical axis) reflect the high - energy barriers associated with rearrangements between native and misfolded or unfolded conformations. extended (unfolded) states (no native contacts and radius of gyration greater than 0.8 nm, red) are sparse (see supporting information figure s4), and are separated by high - energy barriers from the native state (see figure 3 and supporting information figure s4). in fact, starting from the unfolded state, we observe that the pathway to the native state involves a potential energy barrier of around 130 kj / mol (figure 3). overall, the disconnectivity graphs confirm that the conformational landscape of the hairpin in ccl4 is very frustrated with several nonnative conformations separated by high - energy barriers. however, the combination of these results with rexmd suggests that compact structures, not far from the aqueous native state, are the dominant ones for the neutral state of the hairpin. potential energy difference with respect to the native state as a function of the integrated path length for a discrete path connecting an unfolded structure with the native structure for q = 0. the integrated path length is the integrated euclidean distance in cartesian coordinates high energy. snapshots for selected intervening structures are also shown and colored according to the fraction of native h - bond base - pairing interactions as described in figure 2 up to this point we have explored the structure of dna in dry ccl4, but dna is naturally found in water, and it is therefore interesting to study the transfer process and how it affects the structure and energetics of the hairpin. to study the unlikely transfer process (see methods), we first performed a very slow velocity steered md simulation to obtain starting conformations for umbrella sampling free energy calculations. we first pulled the hairpin from one water box to the other, passing through the ccl4 phase (see schematic representation in supporting information figure s5). as seen in figure 4, even for very small pulling velocities, the hairpin crosses the apolar phase hydrated and maintains the general hairpin structure (see supporting information figures s5 and s6), confirming that the structure of dna in solution is a metastable conformation in ccl4. representation of different snapshots obtained in steered md simulations forcing the phase transfer of dna hairpin : (a) from water to ccl4 and back to water for q = 6 dna hairpin ; (b) from water to ccl4 and back to water for q = 0 dna hairpin ; (c) from ccl4 to water for the q = 6 dna hairpin ; (d) from ccl4 to water for the q = 0 dna hairpin. the number of water molecules in the microhydration drop depends on the charge state of the hairpin (see supporting information figure s5), but even for the neutral hairpin there is a small, but nonnegligible, number of water molecules protecting the hairpin from the apolar solvent (see supporting information figure s6). although displacements of the hairpin within the box were as slow as possible, they are still much faster than the relevant experimental time scale, and we can not rule out potential problems in the relaxation of the solvent environment around the dna. to avoid these potential relaxation artifacts as far as possible and to confirm that even a neutral hairpin is hydrated in ccl4, we repeated the pulling procedure but started with a dry hairpin placed in the middle of the ccl4 phase. these complementary simulations confirm that as soon as the hairpin moves from the center of the apolar phase, a stream of water molecules flows in (especially dramatic for the q = 6 charge state), spontaneously generating a microhydration environment (figure 4). this result indicates that the entrance of water molecules in the apolar phase is not a relaxation artifact but a real physical effect. hence we predict that under normal conditions a hairpin (especially a fully charged one) would cross a hydrophobic phase surrounded by a small number of water molecules. the discussion in the previous paragraph suggests that if transferred directly from water, the dna in ccl4 will probably be surrounded by several water molecules. thus, we repeated the rexmd calculations but used microhydrated species (q = 0 and q = 6) obtained from the pulling experiments when the center of mass of the hairpin is placed at the middle of the apolar phase. we found that the impact of the microhydration environment helps to stabilize the folded state (see supporting information figures s7 and s8), shielding the electrostatic repulsion between the charged phosphate groups. as a result, a microhydrated dna placed in the middle of a ccl4 phase maintains a conformation that is not far away for the native structure in water, even when we consider that phase transfer does not imply change in the ionization state of the oligonucleotide. in summary, our simulations strongly suggest that the neutral hairpin maintains well the solution structure when transferred to either dry or wet ccl4. on the contrary, the fully charged hairpin, while remaining close to the aqueous structure in wet ccl4, we used wham and umbrella sampling (from steered - md snapshots ; see methods) to compute the free energy change associated with the transfer of the hairpin across polar and apolar phases. the results displayed in figure 5 (see methods and supporting information figure s9 for details) clearly illustrate the large free energy barrier associated with crossing a hydrophobic phase : around 650 kj / mol for a fully charged hairpin and 150200 kj / mol for the neutral. these numbers are in qualitative agreement with those obtained from a poisson boltzmann calculation (see figure 6) which demonstrated that transfer of a hairpin from water to an apolar solvent, even if microhydrated, is largely disfavored. these results reinforce the notion that decoration of the dna with apolar moieties is desirable to improve permeation. potential of mean force associated with the transfer of the hairpin (q = 6 and q = 0) across a polar / apolar / polar simulation box (see methods and supporting information figure s1). change in the electrostatic solvation energy associated with the transfer waterccl4water of a fully charged (q = 6) and neutral (q = 0) hairpin. finally, we studied the relative weight of neutral and charged states of a hairpin in ccl4. using standard thermodynamic cycles (see supporting information figure s10), we computed the free energy cost of transforming a neutral to a charged state in ccl4, the cost of removing the hairpin charge in water, and the cost associated with the phase transfer of the neutral and charged hairpins. the free energy change associated with the annihilation of the hairpin charge can be determined at neutral ph from the experimental pka in water of a dna phosphate (pka around 2 ; g around 160 kj / mol assuming that all phosphate groups protonate independently of each other). thus, transfer of a hairpin (q = 6) in water into a (q = 0) hairpin in ccl4 is overall disfavored by around 350 kj / mol, while the same transfer assuming q = 6 in both solvents has an associated free energy change around 450500 kj / mol higher. in other words, our simulations strongly suggest that dna in apolar media should have a reduced charge and could even be completely neutral. under these conditions dna dna is a highly polar molecule, which has evolved over millions of years to be stable and functional in aqueous solution. dna will not spontaneously transfer to an apolar phase, such as a biological membrane, which represents a clear advantage for confinement in a given cellular compartment. however, biotechnological and biomedical applications often require the dna to cross biological membranes, be encapsulated in a hydrophobic, environment, or just act as a chemical reactant in a nonaqueous solvent. it is then necessary to study the behavior of nucleic acids in apolar solvents as a preliminary step to design modifications that can favor transfer from water. for this purpose, theoretical approaches are ideal, since they allow us to study dna in such hostile environments. our large - scale simulation effort, described in the present report, illustrates the significant changes occurring in the conformational landscape of a small model system of dna when transferred from water to ccl4. the dna becomes stiffer and the landscape is more frustrated in the apolar phases, which might generate kinetic traps. compact states, probably not far from the aqueous structure, are sampled if the dna is neutralized, while more extended conformations are expected if the dna maintains its ionic state in water. the transfer of dna from aqueous solution to ccl4 is energetically disfavored, especially if the dna maintains its aqueous ionic state. the size of the water droplet increases with the net charge of the dna, but even for a neutral dna a significant number of water molecules appears in our simulations for the apolar phase. a combination of the umbrella sampling potential of mean force simulations (supported by poisson boltzmann calculations) with experimental pka estimates demonstrates that the neutral state is favored, suggesting that transfer of dna could generate a net proton flux across a membrane. overall, neutralization of dna charges seems a key mechanism to improve transfer properties of dna and either changing phosphoric by another acid with higher pkas or neutralizing decorations neutralizing dna charge seems a promising approach to improve dna bioavailability. | the study of nucleic acids in low - polarity environments paves the way for novel biotechnological applications of dna. here, we use a repertoire of atomistic molecular simulation tools to study the nature of dna when placed in a highly apolar environment and when transferred from aqueous to apolar solvent. our results show that dna becomes stiffer in apolar solvents and suggest that highly negatively charged states, which are the most prevalent in water, are strongly disfavored in apolar solvents and neutral states with conformations not far from the aqueous ones are the dominant forms. transfer from water to an apolar solvent such as ccl4 is unlikely to occur, but our results suggest that if forced, the dna would migrate surrounded by a small shell of water (the higher the dna charge, the larger the number of water molecules in this shell). even the neutral form (predicted to be the dominant one in apolar solvents) would surround itself by a small number of highly stable water molecules when moved from water to a highly apolar environment. neutralization of dna charges seems a crucial requirement for transfer of dna to apolar media, and the most likely mechanism to achieve good transfer properties. |
an increase in the consumption of sugar - sweetened beverages (ssbs) and the prevalence of childhood obesity have occurred in tandem. in the united states (us) between 1977 and 2002 the increase in calories consumed from soft drinks and other sweetened beverages increased 230% and 170%, respectively. concurrently, the prevalence of childhood obesity increased threefold in the us, with those in minority and low - income groups experiencing higher prevalence rates [2, 3 ]. during this period, many other factors that increase obesity risk changed as well, such as an increase in sedentary activities, purchase of fast food, and sleep debt [4, 5 ]. however, ssbs are of special concern since the calories contained in this liquid form, for some reason, are not registered by the body, and therefore no dietary compensations are made following intake. instead paradoxically, researchers have found that when youths drink more ssbs it results in an increase in solid food consumption as well, with choices like pizza, burgers, and savory snacks often chosen. mathias. found through analysis of data collected from the national health and nutrition examination surveys (nhanes) conducted between 2003 and 2010 that for every 100 kcalories of ssb consumed by 611-year - olds there was an increase in solid food consumed providing an additional 36 14 kcalories. youths 1218 years of age revealed an intake of an additional 86 10 kcalories in solid food form for every 100 kcalories of ssb product consumed. recently, several reviews have illuminated the strong connection between obesity risk and ssb intake [810 ] ; however weak potency of effect on interventions has called into question the absolute strength of this association. in addition to an increased risk of obesity from consuming liquid calories, the high sugar content in ssbs has been associated with an increased risk of insulin resistance, dyslipidemia, type 2 diabetes mellitus, and cardiovascular disease [1, 6, 12 ]. added sugars consumed from both liquid and solid sources are associated with body weight gain in youths at risk of developing obesity ; however wang. found in their group of 564 youths who were followed for two years that consuming sugar from liquid, but not solid, sources predicted a higher risk of developing impaired glucose homeostasis and glucose resistance. reports spanning the past decade highlight increasing consumption of ssbs among children, adolescents, and teens [1, 4, 14, 15 ]. recent estimates of the mean caloric contribution from ssbs range from 117 kcal / day to as much as 356 kcal / day, with calorie contribution variations based upon age category, sex, and ethnicity [1417 ]. those in minority and low - income groups have been identified as drinking greater amounts of ssbs. it has also been found that approximately 5% of children and 16% of adolescents surveyed are heavy consumers of ssbs, with intakes at or exceeding 500 kcal / day. ssbs are available to youths on the school campus as well as on the home front [17, 18 ]. however, researchers point to data supporting that the majority of ssbs are consumed at home [15, 17 ]. public health experts have made a call for action in the form of educational interventions to address the excessive ssb intake in youths and subsequent adverse health issues [1, 9, 10, 14, 16, 17, 19 ]. the aim of the current study was to gauge impact on knowledge and attitudes regarding ssbs following a hands - on workshop for youths delivered during summer program hours. this experiential workshop addressed the sugar content of commonly consumed ssbs and included preparation and tasting of lower sugar alternatives. a convenience sample of youths who were enrolled in the summer program at a local boys and girls club participated in the nutrition education and blending better beverage options workshop. this program was provided to all attendees of the summer program at one boys and girls club in long island in new york state. the workshop was delivered to approximately 20 participants at a time who were divided into small groups of 68 youths of similar age and were seated at one work table together with two undergraduate nutrition student volunteers. the survey instrument was developed by the study investigators and was based upon current literature [8, 16, 18 ] and designed to explore knowledge and beliefs about ssbs. the survey was modified to be age - appropriate ; one version was created for 59-year - olds and another was developed for those who were 1014 years of age. the same questions were asked, but the language was simplified and smile and frown faces were used for improved comprehension on the survey for the younger children. all participants were offered assistance with completion of the program surveys, and the younger participants were given one - on - one assistance when needed from undergraduate nutrition student volunteers. the survey was completed before the workshop began and following the end of the two - hour program for comparison. each participant was asked to report their usual intake within four commonly consumed beverage categories (soda, sports drinks, sugar - sweetened tea and juice, and energy drinks) before the start of the workshop. for each category the participant was asked to estimate his / her frequency of consumption per week and then to estimate quantity consumed per frequency. sample cans and bottles and representative glassware were displayed at each table to assist the participants in estimating the quantity of ssbs consumed. the survey included four questions regarding knowledge of sugar content (in teaspoon counts) of commonly consumed beverage items (16-ounce bottle of one - half sweetened iced tea and one - half lemonade, 12-ounce can of cola beverage, 20-ounce bottle of sweetened fruit punch, and an 8-ounce can of an energy drink). the participants were asked to select the amount (in teaspoons) of sugar from a list of four choices for each ssb item. the choices for each item were 35 teaspoons, 79 teaspoons, 1012 teaspoons, or 15 or more teaspoons. the survey created for 59-year - olds included assistance in understanding the question by adding qualifying words for each selection with options listed as follows : 35 teaspoons, a small amount ; 79 teaspoons, a medium amount ; 1012 teaspoons, a large amount ; and 15 or more teaspoons, a lot. to record and gauge any change in attitudes held regarding ssb preferences, thoughts about health concerns associated with ssbs, and intention regarding avoidance of ssbs, participants were asked to respond to six statements at baseline and again following the intervention. following each statement, such as i should drink less soda and sweetened beverages, participants were directed to choose from a list of responses : strongly agree, agree, disagree, or not sure. the survey instrument completed by 59-year - olds included the following choices with accompanying faces to help them better understand and choose their response : yes definitely (broad smile), yes (smile), no (frown), and not sure (neutral). on the postprogram survey participants were asked to respond to a question asking whether they had enjoyed participation in the program. attendees were asked to respond to the following statement : i 've enjoyed participating in the beverage workshop. in addition, participants were asked to share their thoughts regarding intention to reduce intake of ssbs in the future by responding to the following statement : i think i will drink less sugar - sweetened beverages like soda because of what i 've learned today. once again the 1014-year - old participants were asked to select from the following responses : strongly agree, agree, disagree, and not sure ; and 59-year - olds chose their answer from yes definitely (broad smile), yes (smile), no (frown), and not sure (neutral), using visual faces to help them better understand and choose their response. each participant took part in a two - hour workshop held during summer program hours that was composed of two separate, yet related, components.educational session revealing the sugar content of commonly consumed beverages and demonstration of adding a similar content of table sugar to water. discussion of the health detriments associated with excessive sugar intake.a hands - on, experiential involvement in blending better beverage options, followed by recipe tastings. a discussion about how to make healthier decisions for beverages.undergraduate nutrition student volunteers assisted participants in completing the program surveys and served as facilitators for the workshop. each volunteer attended a one - hour instructional training session conducted by the principal investigator prior to the start of the program. educational session revealing the sugar content of commonly consumed beverages and demonstration of adding a similar content of table sugar to water. discussion of the health detriments associated with excessive sugar intake. a hands - on, experiential involvement in blending better beverage options, followed by recipe tastings. a discussion about how to make healthier decisions for beverages. after completion of the baseline survey, each table of 68 participants took part in a guessing game and discussion about the sugar content of four popular beverage items led by an undergraduate nutrition student. participants were asked to guess how many teaspoons of sugar were in each of four commonly consumed beverage items. after guessing, the nutrition student revealed the correct answer and asked the participants to count out sugar packets representing the amount of sugar contained in the item. a plate containing all the sugar packages was placed in front of the beverage item to offer a lasting visual image. this process was repeated for each of the four ssbs. when the process was completed for all beverages, the children were asked to view the four items on the table and to consider how much sugar would be consumed if all four ssbs were consumed in one day. then the nutrition undergraduate students at each table led a demonstration showing how much sugar is added to liquid beverages by adding 15 teaspoons of sugar to a 20-ounce glass of water. this item was stirred and passed around for the participants to view the thick, cloudy substance that was created by simulating the amount of sugar often added to ssbs. an interactive discussion regarding the sugar content of ssbs and the health consequences of consuming too much sugar was held. each nutrition undergraduate student was instructed to pose the following questions to the participants.is anybody surprised about the amount of sugar in these beverages?would anyone take a glass of water and add the same amount of sugar to it and then drink it?do you think drinking so much sugar in these types of beverages is harmful to your health?nutrition students were instructed to highlight the association of high sugar intake with weight gain, diabetes, and dental caries.a review of the concerns associated with the ingredients in energy drinks and why children should not drink these products was conducted.would you like to make beverages to drink that are lower in sugar ? would anyone take a glass of water and add the same amount of sugar to it and then drink it ? do you think drinking so much sugar in these types of beverages is harmful to your health ? nutrition students were instructed to highlight the association of high sugar intake with weight gain, diabetes, and dental caries.a review of the concerns associated with the ingredients in energy drinks and why children should not drink these products was conducted. nutrition students were instructed to highlight the association of high sugar intake with weight gain, diabetes, and dental caries. a review of the concerns associated with the ingredients in energy drinks and why children should not drink these products was conducted. participants were led in a hands - on preparation of four recipes : (1) fresh peach and orange infused water, (2) pineapple, mango, peach, and lime slush, (3) cranberry, pineapple, and lime fizzy, and (4) fresh strawberry and banana smoothie. the nutrition undergraduate students were instructed to ask for participant feedback about the taste, acceptability, and ease of preparation of lower sugar beverage alternatives. the importance of preparing beverages using diluted versions of 100% fresh fruit juices was stressed. the study, including instruments, protocols, and consent procedures, received exempt approval by the institutional review board at long island university. written parent consent was not required because the student survey portion of this project was classified as exempt. survey data results were tabulated and compiled into a database and analyzed using stata (se 13) to provide descriptive statistics and analysis. in addition to the standard chi - square tests the analysis includes t - tests for comparing two population proportions. proportions are among the few measures which can be used for summarizing categorical data and provide an additional dimension to the analysis. unlike a chi - square test that tests for the association between qualitative variables using the entire contingency table, the t - test can be applied to test, for example, if the proportion of participants correctly answering the question on the pretest is statistically different from the proportion of participants correctly answering the question on the posttest. the test statistic for comparing two population proportions is t = p1 ^ -p2 ^ -p1-p2/p-1-p-1/n1 + 1/n2, where p1 ^ -p2^ are sample proportions estimates, p1 p2 are population proportions, and p-=x1+x2/n1+n2 is the weighted average of the two sample proportion estimates. all t - tests results are one - tailed tests in order to study if one proportion of respondents is higher than the other, rather than simply being different from each other which would be captured by the two - tailed test. in other words, the tests are to assess if the proportion of participants correctly answering the question on the posttest is higher than the proportion of participants correctly answering the question on the pretest. specific participant sociodemographic data were not obtained due to the study 's exemption status. however, study participants were attendees of the local boys and girls club afterschool program. the attendees of the program are predominately latino and african american and come from single parent (51%) and low - income homes (76% come from families with incomes of less than $ 33,000/year and 74% receive free or reduced fee lunch). a total of 128 surveys were distributed to participants, 100% were returned, and there were no missing responses or surveys that were deemed incomplete. of 128 participants, 81 (63.3%) were male and 47 (36.7%) were female, with an average age of 9.3 years. data were analyzed using the entire sample of 128 participants as well as by two age subgroups : age of 59 years and age of 1014 years. there were 70 participants in the 59-year - old age group (41 male and 29 female) with an average age of 7.6 years and 58 participants in the 1014-year - old age group (40 male and 18 female) with the average age of 11.3 years. the average amount of ssbs consumed per week for the entire sample was 125.6 oz. (17.9 oz. per day), with 113.9 oz. (16.3 oz. per day) for the 59 year old age group and 139.6 oz. (19.9 oz. per day) for the 1014-year - old age group. a two - sample mean comparison t - test found no statistically significant difference in total ssb consumption between the two age groups. in addition, the difference in drinking soda and sugar - sweetened teas and juices was not significantly different between the two age groups. however, the older group was found to drink significantly more sports drinks and energy drinks compared to the younger group (p < 0.05 ; table 1). the drinking habits of males versus females in both the 59-year- and 1014-year - old age groups were not statistically different. however, males in the 1014-year - old age group reported to drink twice as much soda as females in this age group, 31.1 oz. and 15.6 oz. per week, respectively, and are significantly more likely to drink energy drinks, 45% and 11%, respectively (p < 0.05). to evaluate the level of knowledge obtained by attending the beverage workshop pre- and postintervention survey data were analyzed using the standard chi - square tests for the association between two qualitative variables. for the entire sample of 128 participants, the chi - square test for all four knowledge questions rejects the null hypothesis even when the p value is set at p < 0.01. since all the scores have improved, it can be concluded that the intervention was successful in providing information to the participants. the same conclusion is obtained for the 1014-year - old age subgroup. however, for the 59-year - old age group, the chi - square test failed to reject the null for improvement in knowledge on the question about the sugar content in an 8 oz. results of the analyses of knowledge data using t - tests revealed that the proportion of participants who correctly answered the questions on the pretest for the entire sample of 128 participants is statistically different from the proportion of participants who correctly answered the questions on the posttest survey for questions 1 and 2 (sugar content in a 16 oz. serving of sweetened one - half iced tea and one - half lemonade ; correct answer 1012 teaspoons and in a 12 oz. can of cola soda ; correct answer 1012 teaspoons, resp. ; however, for questions 3 and 4 (sugar content in a 20 oz. serving of sweetened fruit punch ; correct answer 15 + teaspoons and an 8 oz. figure 1, panels (c) and (d)), improvement in knowledge was increased, but not significantly. more precisely, for the age group of 1014 years the scores on all four questions improved, while for the age group of 59 year olds only the scores for questions 1 and 3 (sugar content in a 16 oz. serving of sweetened one - half iced tea and one - half lemonade and the 20 oz. serving of sweetened fruit punch, resp., figure 1, panels (a) and (c)) improved significantly. a great majority of participants either strongly agreed or agreed with the statement that they usually choose a glass of water when they are thirsty, that beverages with sugar are not good for them, and that they should drink less soda and sweetened beverages (figure 2, panels (a), (e), and (f), resp.), both before and after intervention. in addition, most participants disagreed with the statement that soda is their favorite drink, that delicious drinks can be made using fresh fruit and beverages without added sugar, and that energy drinks are healthy (figure 2, panels (b), (c), and (d), resp.). the differences between the pre- and postintervention responses to comments addressing attitudes, however, are not statistically different when chi - square tests were applied. in all attitudinal comments posed study participants responded favorably regarding attitudes held on the preprogram survey except when responding to the comment that delicious drinks can be made using fresh fruit without added sugar. the majority of participants disagreed with this comment on the preintervention survey, and although there was an increase in the number of those who strongly agreed or agreed with this comment following the intervention, there was no statistically significant change in response following program completion. responses to one comment went in an unexpected direction for the comment addressing whether an energy drink was a healthy beverage option. the majority of participants disagreed that an energy drink is a healthy beverage option on both the pre- and postintervention surveys ; however, unexpectedly less rather than more participants disagreed with the statement after intervention compared with the preprogram surveys. this result was mostly driven by the change observed in the younger participants in the 59-year - old age group, where more of them either strongly agreed or agreed that energy drinks are healthy for them after the intervention. one possible explanation for this might be due to their unfamiliarity with this type of ssb. this interactive workshop conducted during summer program hours held with youths 514 years of age queried usual intake of ssbs and focused on transmitting knowledge about the amount of sugar contained in commonly consumed beverage items and the potential health detriments associated with overconsumption. the workshop also included a hands - on preparation and tasting of several lower sugar beverage alternatives. in agreement with current literature [8, 14, 15 ], the youths attending this workshop reported regular consumption of ssbs. we found that the large majority of the youths who participated in the workshop reported regularly drinking soda (87%), sports drinks (85%), and sweetened teas and juices (81%). approximately one - quarter of the participants (24%) reported drinking energy drinks, with a significant difference in consumption found in those 59 years of age (16%) in comparison with those 1014 years old (35%). this finding is not surprising given the age of our participants as energy drinks are more commonly consumed by teens and young adults. however, there are many health concerns associated with consuming energy drinks and young children can be influenced by the intense marketing efforts for these products [20, 21 ]. therefore, reports of any intake of energy drinks in youths 514 years of age are of concern and require further investigation focused on this specific group of ssbs. of significance is that males were four times more likely than females to consume energy drinks. based on our results 1014-year - old males, in comparison with younger children and females, were more likely to consume energy drinks and should be targeted in future interventions aimed at eliminating consumption of this problematic beverage in at - risk youth populations. estimated mean intake of soda, sports drinks, sugar - sweetened drinks, and energy drinks per week, rounded to the nearest ounce, was 26, 43, and 44 ounces and 1 ounce, respectively, for 59-year - olds and 26, 63, 41, and 4 ounces, respectively, for 1014-year - olds. we found that the mean intake of fluid ounces of ssbs in our group of participants translated into approximately 17.9 ounces of beverage per day, or a little over two 8-ounce servings. the mean calorie contribution from ssbs found in this study is similar to what others have reported as usual calorie contribution from ssb in children and adolescents [1417 ]. noted in our data, and in agreement with current trends reported, sports drinks are being consumed with increasing frequency. researchers have reported that parents in latino communities may exhibit a misunderstanding regarding sports drinks as some have been found to report that they believe that these drinks are healthy options for their children. this misunderstanding in a community at increased risk of obesity and glucose intolerance is concerning and can promote future health risks. therefore, outreach to parents, particularly in latino communities to inform them about the health risks regarding the sugar content of sports drinks, appears advisable. chi - square tests on the entire sample of participants revealed a significant improvement in knowledge of sugar content for all four commonly consumed beverage items (iced tea / lemonade mix, cola beverage, sweetened fruit punch, and energy drink) between baseline and end of program. since all the scores improved we conclude that the intervention was successful in providing information to participants. results of t - test analysis revealed improvement in knowledge after intervention for all four questions for 1014-year - olds, but significance for 59-year - olds was only established for questions about the sugar content of iced tea / lemonade mix and sweetened fruit punch. these results are expected, since the participants in the age group of 1014 years are more capable of understanding and retaining learned information. in addition, the older participants are also more likely to either consume and/or be familiar with cola beverages and energy drinks. in other words, the younger participants did improve their knowledge regarding the sugar content in all ssbs ; however, for the sugar content in a 12 oz. can of cola and an 8 oz. can of an energy drink ; correct answer 79 teaspoons, this change was not statistically different from the answers they provided in the pretest. it is not surprising that the younger aged participants did not remember the sugar content in energy drinks reviewed during the workshop as this is not a product that is widely consumed in this age category. it is possible that they did not register the information to memory due to lack of interest and familiarity with the product. similarly, the younger participants would be more likely to drink sweetened fruit juice than cola beverages and this could explain the lack of significance found in change in knowledge for these beverage items. analyses of responses to six questions targeting attitudes about ssbs revealed no significant change in attitudes regarding beverage choice preference, thoughts about health concerns associated with ssbs, and wish to reduce ssbs following program participation. one reason why there was not a considerable change found in participant attitudes might be that most participants already selected a favorable response prior to the intervention. baseline surveys revealed that 85.7% of 59-year - olds and 81% of 1014-year - olds reported that they choose water to drink when they are thirsty, and 84.3% of 59-year - olds and 65.5% of 1014-year - olds reported that they knew that they should drink less ssbs. there were 57.1% of 59-year - olds and 74.1% of 1014-year - olds who agreed that they thought beverages with added sugar were not good for them. however, it is possible that this comment may have been misunderstood by some who interpreted not good for them as not good in taste rather than not good for health. there were volunteers who read and explained the concept of the comment to the participants, but there may still have been a misunderstanding of the intention. therefore, response results to this comment need further investigation and when the comment is posed to participants in the future clarity can be enhanced by instead asking for response to beverages with added sugar are not healthy for me. although not statistically significant impact of the program was evident as the results show that, following the intervention, more participants either strongly agreed or agreed with the statement that delicious drinks can be made using fresh fruit and beverages without added sugar (44.5% in the preintervention survey versus 52.3% in the postintervention survey). this trend in change of thought was particularly noticeable for the 1014-year - old age group. a significant percentage of children and adolescents seem to know they should reduce their intake of ssbs ; however this knowledge does not translate into action. the preference for sweets is innate as well as learned, and so this biological response triggered by environmental availability may help to explain the resistance to behavior change in reducing ssb intake. the strong desire for something sweet to drink and desire for a ssb in spite of known health risks were clearly stated by participants in a qualitative study conducted with college students. college students are in an age category older than our participant sample and so even though they are more mature and should be able to better understand the risks of choosing to drink too many ssbs, the desire to drink what they wanted regardless was evident in the narrative captured by researchers. resistance to change in attitude and strong cravings for desired beverages make it difficult to see dramatic behavioral changes in attitudes held following short - term interventions. despite these obstacles, ebbeling. report success in their study where they provided weekly deliveries of noncaloric beverages for 25 weeks to the homes of 53 children, aged 1318 years. compared to the control group the intervention group reduced their intake of ssbs by 84% and experienced a statistically significant reduction in body mass index (bmi) for those participants in the upper tertile for weight at baseline. similarly, james. conducted a year - long, school - based educational program for 644 schoolchildren in england who were 711 years old, called ditch the fizz, and found a small reduction in bmi in the intervention group and a modest reduction in consumption of carbonated drinks report a statistically significant reduction in consumption of carbonated beverages in their seven - month - long, school - based intervention (n = 1134) with 912-year - olds that focused on increasing water intake. the intervention group received classroom activities and water bottles and had promotional banners hung at the school. a statistically significant reduction in bmi was found only in those who were overweight at baseline and only in females. evidently, small successes in reducing ssb intake in youth are achievable. however, long - term interventions that address the home, school, and afterschool environments may be needed to realize greater impact. a convenience sample was used for the study with all the participants coming from one boys and girls club in one community in the us. the participants were predominately latino and african american and therefore study results can not be generalized to other groups. in addition, data collected were self - reported and may be skewed due to participant bias or poor recall. finally and in hindsight, the comment addressing thoughts about whether sweetened drinks were not good for the participant was found to be ambiguously worded and may have been misinterpreted. study strengths include the use of trained nutrition undergraduate students to assist participants with survey completion and the interactive design of the intervention. we engaged youths in the learning process offering an experiential workshop that allowed the participants to prepare and taste alternatives to ssbs and also provided strong visuals to enhance impact and learning. we did not just tell youth participants to avoid ssbs but instead had them prepare and taste no- and low - sugar alternatives. the results from the program evaluation reveal that participants enjoyed participating in this program, as 87.5% either strongly agreed or agreed with this statement. in addition, almost 80% of the participants either strongly agreed or agreed with the statement i think i will drink less sugar - sweetened beverages like soda because of what i learned today ; 81.5% in the 59-year - old age group and 77.6% in the 1014-year - old age group (table 2). weight gain from consuming sugar in liquid form, such as in ssbs, is particularly concerning as liquid calories are not registered by the body and therefore not compensated for with subsequent reduction in food intake. ssbs are ubiquitous and they have made their way into the daily diet of children as they are readily available at home, on the school campus, and at afterschool venues. in this two - hour, hands - on intervention study we found that consumption of ssbs was common in 514-year - olds in three major categories : soda, sports drinks, and sugar - sweetened teas and juices. energy drinks should not be consumed by youths and interventions that address avoiding consumption of energy drinks in this age group are needed. despite providing a relatively brief intervention we were able to show a significant increase in participants ' retention of knowledge regarding the amount of sugar added to commonly consumed ssbs. postprogram data revealed that the large majority of participants enjoyed the program and intended to reduce intake of ssbs following participation in the program. long - term interventions and programs that engage youths and reach out to parents as well as addressing the widespread availability of ssbs are needed in the future to influence resistant attitudes and beverage choosing behaviors in youths. | objective. to reduce intake of sugar - sweetened beverages (ssbs) in youths as a means to reduce obesity risk. methods. youths 514 years old attending a summer program were given a two - hour workshop addressing the sugar content in ssbs, the health risks from drinking ssbs, and hands - on preparation as well as tastings of low - sugar beverage alternatives. data on usual intake of ssbs was obtained at baseline, and pre- and postprogram surveys were conducted to gauge change in knowledge and/or attitudes regarding ssbs. results. there were 128 participants (63% male) in the program. ssbs were commonly consumed with over 80% reporting regular consumption (mean daily intake 17.9 ounces). significant increase in knowledge regarding the sugar content of commonly consumed ssbs was achieved ; however change in attitudes was not significant. the large majority of youths reported enjoying the workshop and intention to reduce intake of ssbs following program participation. conclusion. ssbs are commonly consumed by youths. knowledge regarding the sugar content of ssbs is easier to impart to youths than influencing attitudes held about these beverages. long - term interventions that reach out to parents and address the widespread availability of ssbs are needed to influence resistant attitudes and beverage choosing behaviors in youths. |
the world health organization (who) global status report on road safety 2015 shows that over 1.2 million people die each year due to road traffic crash (rtc) with prolonged morbidity in millions. deaths due to trauma have increased by 62.9% in 2014 as compared to 2004 in india as per the national crime records bureau (ncrb). there is a paucity of literature on prehospital care, emergency care, and epidemiology of traumatic cardiac arrest (tca). hence, the present study has tried to highlight the profile and characteristics of tca patients presenting to emergency department (ed) of a level 1 trauma center of india. a retrospective single - centered cohort study was conducted between 2008 and 2013 in the ed of a level 1 trauma center of india, which has an annual ed visit of 50,00060,000 patients. details of tca patients arriving to ed were retrieved from paper - based registers and computerized patient record system. it represented patients above 1 year of age, of either sex who had an out - of - hospital or in - hospital (ed) tca. the epidemiological variables studied were age, sex, mechanism of injury, prehospital care and transportation, primary arrest rhythms, and outcome in terms of death or discharge from the hospital. the data were analyzed using stata (version 11.2, stata corp, texas, u.s.a) software. about 0.3 million trauma victims presented to the ed of the trauma center from 2008 to 2014 [figure 1 ]. of these, 1061 (0.36%) presented with tca who provided cardiopulmonary resuscitation (cpr) and recruited in the study. a total of 992 (93.5%) patients had out - of - hospital cardiac arrest (ohca) and 40 had cardiac arrest in ed. a total of 909 patients (85.67%) were males and 152 (14.33%) were females (male : female ratio being 5.9:1). the majority were in the age group of 19 - 64 years with median age of 32 years (interquartile range [iqr ] 2345). annual burden of trauma patients in the emergency department age and sex distribution in patients with traumatic cardiac arrest (n=1061) mechanism of injury was available in 872 patients. rtc was seen in 571 (57.16%), fall from height in 185 (18.52%), and assault in 105 (10.51%) patients. the remaining 200 patients had other modes of injury (railway track, gunshot, drowning, electrocution, self - harm, etc.). asystole in 253, pulseless electrical activity (pea) in 11, ventricular fibrillation (vf) in 6 and ventricular tachycardia (vt) in 5 patients were observed [table 2 ]. it was provided by police personnel in 382 (36.59%), ambulance personnel in 110 (10.54%), relatives in 474 (45.40%), and bystanders in 78 (7.47%) patients [table 2 ]. initial arrest rhythms, prehospital care, and transportation data regarding return of spontaneous circulation (rosc) after the first attempt of cpr were available in 933 patients. rosc was present in 69 (7.4%), and 864 (92.60%) patients could not be revived [table 3 ]. in patients with rosc, the burden of trauma in india is increasing as per the ncrb. despite this increase, the outcome of resuscitative efforts is still very poor as highlighted in the present study. prehospital transportation services are run by the government ambulances, private ambulances, and police without any coordination among them. ambulances are often used as merely transport vehicle with limited or non- trained personals manning the ambulances. most of the trauma victims do not achieve access to medical care within 1 h. the present study shows that police personnel were major prehospital service providers. although they played a valuable role in patient transportation, they provided minimal prehospital care. this is because police personnel in india are usually untrained in providing cpr and basic emergency care (bec). husain and eisenberg concluded that providing the police officers with basic cpr skills and training in the use of automated external defibrillator can increase survival rates for ohcas (major link of early defibrillation in the chain of survival). hence, there is an immediate need for training of police personnel and paramedics in bec in india. the authors have taken initiatives in this regard, and the all india institute of medical sciences bec course was designed to address the issue. they have trained medical and nonmedical personnel, including paramedics, police, and paramilitary forces in bec. allison. developed and tested an undergraduate prehospital trauma course in 205 medical students of 4 year of medical school. the feedback was positive and they all felt better equipped to deal with the emergency situations in the future. the authors recommend the training in basic prehospital care for laypersons including school children and medical professionals in india. rosc was achieved in 7.4% cases on the first attempt at cpr in our study. there has been a debate on cpr in traumatic ohca patients citing its futility, wastage of resources and time. battistella. in their study concluded that trauma victims who are pulseless and have asystole or heart rate < 40 beats / min should be declared dead at the scene of injury. reported a bystander cpr rate of 10.2% with on - scene rosc rate of 15.4% in traumatic ohca. leis. after analyzing about 167 tca cases found that rosc was obtained in 49.1%, and 6.6% cases achieved a complete neurological recovery. the high rate of rosc in the above study was attributed to advanced life support (als) on - scene, which included intubation, intravenous access, fluid and drug therapy, point - of - care blood analysis, and procedures such as chest drain insertion, pericardiocentesis, and focused assessment with sonography for trauma. hence, the authors support the view that cpr efforts in trauma - associated ohca patients are not futile and if supplemented by als on scene, may result in increased survival and neurological outcome. the initial rhythms were asystole in 253, pea in 11, vf in six, and vt in five patients. previous studies have identified shockable (vt / vf) or nonshockable (asystole / pea) rhythm on presentation as the most important factor for an increased chance of survival from ohca and have concluded that patients with vf or vt had 5 - 10 times increased chances of survival. about 78.23% patients in our study were in the age group of 19 - 64 years. found that the median age of traumatic ohca patients was 36 years (iqr 25 - 55). the who statistics also supports this trend of young, productive working population being lost each year due to rtc. male preponderance (85.67%) in our study is comparable with deasy., which reported 77.5% of males in traumatic ohca patients. this may be explained by the fact that the major working population outdoors are males who are predisposed to traumatic injury risk factors. this is in accordance with the data from ncrb, india, published in 2014. india accounts for as high as 6% of the world 's rtc although it has just 1% of the world 's vehicles. the rtc rate of 35 per 1000 vehicles in india is one of the highest in the world. hence, the authors stress the need for mass education of public regarding injury prevention strategies. cardiac arrest is a terminal event, doctors and paramedics are not enthusiastic and do not stress upon complete documentation of events. hence, the authors stress the need for electronic recording of data and tca registry. cardiac arrest is a terminal event, doctors and paramedics are not enthusiastic and do not stress upon complete documentation of events. hence, the authors stress the need for electronic recording of data and tca registry. cardiac arrest is a terminal event, doctors and paramedics are not enthusiastic and do not stress upon complete documentation of events. hence, the authors stress the need for electronic recording of data and tca registry. | introduction : there is a paucity of literature on prehospital care and epidemiology of traumatic cardiac arrest (tca) in india. this study highlights the profile and characteristics of tca.methods:a retrospective cohort study was conducted to study epidemiological profile of tca patients 1 year presenting to a level 1 trauma center of india.results:one thousand sixty - one patients were recruited in the study. the median age (interquartile range) was 32 (2345) years (male : female ratio of 5.9:1). asystole (253), pulseless electrical activity (11), ventricular fibrillation (six), and ventricular tachycardia (five) were initial arrest rhythm. road traffic crash (rtc) (57.16%), fall from height (18.52%), and assault (10.51%) were modes of injury. prehospital care was provided by police (36.59%), ambulance (10.54%), relatives (45.40%), and bystanders (7.47% cases). return of spontaneous circulation was seen in 69 patients, of which only three survived to hospital discharge.conclusion:rtc in young males was a major cause of tca. asystole was the most common arrest rhythm. police personnel were major prehospital service provider. prehospital care needs improvement including the development of robust tca registry. |
professional response to the psychological needs of individuals exposed to acute, sudden, and catastrophic natural disasters or terrorism has been the focus of recent research and has become an essential component of comprehensive disaster relief services. however, the human emotional response, resiliency, and potential for chronic psychopathology appear to be different when the disaster is a slow motion, technologically associated event with unremitting long - term impacts on the ecosystem, socioeconomic structure, and health of the resident population. for example, the recent gulf coast oil - spill disaster in the united states (usa) provides a glimpse into the lives of individuals and communities dealing with the early stages of psychosocial stress associated with a massive, long - term environmental event. another case in point, and the focus of this paper, is the delayed psychosocial consequences of a complex and persistent environmental event that is being faced by the community and residents of libby, montana in the usa. libby, a small rural mountain town (population 2600), was contaminated with amphibole asbestos as a result of decades of mining tainted vermiculite ore. in 2002, with the resolve of politicians, medical providers and community members, libby was placed on the national priorities list as a superfund site by the united states environmental protection agency (epa). the community - wide environmental and occupational contamination left libby with a legacy of economic and insidious health - related problems across generations and a reputation as a place with air that kills. in 2009, seven years after designation as a superfund site, the epa declared libby a public health emergency, the first in usa history resulting from an environmental disaster under the comprehensive environmental response, compensation, and liability act (cercla). this determination recognized the serious impact to the public health from the contamination at libby and laid the foundation for a short - term grant to provide needed asbestos - related medical care to persons who were exposed to libby amphibole asbestos. exposure to microscopic asbestos fibers is linked to several malignant and nonmalignant illnesses including lung cancer, mesothelioma, asbestosis, pulmonary fibrosis, and pulmonary effusions. persons at risk for asbestos - related diseases (ard) include (1) former vermiculite workers, (2) household contacts and family members who lived with the workers, (3) community members with no association to the vermiculite mine or mine facilities that lived, worked, or played in libby, and (4) persons living in other communities across the usa where the ore was processed. during 2000 - 2001, medical screenings were conducted by the agency for toxic substances and disease registry (atsdr) of 7,307 persons who had lived, worked, or played in libby for 6 months prior to closing of the vermiculite mine (december 31, 1990). significant increases in asbestos - related pleural abnormalities were also found among residents living in libby after 1990 who were never associated with the mining operation. standardized mortality ratios (smrs) for asbestosis in persons who lived in the city of libby and surrounding areas were 4080 times higher than those for montana and the usa, respectively. in the latest asbestosis mortality statistics, lincoln county, where libby is located, had the highest age - adjusted asbestosis death rate per million people in the usa for residents age 15 and older. as of march 2010, nearly 325 libby residents have died from ard ; thirty - two deaths have been attributed to malignant mesothelioma, a rare form of cancer most commonly associated with exposure to asbestos. because ard has a latency of 10 or more years, more cases are expected to surface. however, the impact of libby amphibole asbestos (laa) extends well beyond this rural community. tainted vermiculite ore was distributed to over 250 processing and distribution plants across the usa, contributing 80% of the world 's supply. it is estimated that the raw ore contained as much as 26% asbestos [7, 14 ]. it is estimated that 35 million homes across the usa alone are insulated with the contaminated vermiculite (zonolite) that came from libby. the first screening of usa workers exposed to libby vermiculite products and living outside libby was carried out in maryville, ohio with a follow - up study conducted 20 years later. pulmonary findings from the maryville study were similar to the libby findings from the 2000 - 2001 health screening. in addition to physical and ecologic damage, technological disasters can affect the psychosocial health of individuals, families, and communities. anxiety, chronic stress, depression, loss of control, and uncertainty among disaster victims have been documented, while communities can be left with an aftermath of controversy, conflict, and social divisiveness. it is reasonable to expect that living with the knowledge of life - threatening risk to health and the potential for continued exposure, as the epa cleanup activities continue, would increase libby residents ' vulnerability to depression and stress. while no one is immune, younger age groups and women have been found to be particularly vulnerable to the psychosocial fallout of an environmental disaster. depression results in sadness, loss of interest in a person 's usual activities, feelings of worthlessness or hopelessness, disturbed sleep or appetite, low energy, and poor concentration [21, 22 ]. occurring at any age and affecting nearly twice as many women as men, depression is one of the most widespread of health conditions, affecting about 121 million people worldwide. depression is expected to be second only to heart disease as the source of global burden of disease by 2020 [24, 25 ]. there is great potential for disrupting the lives of affected individuals, influencing their personal and family relationships, productivity in employment and personal lives, and ultimately, the community at large. as a social health condition, depression is linked to suicide, alcohol and drug misuse, and a variety of chronic, health impairing behaviors. likewise, depression can inhibit the effective management of illness and health promotion behaviors. individuals living in rural areas are particularly vulnerable to depression. according to the 1999 national health interview survey, 2.6 million rural adults in the usa suffer from depression. the prevalence of major depression was found to be significantly higher among rural than among urban populations (6.11% compared with 5.16%). these statistics are particularly troubling given the underdetection and under - treatment of depression in rural primary care and limited access to mental health professionals in the rural usa. any agent or stimulus that challenges adaptive capabilities can be considered a stressor, for example, a traumatic event or an illness. postdisaster stress symptoms are often but not always reported more frequently by women than men. it is important to recognize that uncontrollable and unpredictable stress takes a toll on physical and mental health, and that individuals ' perception of the stressor and how they adapted to the stressful event are important factors that impact ability to cope with the stresses of illness. developing the capacity to manage stress is often helpful in dealing with the assaults of environmental stressors and the illnesses which they may trigger. realistic acceptance of illness is proposed to have a direct influence on how well individuals manage and adapt to living with their health condition. acceptance of illness is not resignation but integration of the disease into one 's overall lifestyle ; that is, getting on with living. illness acceptance restores a sense of personal control by integrating the illness experience into the person 's lifestyle. a strong relationship has been empirically demonstrated between illness acceptance and well - being, improved daily functioning and engagement in normal activities, higher quality of life, and better prognosis in a variety of chronic illnesses. for persons exposed to a deadly toxin, such as those living in areas where amphibole asbestos - contaminated vermiculite was mined and processed, the probability of chronic illness is high along with its negative impact on psychosocial health. thus, it is important to recognize the antecedents of negative psychosocial outcomes, such as depression, stress, and poor acceptance of illness, in this population and identify these individuals so that appropriate interventions and care management can be initiated. the research reported here was conducted to explore the biopsychosocial health status and health service needs of a national cohort of persons seeking care for exposure to libby amphibole asbestos (laa). the purposes of this paper are to (a) describe the psychosocial health status (depression, stress, and acceptance of illness) of the participants, and (b) explore differences in their psychosocial health status based on age, gender, residence, exposure pathway, insurance status, and access, availability, convenience, and financial aspects of health care among this sample. a descriptive cross - sectional design was used for the study. psychosocial health status and demographic data the exposure pathway and physical health data were gleaned from the participants ' health record. approval for the study was granted by the institutional review board at montana state university. data were collected from february through september, 2007, two years prior to the declaration of a public health emergency in libby, montana. written informed consent was obtained, and coded, deidentified data were used in the analysis. participants were clients of a specialty care clinic in libby, montana which has been providing asbestos health screening, follow - up health care, and consultation for exposed individuals since its opening in 2000. at the time of this study, the clinic was caring for more than 1500 patients from 32 states in the usa. to publicize the study, a notice was placed in the clinic newsletter and descriptive posters and brochures as clients presented to the clinic, they were asked to participate in the study. those who consented completed either an electronic (via computer) or paper version of the questionnaire during their clinic visit. in addition, a group of individuals who were exposed to laa and lived elsewhere in the usa, but continued to seek their specialty asbestos care at the clinic in libby, were invited to participate in the study via normal clinic correspondence that included a packet containing a letter describing the study, consent form, and a paper copy of the questionnaire. data collected using the electronic methods were sent through a secure internet connection to a protected database at the university research office. the paper / pencil data and health status information collected from the health record were electronically recorded and transmitted via the secure internet site. summary results from the psychosocial measures were reported back to the clinic weekly as they had a potential for impacting the plan of care. all questionnaire data collected became a part of the client 's health record at the clinic. the measures used to describe the psychosocial health status included center for epidemiological studies - depression scale (ces - d) ; perceived stress scale (pss) ; acceptance of illness scale (aoi) ; patient satisfaction questionnaire (psq - iii). demographic questions were added to gather information on age, gender, education, marital status, and residence (local or distant). the exposure pathway (vermiculite worker / nonvermiculite worker), and primary health insurance coverage were gleaned from the health record. the ces - d is a 20-item self - report designed for initial screening in settings where psychological distress is not routinely assessed and is intended for use with research conducted in the general population. potential scores range from 060 with higher scores indicating higher levels of distress with a reported alpha ranging from.84.90 (current study 0.89). a score of 16 or greater suggests a clinically significant level of psychological distress, but not necessarily clinical depression and indicates that further psychological assessment may be necessary. the pss consists of 14 items that measure the degree to which situations in one 's life are seen as stressful and is recommended by its developers as an outcome measure of levels of stress including that related to dealing with a chronic illness. scores can range from 0 to 56 with higher scores indicative of higher perceived stress. the reported alphas ranged from.84.86, and in this study was 0.87. the 14-item scale includes statements related to acceptance of an illness such as having (this) disease is just part of life and i ca n't conquer (my disease) but i can adapt to it. potential scores range from 14 to 70 with higher scores indicating greater acceptance. the psq - iii is a survey, with seven subscales, designed to evaluate health services delivery. the 12-item access / availability / convenience subscale was developed to measure these factors in relation to hospital, emergency, clinical, primary provider, and specialist care including convenience of services and nonfinancial access. potential scores range from 12 (low) to 60 (high) and reliability is sufficient at.86 (0.85 for this study). the financial aspects subscale of the psq - iii contains eight items designed to address cost of care, health insurance, and ability to pay for care. the purposes of this study were to (a) descriptively examine depression, stress, and acceptance of illness among persons seeking care for exposure to laa, and (b) explore differences in those variables based on age, gender, education, asbestos exposure route, residence, health insurance status, and satisfaction with access and financial aspects of healthcare. for the first purpose, means (observed) and standard deviations were produced to allow for general comparisons with other well and ill samples described in the literature. minimal subgroup analysis based on age, gender, and education allowed for a more complete understanding of observed values. general linear modeling (glm) was used to evaluate the second purpose with main effects as stated and a single a priori determined interaction of agegender. p - values for post hoc tests were adjusted using the bonferroni correction with the exception of the agegender interaction where cell frequencies dropped, for example, n = 22 for women 049 years, n = 76 for women 5064 years, leading to a violation of the equality of variance assumption and the use of the games - howell procedure. a cohort of 386 patients of a specialty care clinic living in the libby area, across montana, and throughout the usa, with a history of environmental or occupational amphibole asbestos exposure, participated in the study. the majority were 5064 years of age (46.9%) ; 17.9% had less than a high school education ; 69.9% were married ; and a slight majority were men (57.3%). most resided in or near libby, montana (73.8%). the primary health insurance was from public sources such as the centers for medicare & medicaid services, veterans administration, and social security administration (24.1%) ; private insurers (29%), and specialty asbestos health insurance called health network america (hna)/libby asbestos medical plan (46.9%). the first study purpose was to describe the psychosocial health status defined by depression, stress, and acceptance of illness among patients (n = 386) exposed to laa. on table 1 are the means and standard deviations for measures of the psychosocial variables for the total sample, as well as subgroups created by age, gender, and education. for depression, stress, and acceptance of illness, the overall means were 13.5 (sd = 10.7), 21.0 (sd = 9.0), and 47.5 (sd = 7.5), respectively. more than one - third (34.5%) of the participants scored 16 or above on the depression scale, indicating a clinically significant level of psychological distress. the intent of the second study purpose was to explore differences in depression, stress, and acceptance of illness based on age, gender, education, asbestos exposure route, residence, insurance status, and satisfaction with access and financial aspects of healthcare. for depression, using an anova analysis (table 2), omnibus differences were found only for satisfaction with healthcare access f(3, 361) = 7.37, p <.05, and the agegender interaction f(2, 361) = 4.96, p there initially appeared to be an omnibus difference in depression based on primary insurance status, but in post hoc tests significant differences were not demonstrated for the three means. multiple comparisons were performed for satisfaction with healthcare access and indicated that those scoring in the lowest quartile were significantly different than the 2nd, 3rd, and 4th quartiles on depression and that the 2nd and 3rd quartiles were different than the highest 4th quartile. similarly, for the agegender interaction, group 6 (women aged 65 +) was significantly different from all other groups of both women and men on depression. it is demonstrated in figure 1 that for women in the oldest age category (65 +), scores on depression dropped significantly from the next youngest age group of women and appear nearly 2 points lower than the lowest scores for men (5064 years). an identical glm procedure was repeated examining stress with the same grouping variables and interaction effect of agegender (table 3). omnibus differences were found for satisfaction with financial resources f(3, 361) = 2.97, p <.05, satisfaction with healthcare access f(3, 361) = 5.48, p <.05, and the agegender interaction f(2, 361) = 4.81, p <.05. multiple comparisons were performed for satisfaction with financial resources as well as healthcare access and indicated that those scoring in the lowest quartile were significantly different from the 3rd and 4th quartiles on stress. additionally, for satisfaction with financial resources, the lowest quartile was different from the second quartile, and for both variables the 2nd and 4th quartiles differed. mirroring the findings from depression, the agegender interaction showed that women in the 65 + age category differed from all other men and women on stress. these differences can be clearly seen in figure 2 where the oldest women appear to score lowest on stress relative to the 5 other age groups. last, the glm procedure was repeated, examining acceptance of illness with the same grouping variables and interaction effect of agegender (table 4). omnibus differences were found for satisfaction with financial resources f(3, 361) = 7.93, p <.05 and the main effect of gender f(1, 361) = 4.20, p <.05. multiple comparisons were performed for satisfaction with financial resources, and scores in the lowest and 2nd quartiles were significantly different from those in the 3rd and 4th quartiles on acceptance of illness. although there was no significant agegender interaction, the main effect of overall differences between men and women can be seen in figures 1, 2 and 3. at every age level, women had higher scores on acceptance of illness. to our knowledge, this is the first exploration of the psychosocial health status of persons seeking treatment for exposure to laa. for depression, stress, and acceptance of illness, no differences in means were found based on residence, exposure pathway, or insurance. gender, age, and satisfaction with financial resources were significantly related to depression, stress, and acceptance of illness. based on the severity, length, and all - prevailing nature of the libby environmental disaster, it could be anticipated that the overall depression level would be high. when comparing the mean depression scores of the libby cohort to those for groups dealing with a serious chronic illness, such as hiv (n = 243 ; 17.4 ; sd = 13.5), rheumatoid arthritis (n = 236 ; 16.6 ; sd = 13.1), and chronic cough (n = 100 ; 18.3 ; sd = 13.2), the libby mean was at least three points lower. while the overall mean depression score for study participants was modest (13.5, sd = 10.7), more than one - third (34.5%) had scores indicating a clinically significant level of psychological distress. this is particularly striking when compared to the percent of americans (5.4%) and montanans (6.7%) who reported being depressed. reported depression levels are often higher for women than for men ; however, in our study the mean scores of women were not higher. it had a lower overall stress score when compared to individuals with parkinson 's disease (n = 70 ; 25.1 ; sd = 7.6) and those with chronic obstructive pulmonary disease (copd) (n = 181 ; 22.8 ; sd = 8.4), and a slightly higher mean score than a group of women with an initial breast cancer diagnosis (n = 113 ; 18.1 ; sd = 6.9). similar to the depression scores, the libby study men and women had comparable mean scores for stress. the impact of the disaster and the subsequent aftermath of resulting chronic illness and deaths in the community appear to be somewhat reflected in the scores on the aoi scale. the libby cohort had a lower mean acceptance score than a group of individuals with post - polio syndrome (n = 1603 ; 53.6 ; sd = 11.9). however, they were considerably higher than persons with diabetes (n = 59 ; 26.6 ; sd = 8.4) and a group with multiple sclerosis (n = 786 ; 34.58 ; sd = 9.0). the libby cohort may be struggling with accepting the effects of exposure to asbestos and ard and integrating the residual limitations into their overall lifestyle because of the progressive nature of the illness. with progressive disease, new losses continue to emerge that must be accepted and integrated into a person 's lifestyle. individuals can not plateau and establish a new equilibrium but must continually struggle with accepting the new state of their illness this score may also reflect the uncertainty about the long - term consequences, stigma, and loss of social support experienced by persons exposed to laa [52, 53 ] and resulting chronic illness. at every age, this difference could be related to the working class rural culture of the community and men 's traditional role and identity of providing for their families. in the multivariable analysis, the analysis for depression yielded two significant findings : those with higher satisfaction with health care access were less depressed ; and age and gender had an interaction effect with older women being much less depressed than older men. for stress, those with higher satisfaction with health care access and with financial resources had lower levels of stress. again, age and gender had an interaction effect with older women being less stressed than older men. for acceptance of illness, men were significantly different than women ; however, differences based on age were not identified. as with stress, those with higher satisfaction with financial resources for health care had higher degrees of acceptance of their illness. in evaluating the findings, it should be recognized that participants were recruited from a single specialty clinic and not from the libby community at large. recruiting from the community at large may have resulted in the inclusion of participants who were not associated with the clinic, were not being treated for laa exposure or illness, and who may have had different health and illness experiences. most participants were local, for example, recruited from lincoln county and across montana potentially limiting the ability to generalize the study findings to other groups of exposed persons. in a study of patients seeking care for exposure to laa, participants demonstrated moderate levels of stress and acceptance of illness however more than one - third (34.5%) had depression scores indicating a clinically significant level of psychological distress. gender, age, and satisfaction with financial resources were significantly related to depression, stress, and acceptance of illness. the use of screening tools, similar to those used in this study, could be implemented in primary care practices serving populations living in or near environmental disaster areas around the world to assess psychosocial health. to take it one step further, incorporating a multidisciplinary team consisting of medical providers, nurses, and social workers could provide interventions to address individual, family, and community psychosocial issues. in our current study, this would include providing outreach, education, and referral services when needed for dealing with depression and chronic stress. since depression can vary significantly across rural and urban areas and may reflect regional differences, demographic characteristics, socioeconomic conditions, and preventable social causes, additional research is necessary to understand the mental health issues facing additional cohorts of persons exposed to laa. of particular interest was the fact that, for a group of people living in an area with serious and persistent environmental contamination along with its associated health risks and potential sequelae, the reported stress levels were not particularly high. further research is needed to gain a better understanding of the impacts of a disaster and its resulting health effects on men that specifically targets their personal identity and social roles. understanding the resilience demonstrated by the women, a quality that might account for their adaptability, could be useful to health care providers and community - based nurses working with clients who live in an area affected by an environmental disaster. studies are also needed that explore the psychosocial health status of exposed persons not affiliated with the specialty clinic in libby to tease out the clinic providers ' influence on the psychosocial health status and access and satisfaction with care. psychological distress identification, prevention, and intervention strategies, including self - management skills, are needed for persons exposed to environmental and workplace contamination such as the laa disaster. with the declaration of a public health emergency in june of 2009, federal funding became available to address health issues resulting from the libby environmental disaster ; however, these resources were set up around a one - year recovery program as opposed to the long - term needs and duration of an ongoing environmental disaster. population - based nurses and other health care providers are uniquely positioned to examine the biopsychosocial human response to an ongoing environmental disaster and intervene appropriately. they are obligated to work with the community to design and integrate best practice self - management interventions to reduce the effect of inordinate adversity from the event 's lingering aftermath. | a cross - sectional exploratory study was conducted to describe the psychosocial health status of persons seeking health care for exposure to libby amphibole asbestos (laa). health indicators including depression, stress, acceptance of illness, and satisfaction with access and financial aspects of care were obtained via electronic and paper - pencil survey. the exposure pathway and demographic data were gleaned from the health record. of the 386 participants, more than one - third (34.5%) demonstrated significant levels of psychological distress. the oldest group of women had the lowest levels of depression and stress and the highest acceptance of illness. gender, age, and satisfaction with financial resources were significantly related to depression, stress, and acceptance of illness. satisfaction with access to care was significant only for stress. no differences in depression, stress, and acceptance of illness were found based on residence, exposure pathway, or insurance status. |
the developing nervous system in utero is exposed to myriad influences with potentially far reaching consequences. most of the research in this area is directed towards understanding the adverse influences and their structural or functional pathogenesis. however, it is also attractive to investigate if foetal neurodevelopment can be positively influenced or enhanced in an analogous manner. there is evidence that appropriate vibroacoustic stimulation by exposure to music alters foetal behaviour and is carried forward to the newborn period [2, 3 ]. music is a noninvasive, culturally acceptable intervention with multiple putative direct and indirect beneficial effects on mother and foetus through the pregnancy and perinatal period. in animals, prenatal music exposure has been shown to improve postnatal spatial learning and memory ; to reduce isolation stress. music has been found to beneficially affect stress response and recovery from critical illness or surgery [5, 6 ]. using optical topography and salivary cortisol as a marker of stress, music has been documented to simulate pleasure and happiness. on a molecular level, music has been shown to alter dominergic neurotransmission and have direct effect on neurotrophic growth factors including brain derived neurotrophic factor and tyrosine kinase receptor b [5, 8 ]. besides direct influence on emotions, behavior, and neurotransmitter systems, there are multiple endocrine effects of music exposure including altered levels of adrenal and gonadal steroids. these changes in a pregnant woman can influence neuroblast proliferation, axonogenesis, synaptogenesis, and neuronal organization with effects on cognitive performance and behavioural gestalt. the present study was carried out to test the hypothesis that music exposure to mother during pregnancy can affect the neonatal behaviour. this was a single - centre, open - label, randomized controlled trial (rct) conducted at a teaching hospital from january 2003 to december 2005. the study was approved by institutional ethics committee and is registered with clinicaltrials.gov (nct01278329). all consecutive primigravida mothers of 19 to 29 years of age with singleton pregnancy attending the antenatal clinic of the study institution first time, at or before 20 weeks of gestation, were eligible for inclusion. mothers with significant coexisting medical diseases or severe to profound hearing loss were excluded (figure 1). mothers were then randomized to music and control groups using a printed random number table. allocation to the groups was concealed from the investigator (ra) performing outcome assessment. mothers randomized to music group were given a cassette player and a prerecorded music audio cassette and were demonstrated their use. garbh sanskar audio cassette (times music inc., mumbai, india) with a running duration of approximately 50 minutes and a cassette player with headphones. this contains a medley of instrumental music, natural sounds, and chants from religious scriptures. they were asked to listen to the recorded music daily in the evening just before going to the bed with a minimum of ambient noise. they were also asked to maintain a record of their music listening activity by making a check mark on a printed calendar. criteria for protocol violation included noncompliance with music listening for more than 2 weeks, development of preeclampsia or eclampsia in the mother, delivery of the newborn at a gestation of less than 37 or more than 42 completed weeks, delivery of the baby by emergency caesarean section, requirement of general anaesthesia even in case of elective caesarean section, neonatal birth weight less than 2500 grams or more than 4000 grams, or presence of significant neonatal disease precluding application of outcome assessment. all healthy term appropriate for date neonates born of spontaneous vaginal delivery or elective caesarean section conducted under epidural anaesthesia were subjected to outcome assessment. outcome measures consisted of the performance on brazelton neonatal behavioral assessment scale (bnbas). the bnbas is a means of scoring interactive behaviour for term and stable preterm infants. the scale consists of 27 behavioural items, each scored on a 9-point scale, and 20 elicited responses, each scored on a 3-point scale. in most cases, the infant 's score is based on the best performance, not an average performance. the bnbas was administered once to each infant in the study on day 2 or 3 of life. the assessment was performed by the investigator (r.a.) who has received prior training in its application, and the items were scored as recommended in the manual. infants were tested midway between feeds in a quiet, dimly lit room with an ambient temperature of 3234c. the items were grouped as recommended by lester into the following 7 clusters : habituation, orientation, motor performance, range of state, regulation of state, autonomic stability, and reflexes. sample size estimation for this study presented many challenges. a prior prospective study with similar design used a sample size of 20 believing it to capture significant differences in fetal behavior. a pilot study was not feasible because of long follow - up period from enrolment of the mother to delivery of the newborn ; lack of single primary outcome measure. hence, it was decided to conduct the study in an open - ended manner limited by time of enrolment (january 2003 to march 2005) rather than number of mothers enrolled. the data was entered in a microsoft excel spreadsheet (ms office version 2003). a total of 352 primigravida females attending antenatal clinic for the first time at a gestation of 20 weeks or less were evaluated for participation. ten females were excluded because of chronic medical diseases including rheumatic heart disease, chronic hepatitis, uncontrolled type 1 diabetes, chronic obstructive pulmonary disease, and vesicoureteric reflux - associated chronic renal failure (1 each). two females were unwilling to participate, and 1 was found to have 90 db hearing loss on audiometric evaluation. the remaining 339 females were randomized to receive music exposure in addition to standard antenatal care (intervention arm, n = 169) and standard care alone (control arm, n = 170). the primary analysis was per protocol, and bnbas assessment was applied to 126 newborns in the music exposure group and 134 newborns in the control group (figure 1). the infants born to mothers exposed to music during their pregnancy scored significantly higher on 5 of the 7 bnbas clusters including habituation, orientation, range of state, regulation of state, and autonomic stability. in all these clusters, the 95% confidence interval (ci) for effect size (es) remained on 1 side of the point of no difference (table 2). the maximal beneficial effect was seen in the clusters of orientation (es 1.13, 95% ci 0.821.44, p 33 weeks) and change in body movements (35 weeks). the authors concluded that processing of complex sounds changes at 33 weeks of gestation. experience in the present study agrees with published literature that music exposure in utero does influence neonatal behaviour this study enrolled mothers in first half of pregnancy and the foetus was exposed to a mean duration of 173 hours of music before birth, whereas other studies have exposed the foetus only for a few hours prior to birth. also, the present study used conventional headphones worn by the mother over her ears instead of the one taped to her abdomen as in other studies. although this would likely have resulted in less direct sonic stimulation of the foetus, the practical implications of this approach are more because of its better adaptability to routine clinical practice. a limitation of the present study was no standardization of the intensity of music stimulus. however, this might be relevant in case of directly applied stimulus over maternal abdomen where it conveys both vibratory and acoustic sensations, but not in present circumstances where it was better to let individual mothers decide about the volume of music as per their convenience. the onset of foetal hearing occurs at about 24 weeks of gestation. in the present study it is not known when the favourable effect of maternal music exposure started, and hence optimal timing for such stimulation in clinical practice can not be ascertained. it is improbable in the present study that music directly had any auditory effects on the foetus. the effects are more likely to be mediated via endocrine changes produced in the mother. music is known to have multiple endocrine effects including increased growth hormone which modulates the production of certain cytokines, increased ovarian steroid secretion, changes in the biorhythms and levels of cortisol, testosterone, and estrogen [5, 16 ]. corticosteroids have several regulatory effects on growth of neuroblasts, myelination, and metabolism in developing brain. they have been demonstrated to influence important enzymes, for example, sodium - potassium atpase, and growth factors, for example, basic fibroblast growth factor (bfgf-2) in developing cerebrum in animals. over 200 steroid responsive genes have been identified in the rat hippocampus involved in axonogenesis, synaptogenesis, cell adhesion, and signal transduction. thus, music exposure in the mother might influence neurogenesis and cerebral plasticity in the foetus through mechanisms mediated by steroids. in conclusion, this study provides preliminary evidence that maternal music exposure beneficially affects neonatal behaviour. a trained clinician can utilize the behavioural organization of the newborn infant to gain insights into the intrauterine experience and the perinatal events which may have influenced the neonate 's cns organization. the present clinical trial was not designed to study these aspects and provides no information regarding the mechanism behind the observed effect. further studies should confirm this observation with a more rigorous design and try to elucidate the direct and endocrine - mediated mechanisms of the effect of music on foetus and newborn. | objective. this study evaluated the effect of antenatal music exposure to primigravida healthy mothers on the behaviour of their term appropriate - for - date newborns assessed using brazelton neonatal behavioral assessment scale (bnbas). methods. this was a single - centre, randomized, open - label controlled trial. primigravida mothers aged 1929 years, free of chronic medical diseases or significant deafness, with singleton pregnancy, with a gestation of 20 weeks or less, were randomized to listen to a pre - recorded music cassette for approximately 1 hour / day in addition to standard antenatal care (intervention arm) or standard care only (control arm). perinatal factors with adverse effect on neonatal behaviour were deemed as protocol violations. outcome measure included scores on 7 clusters of bnbas. primary analysis was per protocol. the trial is registered with clinicaltrials.gov (nct01278329). results. one hundred and twenty - six newborns in the music group and 134 in the control group were subjected to bnbas assessment. the infants of mothers exposed to music during pregnancy performed significantly better on 5 of the 7 bnbas clusters. the maximal beneficial effect was seen with respect to orientation (es 1.13, 95% ci 0.821.44, p < 0.0001) and habituation (es 1.05, 95% ci 0.531.57, p = 0.0001). conclusion. prenatal music exposure to mother significantly and favourably influences neonatal behaviour. |
tumor that extends into pulmonary veins may cause the pulmonary vein flow stasis, and/or vascular injury results in thrombosis generation [25 ]. pulmonary veins infiltration by tumors or compression by affected lymph nodes result in venous stasis is also a potential reason to develop thrombosis [25 ]. impedance of blood flow from the pulmonary vein to the left atrium may cause pulmonary edema or pleural effusion. in the present prospective case series study, we investigate the clinical significance of pulmonary vein obstruction in cancer patients and better characterize the syndrome that we term pulmonary vein obstruction syndrome (pvos). data collected from 1117 patients hospitalized in oncology wards of the chang - gung memorial hospital. the urological cancer was our area of expertise ; most of these patients had urothelial carcinomas. the criteria for pvos diagnosis inclusion / diagnosis are listed as symptoms, chest x - ray findings, and ct findings, and that all 3 were required. the criteria for inclusion were (1) episodes of shortness of breath ; (2) chest x - ray showing unilateral or bilateral abnormal pulmonary hilum shadow with or without presence of pulmonary edema and/or pleural effusion ; (3) ct (computed tomography) scan demonstrating pulmonary vein thrombosis / tumor with or without lesions sticking to the outer vein surface. when dyspnea occurred and chest x - ray shows abnormal hilum shadow, ct scan was traced to detect pulmonary vein thrombosis / tumor. the characteristics of pvos included the presence of acute respiratory distress, combined with other thromboembolic complications and with other paraneoplastic syndromes. acute respiratory distress was described as aggravated by chemotherapy, aggravated by medical / surgical procedures, and subject to diurnal fluctuation in intensity. common thromboembolism - associated complications included consciousness loss / mental change [6, 7 ], paraneoplastic pain, and iliofemoral venous thrombosis symptoms. paraneoplastic pain was defined as breakthrough pain occurring in the absence of an identifiable precipitating cause. cerebral thromboembolic complication and/or paraneoplastic pain in most patients were clinically suspected because of difficulty in definite diagnosis. paraneoplastic syndromes included neoplastic fever (tumor - related fever with good response to naproxen test), cachexia syndrome (simultaneous presence of weight loss > 5% within 6 months, reduced food intake, and muscle wasting). the d - dimer test, complete blood counts, liver function test, renal function test as checked in all patients with pvos, aptt (active partial thromboplastin time) and pt (prothrombin time), calcium, c - reactive protein, blood gas, and pleural effusion study were checked in selected patients when diagnosing pvos. the cutoff d - dimer value was 500 ng / ml. paraneoplastic syndromes included hypercalcemia (serum calcium level more than 11 mg / dl), leukemoid reaction (peripheral count to more than 20,000/l without evidence of infection or leukemia), and prerenal azotemia was defined as bun - to - creatinine ratio greater than 20. chest plain film findings included the location of abnormal pulmonary hilum shadows, presence of pulmonary edema, and presence of pleural effusion. ct scan findings included pulmonary vein obstruction sites, presence of pulmonary embolism, and presence of pleural effusion. echocardiography and lung ventilation / perfusion scan were performed in selected patients when diagnosing pvos. treatment included subcutaneous injection of low molecular weight heparin (lmwh) either fraxiparin (glaxosmithkline) or enoxaparin (sanofi - aventis), intravenous dexamethasone, and intravenous fluids with or without furosemide when pvos with acute respiratory distress occurred. further use of chemotherapy or targeted therapy or hormone therapy depended on the patient 's condition. ct scans were obtained from the hospital picture archiving and communication system (pacs). continuous data (presented as mean standard deviation) were used for d - dimer, c - reactive protein, bun, creatinine, and bun - to - creatinine ratio analysis. the data for 222 consecutive cancer patients (139 men and 83 women ; 2793 years old ; median age, 69) was collected for evaluation of pvos. the patients ' characteristics and important laboratory and imaging findings of pvos were shown in table 1. one hundred and sixty - seven patients (75%) had an eastern cooperative oncology group (ecog) performance status of 2 or greater. common association with thromboembolic complications occurred in 146 patients (66%) : consciousness disturbance (n = 103), paraneoplastic pain (n = 53), and iliofemoral venous thrombosis symptom (n = 16). of them, 62 associated with multiple thromboembolic presentations. of 103 patients with consciousness disturbance, 16 had ct scan- or magnetic resonance imaging- (mri-) evidence of cerebral infarction and/or their angiographic proven. common association with paraneoplastic syndromes occurred in 101 patients (45%) : cachexia syndrome (n = 79), neoplastic fever (n = 23), leukemic - like reactions (n = 18), hypercalcemia (n = 4), and lactic acidosis (n = 3). of them acute respiratory distress (n = 222) was aggravated by chemotherapy (n = 28 ; 13%) and medical / surgical procedures (n = 21 ; 9%) and fluctuated diurnally in intensity (n = 32, 14%). blood gas tests in 126 patients found acidity (ph less than 7.3) in 24 (19%), paco2 > 50 mmhg in 23 (18%), pao2 10,000/l), and 21 patients (9%) with decreased platelet counts (< 100,000/l). aptt (active partial thromboplastin time) was checked in 100 patients and pt (prothrombin time) was checked in 109 patients ; of these, 34 (34%) had aptt values above 36 seconds and 29 (27%) had pt values above 15 seconds. albumin values were below 3.0 g / dl in 78 (41%) of the 189 patients. mean c - reactive protein value was 114 96 mg / l (0.7 to 384 mg of them mean bun value, creatinine value, and bun - to - creatinine ratio were 79.3 41.6 mg / dl (36.3 to 163 mg / dl), 3.7 2.7 mg / dl (0.65 to 12.3 mg / dl), and 21.4 15.5 (6.9 to 60.5), respectively. chest plain x - rays of 222 patients before and at the onset of pvos were shown in figures 1(a), 1(b), 2(a), 2(b), 3(a), 3(b), 4(a), and 4(b). abnormal hilum shadows were bilateral in 175 (79%) and unilateral in 47 (21%) and present in both lobes in 186 (84%), upper lobes in 19 (9%), and lower lobes in 17 (8%). chest plain x - rays showed pulmonary edema in 194 patients (87%) and pleural effusion in 192 patients (86%) (figures 1(b), 2(b), 3(b), and 4(b)). ct scans that revealed tumor / thrombosis located in pulmonary veins were shown in figures 1(c), 1(d), 2(c), 2(d), 3(c), 3(d), 4(c), and 4(d). the separate time between doing ct scan and detecting abnormal hilum shadows by chest films due to pvos was 149 patients (67%) within 1 month, 46 (21%) in 1 - 2 months, 20 (9%) in 2 - 3 months, and 7 (3%) more than 3 months. tumor or atelectatic lesions surrounding the pulmonary vein (figures 4(c) and 4(d)) were seen in 140 (63%). pulmonary vein obstructions were present bilaterally in 204 (92%) and unilaterally in 18 (8%), in both the superior and inferior pulmonary vein in 203 (91%), in the superior pulmonary vein only in 11 (5%), and inferior pulmonary vein only in 8 (4%). chest ct scan detected pleural effusion in 155 patients (70%), pulmonary artery embolism in 70 patients (32%), peripheral tumor / thrombi lesions (figures 2(c) and 2(d)) in 72 patients (32%), and cardiac tumor / thrombi lesions (figures 3(c) and 3(d)) in 7 patients (3%), and lung ventilation - perfusion scan in 5 patients found two (40%) with a pulmonary embolism and echocardiography in 29 patients found 14 (48%) with left atrium enlargement (38 mm). lmwh therapy was given to 170 patients with pvos, including 113 on fraxiparin (3800 iu or 5700 iu daily) and 57 on enoxaparin (6000 iu daily). and intravenous dexamethasone was also used in 133 patients. symptoms and/or image improvement in 113 (66%) including 99 of those treated with dexamethasone. of them, 37 patients continued their lmwh for secondary prevention. of 69 patients who received therapy, including chemotherapy in 59, targeted therapy in 9, and hormone therapy in one, 46 (68%) had disease control. pvos developed again after disease progression in 32 patients (74%), including 20 with their lmwh for secondary prevention. follow - up periods ranged from 1 day to 267 weeks. besides the fact that 12 patients were lost to follow - up, 210 patients could be followed until death or up to the present. three - month, 6-month, 1-year, and 2-year - survival probabilities were 30%, 13%, 9%, and 1%, respectively. for 52 patients not receiving lmwh therapy patients, median overall survival time was 4 weeks. for 170 patients receiving lmwh dexamethasone therapy showed clinical / image improvement in 114 patients. the composite survival rate for those with clinical / image improvement was superior to those without clinical / image improvement. the median survival rate was 11 weeks versus 2 weeks by log - rank test (p = 0.001). the predeath status could be identified in 198 patients as respiratory failure (n = 90 ; 45%) and consciousness loss (n = 108 ; 55%). sixty - three patients (32%) died of septicemia and/or febrile neutropenia, including 34 with respiratory failure and 28 with consciousness loss. trouseau 's syndromein is characterixed by spontaneous, multiple, recurrent, and migratory venous thrombosis, and arterial emboli [1113 ]. a few patients also exhibit other paraneoplastic syndromes related to cytokine production [810 ]. virchow describes the three elements, including venous stasis, endothelial injury, and hypercoagulability that are thought to contribute to venous thromboembolism (vte) [14, 15 ]. cancer cells can activate the hemostatic system through the expression of adhesion molecules, release of inflammatory cytokines, and production of hemostatic factors [1620 ]. activation of blood coagulation results in thrombin generation and intravascular fibrin formation [1620 ]. thrombin - activated tumor cell adhesion to host cells also enhances tumor cell growth, tumor cell seeding, and spontaneous metastasis and stimulates tumor angiogenesis [1619 ]. once cancer cells enter into and/or approach to the pulmonary vein, they result in blood flow stasis and vascular injury. the increase cytokine production among cancer patients have been well recognized in sepsis, surgery / medical procedures and chemotherapy [2022 ]. three lines of evidence are presented in the study to indicate that pvos is a real model of the thromboembolic complication [16, 17 ]. second, acute respiratory distress is aggravated by chemotherapy and medical / surgical procedures and fluctuates diurnally in intensity. cancer itself, its treatments, and its complications can activate cytokine signaling pathways including those of nuclear factor kappa b (nfb) and p38 mitogen - activated protein kinase (mapk). third, d - dimer and crp levels were elevated in 89% and 95%, respectively, of our patients, as occurs in cases of vte and pe [25, 26 ]. elevated d - dimer or crp a rise in hydrostatic pressure occurs due to blood flow through the pulmonary vein to the left atrium stasis. a few patients already had an oncological emergency (i.e., respiratory failure with hypoxemia, hypercapnia, decreased ph, or low sao2). a complex cause of pleural effusion noted in pvos patients, and 69% had leukocyte count less than 500 on the pleural fluids. the reason in part for the low leukocyte count is that, in pvos (like heart failure), hydrostatic pressure is also increased. cancer can induce pulmonary embolism (pe) formation and thrombotic formation and can invade large veins [2830 ]. extensive tumor - associated thromboemboli in the pulmonary microvasculature have been reported in a cancer patient with dyspnea [2830 ]. however, microscopic tumor embolism is rarely recognized before death. pvos can combined with pulmonary artery embolism ; tumor / thrombi from pulmonary veins can extend peripherally [28, 29 ] or enter left atrium. pulmonary artery embolism, peripheral pulmonary tumor / thrombi lesion, and cardiac tumor / thrombi lesions were 32%, 32%, and 3% of our patients, respectively. the appearance of abnormal pulmonary hilum shadows on chest plain x - ray films (which are due to pulmonary hypertension) is essential for raising suspicion of pvos. pulmonary edema and/or pleural effusion on chest plain films indicate severe blockade of pulmonary veins. ct scans are vital for the diagnosis of pvos and can show pulmonary vein thrombosis, tumor, and pulmonary vein stricture especially from their abnormal pulmonary hilum shadows. pulmonary edema and/or pleural effusion may be unilateral or bilateral and may arise from the superior pulmonary vein, inferior pulmonary vein, or both. bilateral lung and pulmonary veins involvement are seen in most of our patients. in the present study, pleural effusion was detected in 86% of our cases on plain chest x - ray films but in only 70% on ct scans. the discrepancy may be due to the timing of the ct scan, which was usually performed before the pvos attack. echocardiography found left atrium enlargement (thought to be due to stenosis of the pulmonary vein distal to the left atrium) in approximately 48% of our cases. in cancer patients, it is important to recognize that early appearance on chest x - ray of abnormal pulmonary shadows can result in a misdiagnosis of lung infection and acute respiratory distress syndrome. cytokine and nfb inhibitors, such as dexamethasone, are used to suppress cytokine formation in acute stage. stress related to cytokines can influence the course of neoplastic diseases. preventing unnecessary procedures and calming the patients can reduce cytokine overproduction. knowing aggravated factors can cause pvos. adequate fluids supplement is necessary for maintaining good perfusion of vital organ (brain, coronary artery, and kidney) [6, 7, 37, 38 ]. the importance of cancer - associated hyper - coagulation is an etiology of acute ischemic stroke [6, 7 ] and probably a leading death in our patients. renal insufficiency had been linked to increased mortality from the literature and prerenal azotemia pattern in our 16% patients. furosemide for preload reduction can provide immediate symptom relief when acute respiratory distress with pulmonary edema occurs. maintenance of lmwh is suggested for secondary prevention [39, 40 ] and probably a survival benefit [4143 ]. clinical / image improved in 66% of our patients after lmwh with or without combination with dexamethasone. the survival rate for those with clinical / image improvement was superior to those without clinical / image improvement. the cause of death was either respiratory failure or consciousness disturbance and related to thromboembolic complications. two - thirds patients died of septicemia and/or febrile neutropenia. in the pathogenesis of sepsis, inflammation and coagulation sepsis increases cytokine production and is considered to be an aggravating factor of thromboembolic complications. first, the data was collected from prospective case cohort study in a single center mainly from a single physician. secondary, image study included chest plain film and ct scan was probably not done simultaneously. third, pulmonary vein thrombosis, tumor, or mixed type was difficult judge from ct scan. fourth, consciousness / mental change related to thromboembolic complication seldom proven by image study. physicians should be alert to pvos when shortness of breath occurs and chest x - ray reveals abnormal pulmonary hilum shadows. symptoms can be relieved by the administration of lmwh, dexamethasone, prevent unnecessary procedures, calm the patients, adequate fluids, furosemide given when there is acute respiratory distress, and antibiotics given immediately when there is an infection. | background. we study the clinical significance and management of pulmonary venous obstruction in cancer patients. methods. we conducted a prospective cohort study to characterize the syndrome that we term pulmonary vein obstruction syndrome (pvos) between january 2005 and march 2014. the criteria for inclusion were (1) episodes of shortness of breath ; (2) chest x - ray showing abnormal pulmonary hilum shadow with or without presence of pulmonary edema and/or pleural effusion ; (3) ct scan demonstrating pulmonary vein thrombosis / tumor with or without tumor around the vein. results. two hundred and twenty - two patients developed pvos. shortness of breath was the main symptom, which was aggravated by chemotherapy in 28 (13%), and medical / surgical procedures in 21 (9%) and showed diurnal change in intensity in 32 (14%). chest x - rays all revealed abnormal pulmonary hilum shadows and presence of pulmonary edema in 194 (87%) and pleural effusion in 192 (86%). ct scans all showed pulmonary vein thrombosis / tumor (100%) and surrounding the pulmonary veins by tumor lesions in 140 patients (63%). pvos was treated with low molecular weight heparin in combination with dexamethasone, and 66% of patients got clinical / image improvement. conclusion. physicians should be alert to pvos when shortness of breath occurs and chest x - ray reveals abnormal pulmonary hilum shadows. |
crimean - congo hemorrhagic fever (cchf) virus is an enveloped rna virus of the family bunyaviridae (genus nairovirus) and causes severe viral hemorrhagic fever. although it is primarily an animal disease, sporadic cases and outbreaks of cchf affecting, humans do occur. cchf outbreaks constitute a threat to public health because of its epidemic potential, high case fatality, the potential for nosocomial outbreaks, and difficulties in treatment and prevention. a 55-year - old, previously healthy female with significant domestic cattle handling, hailing from a rural area around moradabad, uttar pradesh, presented with 15 days history of high grade fever with chills. the patient was admitted to a local hospital near her village, where she was treated with broad spectrum antibiotics and antimalarials. during the course of the hospital stay, the patient developed progressively worsening pancytopenia and developed bleeding from the oral cavity, epistaxis and developed a fine red macular rash all over her body. around the 14 day of her illness, she developed incoherent speech and breathing difficulty and was referred to our center. on presentation, the patient was febrile (39.4c), dehydrated and had severe pallor. she was oriented to time, place, and person, but was very irritable and had lost bladder and bowel control. investigations showed platelet count of 10,000/cmm, hb of 4.8 g / dl, white blood cell 980/cmm, lactate dehydrogenase 34,000 iu / l, serum creatinine 2.4 mg / dl, blood urea 201 mg / dl, serum bilirubin 3.6 mg / dl (predominantly indirect fraction), na 133 meq / l, k : 5.4 meq / l, prothrombin time 23 s, and the international normalized ratio of 3.4. the chest x - ray was suggestive of bilateral alveolar infiltrates and minimal bilateral pleural effusions and ultrasound abdomen revealed mild ascites with hepatosplenomegaly. dengue, malaria, scrub typhus, and leptospira serology tested negative. the patient was treated with oxygen, noninvasive ventilation, intravenous (iv) fluids, and blood products and was started on iv ceftriaxone, artesunate, and oral doxycycline. as her clinical picture matched with the clinical presentation of cchf and her condition deteriorated despite appropriate therapy, blood samples were sent to the national centre for disease control, new delhi for cchf serology testing. she tested positive for cchf disease specific genes by reverse transcription - polymerase chain reaction (rt - pcr). the patient was started on oral ribavirin 1000 mg every 6 h (via naso - gastric tube). over the next 2 days around 20 hospital staff were inadvertently exposed to the patient, of which 16 had close contact with her or had handled blood and urine samples. the drug was poorly tolerated by most and had to be stopped prematurely as most could not tolerate the prescribed dose. contact tracing was carried out, but thankfully none of the family members developed the disease. one of the hospital staff were she was admitted prior to our unit, developed a similar illness, which resolved with supportive management. cchf, is highly contagious and is associated with high mortality, ranging from 10% to 50%. the virus which causes cchf is a nairovirus, which is transmitted by ixodid ticks in animals. humans acquire the virus from direct contact with blood or other infected tissues from livestock having viremia, or they may become infected from a tick bite. the majority of cases have occurred in those involved with the livestock industry, such as veterinarians, agricultural, and slaughterhouse workers. cchf can be transmitted from one infected human to another by contact with infectious blood or body fluids. clinical and laboratory features of patients with cchf mimic the most prevalent diseases, such as dengue, malaria, and scrub typhus which are commonly encountered in routine practice. as cchf is usually not considered as the primary cause of fever with rash and pancytopenia in our patients, it is often diagnosed late. this leads to a higher mortality and exposes more health care workers to the disease. classical clinical features include high grade fever with chills, headache, myalgia, generalized rash, and bleeding. over the next few days, the patient may experience neurological features, such as mood swings, and may become agitated. after 24 days, the agitation may be replaced by mental obtundation and altered sensorium. laboratory diagnosis of a patient with a clinical history compatible with cchf can be made during the acute phase of the disease by using the combination of detection of the viral antigen (elisa antigen capture), viral rna sequence (rt - pcr) in the blood or in tissues collected from a fatal case and virus isolation. later in the course of the disease, in people surviving, antibodies can be found in the blood. general supportive therapy and maintenance of vital parameters is the mainstay of patient management in cchf. both oral and iv formulations seem to be effective. patients with suspected or confirmed cchf should be isolated and cared for using barrier nursing techniques. specimens of blood or tissues taken for diagnostic purposes should be handled using universal precautions. if strict precautions are not taken, there is a risk of nosocomial spread of infection and it is imperative that adequate infection control measures be observed to contain the infection. healthcare workers who have had contact with tissue or blood from patients with suspected or confirmed cchf should be followed up with daily temperature and symptom monitoring for at least 14 days after the putative exposure. outbreaks of cchf have been reported from india and other regions of the south east asia earlier and have been associated with high mortality. most of these cases, like our patient, were diagnosed late, as cchf was not initially considered as a differential diagnosis. cchf should be considered in patients with compatible clinical and laboratory features, so that early ribavirin therapy may be started, and appropriate control measures are taken to prevent nosocomial and community spread of the disease. | we present the case of a 55-year - old female, who presented with 15 days of fever with rash, pancytopenia, and altered behavior. she was investigated for routine causes of fever with rash and multi organ dysfunction and treated for the same. as she tested negative for all routine causes of such an illness and did not show improvement to therapy, she was investigated for crimean - congo hemorrhagic fever and tested positive for the same. she was started on ribavirin, but eventually succumbed to her illness. this disease has rarely been reported from the northern india and we need to have high clinical suspicion for this deadly disease so that appropriate therapy can be started in time for the patient and prophylaxis given to all inadvertently exposed. |
preterm premature rupture of membranes (pprom) occurs in approximately 3% of all pregnancies, accounting for nearly 1/3 of all preterm births [15 ]. moreover, pprom is a contributor to perinatal morbidity and mortality primarily due to its association with delivery remote from term, chorioamnionitis, placental abruption, and umbilical cord compression or prolapse [1, 2 ] management of women with hiv infection who develop pprom poses an obstetric dilemma. limited data are available to direct optimal management of hiv - positive women who develop pprom. previous investigations have demonstrated that in term pregnancies complicated by maternal hiv infection, duration of membrane rupture > 4 hours increases the risk of intrapartum vertical transmission [1217 ]. by extrapolation of this data, some authors suggest that women with hiv who undergo pprom should not be managed conservatively [1, 1214 ]. however, in instances near the limits of viability, conservative management of pprom may be justified because of the extremely high risk of mortality and chronic morbidity when immediate delivery is prompted. we and others have been unable to identify any published manuscripts to date reporting on conservative management of pprom in the setting of hiv (medline search 1 january 19661 september 2005 : search terms : preterm premature rupture of membranes, human immunodeficiency virus infection, acquired immunodeficiency syndrome). given an absolute lack of published reports, we sought to provide an initial report on the management and outcomes of a series of hiv - infected women whose pregnancies were complicated by preterm premature rupture of membranes (pprom). discussion of management options in each instance, a thorough and complete review of the relative risks of prematurity and pprom remote from term and potential for vertical transmission with prolonged rupture of the membranes was reviewed with the patient, and documented in the patient chart. these discussions involved obstetrician / gynecologists, maternal - fetal medicine specialists, neonatologists, and/or infectious disease specialists, with language interpreters as indicated. at hennepin county medical center (hcmc), the hiv - infected women were identified through the hcmc hiv database encompassing the interval from 1990 to 2004, and the electronic medical records (emrs) system was reviewed to identify whether they had been seen through the obstetrics and gynecology clinics, high - risk obstetrics clinics, or on labor and delivery. for those with identified encounters, exhaustive review of both the emr and patient chart was undertaken to ascertain whether a pregnancy had occurred, and whether the pregnancy had been complicated by pprom remote from term. at the university of alabama at birmingham (uab), an electronic obstetric database (obar) of women who obtained their prenatal care and delivered at uab from 1980 to 2004 was searched. women with hiv having undergone prom were identified and for those women in whom latency was greater than 24 hours, an exhaustive chart review was performed. pprom remote from term was defined as preterm, premature rupture of the membranes prior to 32-week gestation confirmed by ferning, pooling, or indigo - carmine amnioinfusion ; dating was by previously published american college of obstetricians and gynecologist (acog) criteria and employed last menstrual period with fetal biometry by ultrasonographic determination. maternal hiv - positive status was defined as documentation of hiv-1 or hiv-2 seroconversion, confirmed by western blot analysis. aids was defined as an absolute cd4 +, cd3 + lymphocyte count less than 200 10 cells / l. infants were considered infected if they were hiv - seropositive, confirmed by western blot at > 18 months of age, had two or more positive pcr tests or viral cultures at any age, had an aids - defining illness, or if they died with an hiv - related condition. once a pregnancy was identified as having been dually complicated by hiv - positive maternal status and prom remote from term, data was uniformly extracted from the patients chart with respect to maternal characteristics (age, ethnicity, parity, year of hiv - positive diagnosis, past medical history, polysubstance abuse history, and infectious comorbidities), prenatal care (gestational age and dating criteria, gestational age at initiation of prenatal care, gestational age at initiation of haart / therapy, number of prenatal visits), hiv characteristics (genotype, aids, infectious comorbidities [gbs, trichomonas, gonorrhea, chlamydia, ppd, hepatitis a, b, or c ]), pprom characteristics (means of confirmation, most recent viral load and cd4 count prior to pprom, antiviral therapy at pprom, gestational age at delivery, latency interval, indication for delivery, clinical or histologic evidence of chorioamnionitis), use of antiviral and antimicrobial therapy (haart / intrapartum azt, duration of use, utilization of antibiotics for the prolongation of latency), maternal peripartum complications (sepsis, chorioamnionitis, endometritis), and fetal and infant characteristics (administration of steroids for the promotion of fetal lung maturity, infant sex, birth weight, apgars, cord gasses, nicu admission and duration of nicu stay, neonatal sepsis, rds, ivh, necrotizing enterocolitis, vertical transmission, other complications or medical morbidities). at hcmc, the hiv database for women with hiv is supported by infectious disease hiv clinic with irb approval. as this represented a case series, no further irb approval was deemed necessary. at uab patient characteristics (table 1) over the indicated study intervals at hcmc and uab, birmingham, we identified 291 pregnancies having occurred in the cohort of hiv - positive women. of these pregnancies, 7 (2.4%) developed pprom with subsequent delivery from 25-to 32-week gestation. with the exception of one patient (patient 1), all women received prenatal care in their first or early midtrimester. for those who received adequate prenatal care, medical comorbidities reported reflect those found in the at - large hcmc and uab populations. hiv and infectious morbidity (table 2) with the exception of two patients (patients 2 and 6), all women were aware of their diagnosis of hiv prior to the pregnancy. note that the diagnosis of hiv seroconversion (for patient 1) occurred in a prior pregnancy, but treatment was never initiated secondary to failed followup ; this was in spite of multiple attempts to encourage compliance with care. each of the patients who received prenatal care at hcmc was initiated on haart in the early midtrimester ; two of these patients continued a modification of their prepregnancy therapy (table 2). at uab, both pregnancies occurred (1993) prior to the use of haart in pregnancy. one woman (patient 6) was part of the pactg 076 trial and was randomized to placebo. all patients were screened for viral and bacterial infectious comorbidities on initial presentation ; all were rescreened on admission. none were hepatitis b surface antigen - positive, one was hepatitis c positive (patient 7) ; one (patient 3) had been previously exposed to hepatitis a but was without active disease. three of the 7 patients had trichomonas vaginalis detected and treated, and one woman (patient 1) was positive and treated for trichomonas, c trachomatis, and n gonorrhea at the time of admission. two women (patients 3 and 7) had a history of tuberculosis, and had previously received therapy accordingly. pprom and delivery characteristics (table 3) each patient in our series was distinct from the others with respect to gestational age at the time of pprom ; however, the overall profile typifies that seen among hiv - negative women with pprom. in one instance (patient 2), pprom occurred prior to viability and extensive counseling regarding the risks of oligohydramnios and prolonged ruptured membranes was undertaken. the patient elected to pursue expectant management. with respect to viral load at the time of pprom, one woman (patient 1) was hospitalized for preterm labor when rupture of membranes occurred. with respect to patients 2 and 5, noncompliance with however, with inpatient management (patient 5) and frequent outpatient visits (patient 2), compliance improved and the viral load declined accordingly (table 3). as per protocol standard at the time of presentation, viral loads were not obtained in the women that delivered at uab. all patients with ongoing pregnancies at hcmc received intravenous azt for a maximum of 48 hours following membrane rupture, with repeat administration of therapy proximal to delivery. during the same interval, the single exception to optimal utilization of intrapartum azt use was patient 3, as she had experienced a prior episode of neutropenic sepsis with ards. at uab, intrapartum azt was not administered to one woman (patient 6) as per study (pactg 076) protocol following randomization to placebo. in another patient, intravenous azt was administered for 60 hours during her prolonged latent phase (from the time of admission to the time of delivery). the mean latency in this cohort was 17.1 days with a median of 5 days ; one woman (patient 2) had a latency of 92 days. indications for delivery typified that of pregnancies complicated by pprom, and mode of delivery was determined by obstetrical indications (table 3). maternal complications (table 3) clinical or histologic evidence of chorioamnionitis was prevalent ; endometritis ensued in four cases. there was one instance of maternal sepsis with positive blood cultures for peptostreptococcus. otherwise, the duration of postdelivery hospitalization mirrored that of the general obstetric population at the respective institutions. infant sequelae and complications (table 4) neonatal outcomes of the pregnancies in our series are shown in table 4. vertical hiv transmission was noted in 2 of the possible 6 cases (33%). in the two instances of vertical transmission, one woman and infant pair received no antiretroviral therapy as per study (pactg 076) protocol. in the second instance, azt was the only antiretroviral employed and viral loads were not used to guide delivery practice. all infants (except for patient 6 (pactg 076 protocol)) received antiretroviral therapy in the neonatal period as per standard of care. of the cases without vertical transmission in our series, all women received haart at the time of pprom with cesarean delivery for those with a viral load > 1000 copies / ml. in our series, there were no instances of vertical transmission in women with pprom who were receiving multidrug therapy or haart despite prolonged intervals of rupture of membranes. the only two cases of vertical transmission observed were in one woman who did not receive antepartum or intrapartum antiretroviral therapy, and in another who received only azt in the antepartum period. our observations suggest that in the era of haart, one might consider whether or not immediate delivery should be undertaken in pregnancies complicated by pprom at an early gestational age. alternatively, in pregnancies that may lend themselves to expectant management, such a strategy should be entertained. in pregnancies complicated by maternal hiv infection, duration of rupture of membranes > 4 hours, in term pregnancies appear to increase the risk of intrapartum vertical transmission [1217 ]. in one study, prolonged rupture of membranes in term (> 37 weeks) relative to preterm (4 hours. these authors attempted to delineate whether vertical transmission occurred in the intrauterine or intrapartum period and came to the conclusion that preterm infants are at increased risk of acquiring infection at the time of delivery, however, transmission risk was not stratified by use of antiretroviral therapy, gestational age, nor mode of delivery. as with this study, other studies [1217 ] exploring the relationship of duration of membrane rupture and perinatal hiv transmission have failed to account for the use of haart and viral load at the time of delivery. one recent analysis investigated the risk of perinatal hiv transmission in women on combination antiretroviral therapy with viral loads 4 hours. interestingly, monotherapy (or 6.3, ci 2.416.6) or lack of antiretroviral therapy (or 17.4 ci 6.745.5) and viral load > 1000 copies / ml (or 9.9, ci 1.284.5) were independently associated with neonatal infection. our case series suggests that this relationship between combination antiretroviral therapy and reduction in the rate of vertical transmission may apply to pregnancies complicated by prolonged premature preterm rupture of membranes and hiv. we recognize that the primary limitation of this study is its limited sample size and its heterogeneous population. our series does not claim to provide any statistical risk assessment for counseling patients on the relative merits or risks of transmission in the setting of pprom remote from term. rather, it is our hope that this novel series will provide an impetus to further reports and larger multi - institutional investigations to address this important clinical issue. we report a novel case series describing management and outcomes in human immunodeficiency virus - infected women who developed preterm premature rupture of membranes remote from term. pnc : prenatal care ; ards : adult respiratory distress syndrome ; azt : zidovudine ; aids : acquired immunodeficiency syndrome ; n / a : data not available. hiv and infectious morbidity. hiv : human immunodeficiency virus ; aids : acquired immunodeficiency syndrome ; haart : highly active antiretroviral therapy ; azt : zidovudine ; inh : isoniazide ; pactg : the pediatric aids clinical trials group ; tb : tuberculosis ; s / p : status - post. pprom : preterm premature rupture of membranes ; haart : highly active antiretroviral therapy ; azt : zidovudine ; c / s : cesarean delivery ; iufd : intrauterine fetal demise ; nsvd : spontaneous vaginal delivery ; n / a : data not available. nicu : neonatal intensive care unit ; rds : respiratory distress syndrome ; ivh : intraventricular hemorrhage ; nec : necrotizing enterocolitis ; iufd : intrauterine fetal demise ; vsd : ventriculoseptal defect. | objective. to evaluate the management and outcomes of a series of human immunodeficiency virus-(hiv-) infected women whose pregnancies were complicated by preterm premature rupture of membranes (pprom). study design. we conducted a retrospective chart review of all women with confirmed hiv infection who had a pregnancy complicated by pprom remote from term. pprom remote from term was defined as rupture of membranes prior to 32-week gestation. collective cases from two centers (hennepin county medical center and the university of alabama at birmingham) were reviewed and data on management and outcomes were abstracted. results. of the hiv - positive women, we identified 291 pregnancies having occurred in the study interval from two institutions. of these pregnancies, 7 (2.4%) developed pprom remote from term with subsequent delivery from 25- to 32-week gestation. vertical hiv transmission was noted in 2 of 6 children whose long - term followup status was confirmed (33%) of these cases. however, both of these cases occurred in women with either no antepartum / intrapartum antiviral therapy or where only zidovudine monotherapy was used. importantly, in spite of expectant management, no cases of vertical hiv transmission occurred in women who were receiving either multidrug or highly active antiviral therapy (haart) at the time of pprom and who had a cesarean delivery in cases where the predelivery viral load > 1000 copies / ml. conclusion. our limited observations raise the question as to whether in the current era of multidrug therapy immediate delivery should be undertaken in hiv+ pregnancies complicated by pprom at an early gestational age. this case series further suggests that in those pregnancies that lend themselves to expectant management, such a strategy may be considered appropriate. |
vitelliform lesions (vls) correspond to an accumulation of yellowish material in the subretinal space. [1, 2 ]. these lesions may evolve over time and may be classified in the following stages : stage i (previtelliform) : normal or only subtle rpe changes (tiny, central honeycomb structure centrally), normal vision.stage ii (vitelliform) : classic egg - yolk lesion, normal vision or mild vision loss.stage iii (pseudohypopyon) : layering of lipofuscin, normal vision or mild vision loss.stage iv (vitelleruptive) : breakup of material gives scrambled egg appearance ; vision may be mildly decreased.stage v (atrophic) : central rpe and retinal atrophy ; vision may range from 20/30 to 20/200.stage vi (choroidal neovascularization, cnv) : in about 20% of patients ; vision often decreased to 20/200 or worse [1, 2 ]. stage i (previtelliform) : normal or only subtle rpe changes (tiny, central honeycomb structure centrally), normal vision. stage ii (vitelliform) : classic egg - yolk lesion, normal vision or mild vision loss. stage iii (pseudohypopyon) : layering of lipofuscin, normal vision or mild vision loss. stage iv (vitelleruptive) : breakup of material gives scrambled egg appearance ; vision may be mildly decreased. stage v (atrophic) : central rpe and retinal atrophy ; vision may range from 20/30 to 20/200. stage vi (choroidal neovascularization, cnv) : in about 20% of patients ; vision often decreased to 20/200 or worse [1, 2 ]. when detected in younger patients, vls usually occur in the setting of best macular dystrophy (best disease), an autosomal dominant disease associated with a mutation in bestrophin 1 (best1) [3, 4 ]. adult - onset foveomacular vitelliform dystrophy (aofvd) is a subtype of macular pattern dystrophies, being associated with mutations in the peripherin 2 (prph2) gene, either sporadic or inherited in an autosomal dominant manner [5, 6 ]. the age of onset is typically between 30 and 50 years, and it is recognized as a pleomorphic disease, with great variability in size, shape, and distribution of the vitelliform material [1, 5 ]. acquired vitelliform lesions (avl) are a different type of vl associated with multiple retinal diseases such as age - related macular degeneration (amd), cuticular drusen, tractional maculopathies, pseudoxanthoma elasticum, and central serous chorioretinopathy [1, 7 ]. all the aforementioned diseases share the lack of direct apposition between the photoreceptor outer segments and the retinal pigment epithelium (rpe), which could delay the phagocytosis of shed outer segment photoreceptor tips, and lead to the accumulation of yellowish subretinal vitelliform material [2, 69 ]. whereas best disease may be easily distinguished from other vls by the age of diagnosis or electrophysiological study, the differential diagnosis between aofvd and avl may be difficult in patients without familial history of disease or with simultaneous macular diseases. in this study we characterize the vl in aofvd and avl patients using multimodal imaging analysis, exposing similarities and differences that could help in differential diagnosis between the two entities in clinical practice. this is a retrospective study of patients diagnosed with avl or aofvd, who were evaluated in the department of ophthalmology of hospital so joo, a tertiary health care center, at porto, between june 2011 and december 2013. the tenets of the declaration of helsinki were followed and local ethics committee approval was obtained. only eyes having previously undergone multimodal imaging analyses (color fundus photographs, fundus autofluorescence (faf), fluorescein angiography (fa), and spectral - domain optical coherence tomography (sd - oct)) were included. color fundus photographs were obtained with trc-50ex mydriatic camera (topcon medical systems, tokyo, japan). spectral - domain optical coherence tomography, faf, and fa were performed using spectralis hra + oct system (heidelberg engineering, heidelberg, germany). for each patient we recorded age, type and laterality of vl, associated macular lesions, baseline and final best - corrected visual acuity (va) using etdrs charts, and follow - up period. vitelliform lesions were evaluated for their stage, integrity of external limiting membrane and the assumed inner segment / outer segment junction (classified as present, absent, or disrupted), intraretinal fluid, and hyporeflective area in the subretinal space both at the baseline and at the final consult. the shape of vl was classified as round or irregular and the location was determined by the foveal topographic relationship and symmetry, being categorized as central or eccentric to the fovea. quantitative measurements of anatomical features (greatest linear dimension (gld) and lesion height (lh)) were recorded using the calipers provided by the review software of the spectralis hra + oct, similar to the method used by freund.. patients were divided into two groups, aofvd and avl, according to familial history and the absence of other previous retinal diseases in the former. in both groups, no patient has received any treatment, such as cataract surgery or cnv intravitreal injections. statistical calculations were performed using statistical package for social sciences (version 20.0 ; spss, inc., categoric variables were compared using the chi - square test, and differences for quantitative variables were analyzed by mann - whitney u or kruskal - wallis test. the association between va and quantitative measures of lesion was analyzed by kendall 's tau test. thirteen patients were diagnosed with avl (16 eyes) and six with aofvd (12 eyes). the median follow - up was 10 months, being the 25th75th percentiles [529 ]. the groups were followed up by a similar median (p = 0.782)the avl group was followed up by a median of 9 [7 ; 22 ] months and aofvd by 11 [5 ; 28 ] months. a summary of patient characteristics in each group is presented in table 1. in our case series, vls were more frequent in patients in their eighth decade and a slight female predominance was observed. we found no statistical differences in the demographic characteristics between the two groups. at presentation, the majority of lesions were stage ii, with the classic egg - yolk pattern being present in 8 eyes (50%) of avl patients and in 7 eyes (58.33%) of aofvd group. pseudohypopyon was observed in 5 patients (31.25%) in avl group, compared with 2 cases (16.7%) in aofvd, which also represented a nonsignificant difference between groups (p = 0.662). the presence of vitelleruptive stage was also evenly distributed between groups (3 eyes versus 2 eyes, p = 0.662). bilateral vls were found in 3 avl patients (23.1%) and in 6 aofvd patients (100%), being significantly associated with the adult - onset disease (p = 0.013). regular and centered lesions were associated with adult - onset type (p = 0.004 and p = 0.016, resp.), whereas irregular and eccentric lesions were more frequent in acquired disease (figures 1 and 2). vitelliform lesions observed in the fundoscopic examination were topographically correspondent to hyperreflective material localized between rpe and ellipsoid zone or elm in sd - oct (figures 1 and 2). a disrupted ellipsoid zone was statistically associated with adult - onset type of disease both at first and at last clinical evaluation (p = 0.020 and p = 0.049). in acquired form, either the integrity or the absence of this line was more common than a disruption. there was no relation between the integrity of ellipsoid zone and the initial or final va (p = 0.131 and p = 0.384, resp.). initial median lh was 142.5 m in avl patients and 118.5 m in aofvd cases. this intergroup difference did not achieve statistical significance (p = 0.452). at the last follow - up visit, however, in avl group, when comparing the initial and final measurement of lh, there was significative shrinkage (142.5 versus 125.5, p = 0.009), which was not the case in aofvd group (118.5 versus 189, p = 0.348). greatest linear dimension of vl was also similar when we compare the two groups, at both the initial and the last follow - up visit (p = 0.336 and p = 0.213). once again, in the avl group there was shrinkage, with initial and final measurements being statistically different (864 versus 761.5, p = 0.001), while in aofvd group growing was not significant (736 versus 1355, p = 0.188). we were not able to measure vl of 10 eyes in the final observation : 4 patients lost follow - up (6 eyes), 1 developed cnv (1 eye), 2 evolved to vitelli - disruptive - like stage of the disease (2 eyes), and 1 aofvd case presented an atrophic - like stage of the disease in one eye. vitelliform lesions were hyperautofluorescent in faf exams (figures 1 and 2). in some cases with pseudohypopyon, faf revealed a hypoautofluorescent superior half and a hyperautofluorescent bottom half. some cases evolved to vitelli - disruptive stage and one aofvd case presented the atrophic stage of the disease. in either case, residual macular lesions were hypoautofluorescent, revealing damage to the rpe. in each patient, fa showed early hypofluorescence of the yellowish subretinal vl (figure 1), occasionally with a halo of hyperfluorescence (figure 2). a progressive late staining of vl was observed during the angiogram (figures 1 and 2). concerning avl group, early amd was the most frequent concurrent disease, being found in 7 cases. we found a correlation between loss of va and increasing gld (p = 0.034) and a tendency to an increase in thickness (p = 0.058). this study exposed both similarities and differences between avl and aofvd patients in a multimodal assessment. bilateral vls with regular shape, central location, disrupted ellipsoid zone, and the absence of other fundoscopic findings were significantly associated with aofvd. on the other hand, va, lh, and gld did not appear to be distinctive features between these two different clinical entities. regard, sd - oct was determinant to localize vitelliform material in the subretinal space, between rpe and ellipsoid zone [3, 8 ]. fundus autofluorescence also proved to be useful in the controversial theme regarding the composition of vitelliform material, since typical hyperautofluorescence of vl was consistent with previous histopathological findings of outer segment material, melanin granules, and fluorescein angiography is a keystone in the diagnosis of retinal disease, and it has been fundamental to identify several distinct entities associated with vl, particularly in avl patients. it is well known that all vls share a yellowish appearance in fundoscopic evaluation, a subretinal location and homogeneous hyperreflectivity in sd - oct, hyperautofluorescence in faf, and a similar behavior during fa [2, 4, 6, 9, 10 ]. theoretically, we can distinguish these patients by the age of diagnosis, familial history, and the presence or absence of associated diseases. however, in clinical practice, this distinction is often difficult since we frequently consult patients for the first time in their sixth to eighth decade, with simultaneous retinal diseases and unknown familial ophthalmological history. in fact, the age of diagnosis in our series was not significantly different between the avl and aofvd group. bilateral disease, although not exclusively, was associated with aofvd and unilateral disease was present only in the avl group. the association found between avl and simultaneous retinal diseases, namely, early amd, was previously reported by several authors and expected according to the diagnostic criteria used for the two groups [2, 6, 9, 11, 12 ]. we found no statistical difference in va, lh, and gld between groups, both at initial and at final observation. however, during the follow - up, we recorded a significant decrease in median lh and gld in the avl group. on the other hand, aofvd lesions became larger and thicker at an apparent larger scale but failed to demonstrate a statistically significant change. this finding could be explained by the different etiologies of these two clinical entities. in avl patients, vls are secondary to an underlying retinal disease and could tend to evolve earlier to atrophy. contrarily, in aofvd patients vls occur as a result of a genetic predisposition, becoming atrophic only at later stages. further studies are necessary to validate this different clinical course between the two entities, namely, the earlier progression to atrophy in avl patients. nevertheless, one must add that pseudohypopyon was observed in 31% of avl patients, a finding considered to be rare in these patients. a recent report by gonalves. also presented a case of avl in a patient with cuticular drusen diagnosed in pseudohypopyon stage. this patient evolved to a stage similar to vitelli - disruptive stage described in best disease, maintaining a good va, probably due to a preserved ellipsoid zone [1, 2 ]. in our series, we did not find an association between va and the integrity of ellipsoid zone. contrasting with previously published series, we found a statistically significant correlation between loss of va and increasing gld. the main distinctive features we found between avl and aofvd groups in multimodal imaging were morphological, namely, the shape of vls and their topographic relationship with the fovea. there was a strong association of centrally located and regular ovoid vl in aofvd patients, as opposed to irregular shaped and eccentrically located vl in avl cases. in 1974, gass first described retinal findings in aofvd as round or oval, yellow, subretinal lesions in the foveal area of each eye. on the contrary, later studies reported significant variability in size and shape of vl both in aofvd and in avl patients [1, 2, 5, 7, 15 ]. the size of our sample could partially explain the statistical significance of our morphological findings. however, since acquired lesions develop from previous retinal diseases, this could define their variable location and shape, leading us to believe that in clinical practice more vls will be eccentric and irregular in avl patients when compared to aofvd. the sample size was also small, limiting our ability to detect further statistic correlations or differences. we included both eyes of some patients in the analysis, which may influence the results, though it is partially controlled by the statistical methods used. we measured vl with the caliper function of sd - oct, which, despite being used in other studies, could introduce some variability in lh and gld values. when consulting a patient presenting a vl with unknown age of onset, familial history, or previous retinal diseases, some aspects of multimodal imaging assessment may lead the ophthalmologist to a correct diagnosis of either avl or aofvd. these features may comprise laterality, shape, vl location in relation to fovea, and its size evolution. | purpose. to characterize vitelliform lesions (vls) in adult - onset foveomacular vitelliform dystrophy (aofvd) and acquired vitelliform (avl) patients using multimodal image analysis. methods. retrospective study of twenty - eight eyes from nineteen patients diagnosed with avl or aofvd. they were evaluated by color fundus photographs, fundus autofluorescence (faf), fluorescein angiography (fa), and spectral - domain optical coherence tomography (sd - oct). results. bilateral vls were associated with aofvd (p = 0.013). regular and centered vls were associated with aofvd (p = 0.004 and p = 0.016), whereas irregular and noncentered lesions were more frequent in avl patients. visual acuity, greatest linear dimension (gld), lesion height (lh), and pseudohypopyon were similar between groups. whereas median lh and gld in avl group diminished significantly during follow - up (p = 0.009 and p = 0.001), aofvd lesions tended to become larger and thicker. conclusions. when consulting a patient presenting a vl with unknown age of onset, familial history, or previous retinal diseases, some aspects of multimodal imaging assessment may lead the ophthalmologist to a correct diagnosis. |
indoor concentrations of chemicals and resulting human exposures often substantially exceed corresponding outdoor concentrations, mainly because there are significant indoor emission sources and with much lower dilution volumes. for example, typical concentrations measured for tetrachloroethylene and formaldehyde in the ambient environment are smaller than 9 (1) and 24.6 g / m(2), respectively, whereas they are several orders of magnitude higher in many industrial or household settings (2,3). moreover, people spend most of their time indoors, which for industrial countries amounts to more than 20 h a day on average when considering both time spent at home and at the workplace or school (4). both aspects often give rise to indoor emission intakes of up to several orders of magnitude higher than outdoor emission intakes (46). nevertheless, health effects from indoor exposure are generally neglected in life cycle assessment (lca). such an omission is an important shortcoming, as it may result in product or process optimizations at the expense of workers or consumers health. recently, there have been significant efforts to integrate indoor exposure models within environmental models commonly applied to lca. for instance, meijer. (7,8) developed a model for the assessment of household exposure to chemicals and radiation emitted to indoor air. (9) used bulk - mixing models for occupational exposure in conjunction with multimedia models for the assessment of cumulative chemical exposure from ambient and indoor environments. both studies illustrate that indoor exposure models are compatible with environmental models used in lca. moreover, they reveal the significance of health effects associated with occupational and household exposure in comparison to the total human - toxicity potential from all pathways. to capture potentially relevant effects to human health, indoor exposure to pollutants should be considered within lca. this paper summarizes the work of an international expert group on the integration of indoor and outdoor exposure in lca, within the unep / setac life cycle initiative (http://lcinitiative.unep.fr), which is taking up recommendations and conclusion toward the enhancement of the current lca framework. the goal of this paper is to develop a general methodological framework that allows for the assessment of indoor emissions and human exposure in combination with commonly used outdoor fate and exposure models in lca. to achieve this objective, (a) we review and evaluate existing indoor exposure models for households and occupational settings concerning their use in lca, (b) we use these models to provide a methodological framework for the routine assessment of indoor exposure to chemicals within lca studies, (c) we gather a sample set of model parameter values and illustrate the application of the methodological framework, and (d) compare examples of human intake fractions of indoor and outdoor exposure for a number of chemicals. intake fractions are defined here as the mass of pollutant inhaled by humans per mass unit of chemical emitted (10). in the field of indoor air quality for both residential and occupational environments, a large number of models has been developed for assessing exposure to indoor pollutants, ranging from semiquantitative models (1115) to physical - process models (1618). in this paper, we address only the latter type of models, as these are also commonly used for outdoor fate and exposure models, to allow for quantitatively comparing assessments of exposure. a model review is performed to enable the lca practitioner to make an informed choice among suitable model alternatives for use in lca, depending on the level of detail needed. the first column of table 1 displays an overview of the model types available for indoor exposure assessments. these models range from simple bulk mixing models (zero - ventilation model, one - box model, two - zone model) to diffusion - based models (eddy diffusion model, gaussian plume dispersion model) and complex computational fluid dynamics models. a brief description of the model principles is presented in column 2 of table 1, together with some selected references for in - depth explanations in column 3 (a detailed overview is also presented in (16,19)). all models not marked in bold were considered to be compatible with conventional exposure models for the environment used in lca (see last column for explanation). variables : pvap is the saturation pressure [pa ], patm the ambient atmospheric pressure [pa ], c the indoor concentration [ppm or mg / m ], q the ventilation flow [m / s ], g the emission rate [mg / s ], m the mixing factor, cnf, ff the uniform concentration of the near and far field [mg / m ], the airflow rate between near and far field or between different rooms [m / s ], d the eddy diffusivity [m / s ], and r the distance from the emission source [m ]. further references for model applications and comparisons to measurements are provided in tables s1 and s2 in the supporting information. model performance was evaluated qualitatively, with regard to the capabilities and limitations of each alternative and its appropriateness in the context of lca. the following criteria were set in assessing the models : accuracy and precision (reliability of the model), transparency (the ability of the model to communicate emissions / exposure relationships), data requirements, and ease of use. in addition, a literature survey was performed on case studies, mainly from occupational hygiene, that compared model calculations to measured concentrations (see the supporting information). the purpose of this screening assessment was to narrow down the choice of models to those that are compatible with the environmental fate and exposure models used in lca. the screening assessment was performed by a team consisting of lca and risk assessment experts as well as occupational hygienists (unep / setac working group on the integration of indoor exposure assessment within lca, with the authors of this paper and the persons mentioned in the acknowledgment as members). equation 1a shows the calculation of intake fractions resulting from indoor pollutant inhalation using the example of the one - box model without eq 1a and with eq 1b correction for incomplete mixing, assuming a constant exposure time. where if is the population intake fraction of a chemical (), ix is the daily intake of a chemical by an individual (kg / day), ir is the daily inhalation rate of air of an individual (m / day), n is the number of people exposed, cx is the chemical concentration in air (kg / m), gx is the emission rate of chemical x (kg / day), q is the ventilation rate in the exposure area (m / day), v is the volume of the exposure area (m), kex is the air exchange rate of the volume in the exposure area, and m is the mixing factor. with regard to the models that were considered appropriate to calculate human intake fractions, a decision tree (figure 1a) was elaborated based on the model assessment, providing rough guidance for model choice in specific situations. all dashed lines lead to the models that allow for the assessment of near - field and far - field exposure (for model characteristics see table 1) ; (b) nesting the indoor model into the environmental fate and exposure model usetox (adapted from ref (21)). the great variability between results of current toxicity models often impedes a wide consideration of toxicity impacts in comparative studies. to provide an agreed, consistent, and stable toxicity assessment method for comparative environmental assessments, the usetox model (20,21) usetox is based on a scientific consensus and thus parsimoniously built from only the most influential model elements identified via an extensive model comparison (20,21). it is currently being reviewed for endorsement by unep / setac as the globally recommended model and source to address human - toxicity impacts within lca. as shown in figure 1b, the model covers an urban and continental scale that is nested into a global scale accounting for impacts outside the continental scale. the human intake fractions include exposure through inhalation of air, and ingestion of drinking water, leaf crops, root crops, meat, milk, and fish from freshwater and marine aquatic compartments, for the total human population. the indoor exposure model was nested into the fate, exposure, and effects model usetox (21), in order to quantify indoor and outdoor exposure on the same methodological basis. the combined usetox model with an indoor compartment embedded will be made publically available in 2009. value ranges of parameters needed to run the models and calculate intake fractions were retrieved from a literature review and personal communications with authorities and building insurance offices (see the supporting information). intake fractions from both indoor and outdoor exposure are part of the characterization factor (cf), within the same impact category human toxic effects, following the usetox methodology (20,21) (eq 2) where if is the intake fraction [kgintake / kgemitted ] and ef the effect factor [cases/ kgintake ]. in the life cycle impact assessment phase, the characterization factors are multiplied to the emissions reported in the inventory phase to determine an overall impact score for potential human - toxic effects. as shown in table 1, five of the existing indoor models (one - box model, one - box model with mixing factor, two - zone model, multibox model, and eddy - diffusion model) were considered compatible to the general principle of environmental exposure models, used for the assessment of human health effects in lca. therefore, these models could be connected to the environmental exposure models, in order to assess human - health effects from indoor as well as outdoor exposure, using the same methodological basis. bulk - mixing models are particularly easy to integrate in the current lca framework, because the models are conceptually very similar to the existing environmental models. although not yet applied within the context of lca, the steady - state version of this model could easily be integrated with environmental multimedia models. the eddy - diffusion model has the advantage that it can model spatial concentration profiles in indoor settings more accurately than bulk - mixing models. however, within the context of lca it is open to discussion whether such level of detail is necessary or even possible. for instance, even if the information on the eddy - diffusitivity parameter is available (which is often not the case), the distance of all humans exposed to the emission source must be known to calculate intake fractions. the zero - ventilation model assumes complete saturation without considering any pollutant sinks, such as ventilation. therefore, this model may be applicable for assessing worst - case scenarios, for instance within a risk assessment, but not within the framework of lca. on the other hand, the gaussian plume and computational fluid dynamics models require too much input information, e.g. the specific airflows within the room (table 1), which is not available within standard lca studies. the one - box model can be used as default model, as it matches the model principles and the level of detail of exposure models for the environment (figure 1b). thus, the focus of the current paper will be put on the one - box model. however, in some situations, a higher level of detail is necessary, and a more sophisticated model may be needed to refine the analysis and model spatial concentration patterns. in order to outline in which exposure situation which model is most appropriate, a decision tree was set up by the expert team (figure 1a). for instance, if there are multiple sources in the room or if the ventilation conditions are very good, the one - box model is often a valid choice. on the contrary, if the room volume is big and if the ventilation conditions are poor, other models that allow for the assessment of near - field exposure are more appropriate. the models shown in figure 1 require information on parameter values with varying extends (see eq 1a and equations in table 1). table 2 displays the results of a literature review concerning value ranges for a selection of exposure parameters. to avoid double counting, the consideration of exposure time in all compartments is necessary when assessing exposure in indoor and outdoor settings. the number of people exposed, n, and the building volume vary throughout and within industrial sectors (see the supporting information). figure 2 shows a comparison of outdoor and indoor intake fractions for households and for several industrial settings and chemicals. the intake fraction is independent from the amount emitted (as opposed to, for example, concentration or dose) and expresses the marginal increase in exposure due to an increase in emission. it can be seen that the amount taken in by humans per kilogram of emission is several orders of magnitude higher for indoor emissions at workplaces and in residential settings than for outdoor emissions. therefore, if, for example, a substance is emitted in an indoor setting and is eventually transferred to outdoor air by ventilation (neglecting for degradation), the major part of the impact is likely to occur in the indoor setting. examples for intake fractions found in indoor industrial and residential settings and in the ambient environment. in contrast to outdoor intake fractions, intake fractions related to indoor environments do not depend on the chemical, as ventilation was assumed to be the primary removal pathway, neglecting substance - specific degradation and adsorption (see discussion). the assessment of indoor exposure needs to be facilitated by including emission factors, intake fractions and human - toxicological effect factors for indoor air sources into existing lca software tools and databases. the present study helps by providing the methodological framework to estimate intake fractions in indoor settings in a structured, transparent and consistent manner. however, indoor emission data also need to be provided in inventory databases, similar to those available for outdoor emissions. this could be achieved by including an indoor air compartment, in addition to the existing air, water, and soil compartments in life cycle inventory databases, e.g., ecoinvent (51). further work is planned to establish a ready - to - use list of relevant emission factors that can be incorporated in the life cycle inventory analysis. in lca, this will often restrict the choice of the model to the one - box model. the one - box model seems to be a good default choice, as the level of detail matches that of environmental models used in lcia. more sophisticated models with indoor spatial differentiation may be used as well, if specific information, for instance on the spatial distribution of sources and people in the room, is available (table 1). however, in this case, the level of detail would deviate from the environmental fate and exposure models commonly used, as the latter do not consider inhomogeneous mixing within the environmental media. setting up a list of recommended values for exposure parameters may be, in general, difficult. in this paper, we tried to provide ranges of parameters for the models that were considered suitable for use in lca (tables 1 and 2). however, these ranges are very broad. especially with regard to the more abstract, but very sensitive parameters, such as the air exchange rates between the conceptual inner and outer boxes in a two - zone model or the eddy - diffusion constant, it is often difficult to find representative values. contaminant dispersion phenomena within the rooms can be influenced by complex interactions between variables such as the room geometry, the direction of the principal air flows, and the presence and movements of occupants (5257). the models identified suitable for use in lca (table 1) do not take into account such detailed information. further, the intake fraction in indoor environments was assumed to not depend on the chemical. this is a valid assumption if removal by ventilation is large in comparison to adsorption to surfaces and degradation (which is often the case in household and occupational settings). however, intake fractions may be reduced significantly by sorption to indoor surfaces (58), especially for strongly sorbing substances in furnished residential homes. for generic lcas, a good approach is to calculate intake fractions for several generic workplace and household environments, which are characterized by air exchange rates, volumes, and numbers of people exposed. the parameter values for the indoor models (i.e., room volumes, air exchange rates, etc.) may vary geographically, e.g., because of climate conditions, cultural aspects, or different ventilation practices. for instance, the number of people per cubic meter working in the chemical industry in countries with cheap labor costs, such as china or india, is probably much higher than in industrial countries with a high degree of automation. therefore, an important requirement for the final implementation of the model within the usetox model is that the user can adapt the parameter values to his or her specific circumstances and that default parameter lists for various workplace settings and geographical regions are provided to facilitate the application. the results assessed with the generic characterization factors based on the one - box model calculations will give only an indication of whether indoor exposure may be important. in such cases, it is advisable to refine the model parameter values or even change the model according to figure 1a. the implementation of various indoor model options into the usetox model, among which the user can choose, will make this recommendation feasible as well for lca practitioners with limited time availability. the use of a model can be circumvented in the case that monitored concentration values and production volumes are available. instead of multiplying the emissions to the intake fractions, as usually done in lca, the amounts taken in by the people exposed would be directly calculated from the monitored concentrations and the number and inhalation rates of people. this approach requires that pollutant concentrations can be directly linked to the functional unit (source appointment), which is possible in some indoor settings. in a later stage, indoor exposure to radioactive gases such as radon radiation in lcia methods such as eco - indicator 99, similarly to the framework shown in this paper. this is especially important for household settings, where radon can be an important factor for the total health damage as a result of indoor exposure. the framework suggested in this paper is the first in putting forward a general procedure for indoor exposure assessment within lca. from a practical point of view this is relevant, as the model results suggest that intake fractions from indoor emissions are often larger than intake fractions from outdoor emissions. this finding is confirmed by previous studies (6,59) showing that indoor chemical concentrations often surpass outdoor concentrations by many orders of magnitude. it could even lead to human toxicity becoming a dominant impact category for certain products such as paints, furniture, or carpets. a routine assessment of indoor exposure in lca will be facilitated by including the indoor model in the usetox model (20,21). similar developments can be anticipated for the field of risk assessment, as european reach legislation also calls for exposure scenarios, including worker or consumer exposure, for example. such integrated assessment will point to the most important exposure pathways and improvement potentials, considering the whole life cycle of chemicals (60). moreover, the past has witnessed several cases in which chemicals were banned for one reason, such as ecological impacts, but got substituted by chemicals with other problems, i.e., occupational health effects (e.g., the market introduction of n - hexane / acetone based brake cleaning products due to air quality rules in california in 1990 (61)). such trade - offs between the various possible effects of chemicals can be revealed when applying integrated models for indoor and outdoor exposure and, ultimately, such problem shifting may be avoided. | neglecting health effects from indoor pollutant emissions and exposure, as currently done in life cycle assessment (lca), may result in product or process optimizations at the expense of workers or consumers health. to close this gap, methods for considering indoor exposure to chemicals are needed to complement the methods for outdoor human exposure assessment already in use. this paper summarizes the work of an international expert group on the integration of human indoor and outdoor exposure in lca, within the unep / setac life cycle initiative. a new methodological framework is proposed for a general procedure to include human - health effects from indoor exposure in lca. exposure models from occupational hygiene and household indoor air quality studies and practices are critically reviewed and recommendations are provided on the appropriateness of various model alternatives in the context of lca. a single - compartment box model is recommended for use as a default in lca, enabling one to screen occupational and household exposures consistent with the existing models to assess outdoor emission in a multimedia environment. an initial set of model parameter values was collected. the comparison between indoor and outdoor human exposure per unit of emission shows that for many pollutants, intake per unit of indoor emission may be several orders of magnitude higher than for outdoor emissions. it is concluded that indoor exposure should be routinely addressed within lca. |
obesity has become recognized as a worldwide health threat and a major public health challenge. there is currently a lack of simple and effective therapies or preventative treatments against obesity, and the mechanisms involved in the onset of diet - induced obesity remain unknown. there is growing evidence that cellular leptin resistance in the hypothalamus is important in maintenance of obesity but is unlikely to have a causative role in the onset of obesity. there is growing evidence that altering the strength or sensitivity to the hedonic attractiveness of food, availability of food, learned preferences, or signaling from the gut may be involved in initiating diet - induced obesity. since its identification in 1994, leptin has attracted much attention as a key central and peripheral signal involved in energy homeostasis [68 ]. global deficiency in leptin or leptin receptor (lepr) results in an increase in appetite, hyperphagia, and morbid obesity in both humans and rodents [911 ]. few cases of obesity have been attributed to leptin deficiency ; rather hyperphagia and obesity are associated with cellular resistance to leptin and the consequent lack of anorexigenic action of leptin. considerable attention has focused on leptin resistance in arcuate neurons of the hypothalamus as a key event in development of hyperphagia and obesity. however, in rodent models of diet - induced obesity, leptin resistance in arcuate neurons does not develop until after food intake, body weight and adiposity increase, calling into question whether leptin resistance in hypothalamic neurons drives the initial hyperphagia and obesity. other populations of neurons important in regulation of food intake express the leptin receptor, including vagal afferent neurons (van) and neurons in the nucleus of the solitary tract, the site of central termination of van. we have shown that within 6 weeks of feeding a high - fat diet in rats, van become leptin - resistant ; this leptin resistance coincides with the development of hyperphagia without any measurable change in leptin signaling in the hypothalamus. leptin is a gut and adipose tissue - derived hormone that regulates a range of biological functions and processes, including energy intake and expenditure, body fat, neuroendocrine systems, autonomic function, and insulin and glucose balance. multiple splice variants of the lepr (lepra - f) have been identified with identical extracellular, transmembrane, and proximal intracellular domains. only leprb, the long isoform containing a 300 amino acid intracellular tail can mediate the physiological effects of leptin. binding of leptin to leprb results in the activation of janus tyrosine kinase 2 and leads to the phosphorylation of signal transducer and activator of transcription 3 (stat3). mice with a neuron - specific disruption of neuronal stat3 are hyperphagic, obese, diabetic, and infertile. van express a plethora of receptors and carry the bulk of the information about the nutritional content of a meal from the gastrointestinal (gi) tract to the brain. van have been implicated in short term control of meal size and duration, but whether inputs from the gut via van play a role in the long term regulation of food intake and body weight is not clear. in the current study, we test the hypothesis that leptin resistance in van is an initiating factor in the development of hyperphagia and obesity. using a nav1.8cre - loxp system we developed a conditional knockout mouse that lacks leptin receptor only in primary afferent neurons. the conditional leptin receptor allele has been used previously to generate liver- and brain - specific ko mice. lox p sites flank either side of the first coding exon of lepr (leprlox), which includes the signal sequence ; thus cre - mediated recombination deletes all splice variants. leprlox mice were bred with mice expressing cre driven by the nav1.8 promoter to generate selective deletion of leptin receptor in primary afferent neurons (figure 1a). cre - negative, lepr, and lepr littermates (wt) were used as controls in all studies. both wt and nav1.8/lepr mice were born at the expected mendelian frequency, survived to adulthood, and were fertile. the average litter size was 6 for both genotypes and ranged from 2 to 13/litter in nav1.8/lepr mice and 113/litter in wt mice. nav1.8 has previously been demonstrated to be exclusively expressed in sensory neurons, and was actively found to be absent from the cortex, cerebellum, and hippocampus in the brain. here we report that lepr expression was unchanged in both hypothalamus and whole brain extracts of wt and nav1.8/lepr mice by real - time quantitative pcr analysis (figure 1b). we confirmed by immunohistochemistry that there was no ectopic cre recombinase in discreet neurons of the arcuate nucleus or nucleus of the solitary tract (sup figure 1). in addition we demonstrated that other organs that do not express nav1.8, including liver, spleen, muscle, white adipose, heart, lung, and kidney had similar lepr expression in both nav1.8/lepr mice or wt mice (sup figure 1). we did observe a significant decrease (93%) in lepr mrna in neurons of the nodose ganglia in nav1.8/lepr mice compared with wt mice (figure 1c). in contrast, there was no significant decrease in lepr expression in populations of other primary afferent neurons that express nav1.8 including the trigeminal ganglia (tg), dorsal root ganglia (drg), spinal cord, and superior cervical ganglia (scg) in nav1.8/lepr mice compared with wt mice. we suggest that this was at least in part due to the overlap between lepr and nav1.8 expression within subsets of sensory neurons. approximately 70% of van express leptin receptor (lepr) and a similar percentage of these neurons express nav1.8. the large reduction in lepr expression in nodose ganglia suggests that there is significant overlap between lepr and nav1.8 expression in these neurons. around 70% of drg neurons are positive for nav1.8, but only a small population of drg neurons express lepr. there was a small decrease in lepr expression in drg of nav1.8/lepr mice that did not reach statistical significance, suggesting that spinal afferent neurons expressing nav1.8 are a different subpopulation to those expressing lepr. although tg neurons express high levels of lepr protein, very few are nav1.8 positive ; we found no difference in lepr expression in the tg. there are currently no reports in the literature demonstrating that scg neurons express lepr ; although it has been proposed that cultured scg neurons may be responsive to leptin, suggesting that at least a proportion of these neurons may express the lepr gene. here we report that scg neurons do express lepr, although in lower concentrations than in ng, drg, and tg (figure 1c) ; no change in lepr expression was found, presumably as a result of low nav1.8 expression in these neurons. to demonstrate that the lack of lepr mrna results in loss of lepr protein we stained nodose ganglia with a lepr antibody. wt mice express lepr on the plasma membrane, while nav1.8/lepr mice little to no lepr staining (figure 1f). to confirm that the absence of lepr results in the absence of functional responsiveness to leptin in van, we measured the ability of an intraperitoneal administration of leptin to induce nuclear translocation of phosphorylated stat3 (pstat3), a known mediator of leptin signaling downstream of leprb (figure 1d, e). in wt mice, intraperitoneal leptin (80 g / kg) increased nuclear pstat3 in van compared with saline (27.6 3.3 vs. 8.4 1.8% ; p 0.05). the 93% reduction in lepr expression results in an 80% reduction in pstat3 nuclear expression in van. these data confirm that the lepr deletion was specific to vagal afferent neurons and we conclude that any phenotypic alteration observed in nav1.8/lepr mice is due to the loss of lepr in van. to determine the importance of endogenous leptin signaling in van on the regulation of energy homeostasis, we monitored the body weight of wt and nav1.8/lepr mice. deletion of lepr in van of chow - fed mice led to a small but significant increase in body weight at 10 weeks (p 0.05) ; however, adiposity increased 40% in nav1.8/lepr mice compared to wt mice (figures 2b and 3b). the weight of subcutaneous, retroperitoneal, mesenteric, and epididymal fat pads were increased in nav1.8/lepr mice compared with wt mice (figure 2d) as a result of increased adipocyte cell size (figure 2f i). when the fat mass was adjusted for body weight we determined that there was a redistribution of fat pad mass to mesenteric and retroperitoneal depots in the nav1.8/lepr mice compared with wt mice (figure 2e). this is consistent with previous studies in which disrupting vagal afferent signaling altered visceral fat depots [3739 ]. however, the mechanism remains unclear since there appears to be little parasympathetic supply to white adipose tissue. interestingly, despite the very significant increase in adiposity, circulating plasma leptin concentrations were indistinguishable between genotypes at 6 or at 12 weeks (figure 2c). dissociation between circulating leptin and adiposity has been reported in female wistar rats fed a moderately high - fat diet and was suggested to contribute to weight gain. it is possible that the lack of feedback from the adipose tissue in the nav1.8/lepr mice contributes to the weight gain although this needs further investigation. to determine the mechanism by which lepr knockout in van increases body weight, wt and nav1.8/lepr mice (12 weeks old, n = 8) were randomly selected to be placed in metabolic cages to measure food intake, meal patterns, indirect calorimetry, and locomotor activity ; based on their body weight these mice were representative of the whole population. whole body composition analysis revealed that nav1.8/lepr mice weighed significantly more than wt mice as a result of increased fat mass, with no change in lean mass (figure 3a d). energy expenditure and meal patterns were evaluated using a comprehensive lab animal monitoring system in which animals were fed powdered labdiet 5058. there were no changes in energy expenditure (figure 3e g), activity, or dietary fuel oxidation between the groups (figure 3h i). there was a modest increase in energy expenditure, activity (data not shown) and respiratory quotient in all animals during the dark cycle, reflecting their nocturnal behavior. overall cumulative daily food intake trended to increase in nav1.8/lepr mice but this did not reach statistical significance (p = 0.07) ; however, nav1.8/lepr mice ate significantly more than wt during the dark cycle (+ 22% ; figure 3j l ; p < 0.05). the increase in food intake occurred predominantly in a 3 h window at the onset of the dark cycle (figure 3l). meal patterns over 24 h and in the light phase were not significantly different between groups, but we found prolonged meal duration and increased meal size in the dark phase, especially in the first few hours of the dark phase. meal pattern analysis revealed that nav1.8/lepr mice ate longer meals (+ 26%) compared to wt mice in the early and total dark cycle (p < 0.05 ; figure 4c, d). the increased meal duration in the dark, but not the light phase, led to a trend towards increased 24 h meal duration (p = 0.06 ; figure 4a, b). the quantity of food ingested in each meal during the total dark phase trended to increase (+ 16%) in nav1.8/lepr mice compared with wt (p = 0.05 ; figure 4 g), and was significant in the early part of the dark phase (p < 0.05 ; figure 4h). nav1.8/lepr mice had a smaller satiety ratio than wt mice in the early dark phase (p < 0.05 ; figure 4l). the satiety ratio correlates meal size with the time to the subsequent meal ; the smaller satiety ratio indicates that nav1.8/lepr mice are less satiated by a meal than wt mice. no difference in meal number, intermeal interval, rate of ingestion thus, deletion of the leptin receptor in van produces a significant effect on dark phase calorie consumption and meal patterns independent of energy expenditure. these findings indicate that disruption of leptin signaling in van ablates a physiological satiety mechanism that controls meal termination. notably, this mechanism primarily operates during the first hours of nocturnal feeding when rodents eat the first and largest of their daily meals. intestinal feedback inhibition of food intake is mediated by cck - induced activation of the vagal afferent pathway and comprises of a decrease in meal size and duration. we hypothesized that a reduced sensitivity of van to cck may be responsible for increasing meal size and duration in the nav1.8/lepr mice. leptin and cck synergism is well established [4345 ] although the site of synergism remains unclear. cck predominantly mediates its effects on food intake by activating cck1 receptors on vagal afferent terminals innervating the gut. there is evidence that leptin is required for cck to signal in van : in cultured van, leptin increases cck signaling and leptin resistance in van reduces cck - induced satiation. to test whether the absence of leptin signaling in van could inhibit cck - induced satiation, we compared feeding responses to peripheral injections of exogenous cck in wt and nav1.8/lepr mice. cck (0.3 g / kg or 3 g / kg, ip) inhibited 2 h food intake in wt mice (figure 5a), but failed to have any effect in nav1.8/lepr mice (figure 5b). a higher dose of cck (30 g / kg, ip) significantly reduced food intake in both wt and nav1.8/lepr mice (figure 5a, b). thus, the absence of leptin signaling in van significantly reduces the ability of cck to inhibit food intake. this deficit in cck - induced signaling in the nav1.8/lepr mice reduces vagal afferent signaling of intestinal feedback inhibition of food intake, leading to hyperphagia, increase in body weight and a redistribution of fat to visceral depots. in a previous study, leptin (120 g / kg ; ip) was demonstrated to significantly reduce food intake over 7 h in fasted c57bl/6j mice compared with saline. we confirmed previous reports that peripheral leptin significantly reduces food intake after 7 h in wt mice (figure 5c, d). in the nav1.8/lepr mice, leptin failed to significantly reduce food intake compared to saline (figure 5c, d). at least at this dose, peripheral administration of leptin appears to mediate feeding behavior predominantly via a vagal afferent pathway. it should be noted that the trend in reduced rate of food intake observed over the course of the 7 h in the nav1.8/lepr mice may be a result of exogenous leptin crossing the bbb and acting on leptin receptors in the nts or hypothalamus, since lepr expression remains intact in the cns. nerve damage or inflammation alters gene expression, changing sensitivity and excitability of van [4648 ]. more recently, nutrient availability in the gut has been association with changes in expression of gpcrs and neuropeptide transmitters in van of rodents and humans. the neurochemical phenotype of van reversibly switches from an anorectic phenotype post - prandially to an orexigenic phenotype under fasting conditions. since cck is a predominant mediator of this switch in phenotype and the absence of the leptin receptor expressed by van markedly compromises cck - induced activation of van, we hypothesized that changes in van phenotype between fasted and fed conditions would be attenuated or abolished in the nav1.8/lepr mice. expression of the neuropeptide transmitter cocaine and amphetamine regulated transcript (cart ; figure 6a) and peptide yy receptor type 2 (y2r ; figure 6c) were high, while the transmitter melanin concentrating hormone (mch ; figure 6b) and cannabinoid receptor type 1 (cb1r ; figure 6d) expression were low in 2 h refed compared with fasted wt mice, as previously described in lean rats [5052 ]. conversely, food withdrawal decreased y2r and cart expression and increased cb1r and mch expression. y2r and cart expression was constitutively low in van of ko mice and the expression of the cb1r and mch in van was high. thus, the inability of van to respond to leptin results in a loss of cck - induced neuronal plasticity. to determine whether leptin resistance in van is necessary for the onset of obesity we chronically fed nav1.8/lepr and wt mice with high fat diet. at 9 weeks, when all the animals still weighed the same (figure 7a), mice were either kept on a chow diet or switched to a 45% high fat diet. as expected wt mice gained more weight on a high fat diet than on a chow diet (figure 7b). after 21 weeks on chow nav1.8/lepr mice weigh more than wt mice (figure 7b, c). crucially, nav1.8/lepr mice failed to gain additional weight despite chronic ingestion of an hfd for 12 weeks (figure 7c), and weighed less than high fat fed wt mice (figure 7b, c). we demonstrate that leptin resistance in van is sufficient to promote weight gain in the absence of a high fat diet, and that consumption of a high fat diet fails to increase weight gain in nav1.8/lepr mice. we infer from this data that other factors are involved in high fat diet - induced weight gain and that they are downstream of leptin resistance onset in van. we postulate that ko mice acquire compensatory mechanisms to deal with the loss of lepr in van during development which prevents them from gaining further weight on a high fat diet. adiposity comparisons in 21-week - old mice fed their respective diets for 12 weeks, revealed that nav1.8/lepr mice gained significantly less fat than wt mice when fed a high fat diet (figure 7d). as expected adiposity was significantly increased in wt mice fed a high fat diet compared with chow fed wt mice, however, despite a large trend there was no statistical difference in adiposity between nav1.8/lepr mice fed chow or a high fat diet (figure 7d). when studied at the individual fat pad level, we observed significant increases in epididymal and retroperitoneal fat pads of high fat fed nav1.8/lepr mice compared with chow fed nav1.8/lepr mice (figure 7e). considerable evidence has accumulated to suggest that the inability of leptin receptor - bearing neurons to respond to leptin plays a pivotal role in the development and/or persistence of an obese phenotype. we have developed and utilized a powerful new tool which allows the first targeted approach to determine the functional role of specific proteins in gut - brain signaling. using the cre - lox method, we conclusively demonstrate that knocking out lepr in vagal afferent neurons is sufficient and necessary to increase food intake, weight gain and adiposity. there is evidence that lepra, leprb and lepre splice variants are expressed in vagal afferent neurons. in this study we have deleted all isoforms of lepr ; however, based on the fact that leprb is currently the only isotype found to be involved in the control of food intake, and that leprb signaling (i.e. stat3 activation) is severely blunted in nav1.8/lepr mice, our data suggests that deletion of leprb is responsible for the phenotype of the nav1.8/lepr mice. the data also show that the lack of lepr expression in vagal afferent neurons leads to hyperphagia via a mechanism involving the reduction in sensitivity to gut hormones. taken together with our previous findings that leptin resistance in vagal afferent neurons develops early in diet - induced obesity and coincides with hyperphagia, these findings demonstrate that the leptin receptor signaling in van mediates the hyperphagic response to chronic ingestion of a high fat diet. the finding that van are important in the pathophysiology of diet - induced obesity by initiating overconsumption of food is particularly significant given that the vagal afferent pathway has largely been discounted as a putative mechanism for the onset of obesity and has only been thought to be involved in short term, meal - to - meal regulation of food intake. van are well known to carry the bulk of the information about the nutritional content of a meal from the gastrointestinal tract to the brain, and lead to meal termination. although we did not specifically study lepr expression in van innervating the gastrointestinal tract, nav1.8-cre mice have been demonstrated to have extensive vagal innervation of the gastrointestinal tract. furthermore, retrograde tracing experiments have established that van innervating the gut are located in the caudal portion and express cck1 receptor. we report here that lepr immunostaining is lost in the caudal region of the nodose ganglia and that cck signaling is blunted in the nav1.8/lepr mice. therefore, this is the first conclusive evidence that chronic disruption of gut - brain signaling via a vagal pathway reduces satiation over multiple meals leading to hyperphagia and obesity. we propose that the absence of weight gain in nav1.8/lepr mice fed a high fat diet suggests that leptin resistance in van is a necessary initiating step in the development of diet - induced obesity. we demonstrate that ingestion of a high fat diet leads to weight gain in wt mice, and that deletion of lepr in van leads to weight gain, but that adding high fat diet to nav1.8/lepr mice does not cause additional weight gain. since we know that leptin resistance in van develops as a result of chronic ingestion of a high fat diet, we conclude that leptin resistance in van is a necessary initial step in diet - induced obesity. we suggest that the nav1.8/lepr mice have acquired compensatory mechanisms to deal with the loss of leptin receptor in van during development which prevents them from gaining further weight on a high fat diet. wt mice fed a high fat diet develop leptin resistance which initiates weight gain, and secondary mechanisms promote further weight gain. the compensatory mechanisms acquired by the nav1.8/lepr mice prevent the secondary mechanisms from promoting further weight gain in response to a high fat diet. we suggest that preventing acquisition of compensatory mechanisms by knocking out lepr in van during adulthood would result in more pronounced weight gain on a chow diet and increased susceptible to additional weight gain in response to a high fat diet. the data show that the hyperphagia observed in the conditional knockout mice occurs as a result of reduced meal termination rather than meal initiation. the leptin receptor knockout in van increases intake in the early dark phase when the animals consume the majority of food. we observed prolonged meals (reduced meal termination) with no increase in meal numbers or a reduction in the intermeal interval (meal initiation). this suggests that leptin signaling in van is involved in meal termination, and that knocking out leptin receptor reduces satiation. the data is consistent with previous findings that van are involved in meal termination and that gastrointestinal hormones released post - prandially (i.e. cck, pyy, glp-1) activate van to mediate satiation. here we report that leptin signaling in van is required for cck - induced satiation. we have previously demonstrated that during fasting, when there is little nutrient content in the proximal gut, the neurochemical phenotype of van is to express orexigenic peptides (e.g. mch) and receptors (e.g. cb1r). post - prandial release of cck induces a switch in the phenotype of van shown by an increase in expression of anorectic peptide (e.g. cart) and receptors (e.g. y2r). the reduced sensitivity of van to cck in mice lacking leptin receptor in van results in loss of this plasticity ; expression of the peptide transmitters, cart and mch, and expression of the receptors, y2r and cb1r, fail to change in response to feeding or fasting in the nav1.8/lepr mice. there is some evidence that at least cart is released from cultured van and that cart can prolong satiation in vivo. furthermore, knocking down cart expression in van of freely behaving rats has been shown to increase food intake in short term studies. together these data suggest that cart may act as a neuropeptide transmitter involved in inhibiting food intake. thus, the reduction in cart expression in van of nav1.8/lepr mice may account for the reduced satiation and consequently hyperphagia. we have developed and utilized a novel tool, namely a mouse with a conditional knockout of the leptin receptor, which allows the first targeted approach to determine the functional role of vagal afferent neurons. using this method we have been able to show for the first time that the vagal afferent pathway influences food intake, adiposity, and body weight over the long term. this result contrasts to surgical and chemical ablation studies in which long term effects on body weight or adiposity have not been reported. total subdiaphragmatic vagotomy ablates both afferent and efferent pathways ; the more selective subdiaphragmatic deafferentation ablates 50% of the efferent fibers, in addition to afferent fibers. perivagal application of capsaicin, thought to cause degeneration of afferent c fibers may also damage efferent neurons. more recently, this cre - lox system does not discriminate between vagal afferent and vagal efferent neurons, and is also expressed in enteric neurons of the intestine, central noradrenergic neurons of the nucleus of the solitary tract, neurons of the area postrema, in most of the rhombencephalon (caudal to r1) at least during development, in a subset of sympathetic neurons, and extensively in neurons of the iiird, ivth, viith, ixth, xth and xith cranial ganglia. thus, the current study is the first to selectively target vagal afferent signaling to determine their role in food intake and body weight. it should be noted that nav1.8 cre targets drg and scg neurons in addition to van ; however, because few drg and scg neurons express lepr, the method provides knock down of leptin receptor specifically in van. it is interesting to note that deleting all the splice variants of lepr using phox2b - cre or nav1.8-cre produces similar phenotypes. both mice lines increase weight gain on a chow diet, increase food intake at 12 weeks, had no change in respiratory quotient, and were satiated in response to a high dose of cck. finally, similarly to our nav1.8/lepr mice the phox2b - cre lepr mice failed to gain weight on an hfd. the similarity in phenotype suggests that the phox2b - cre lepr is more likely a result of lepr deletion from van rather than nts neurons. vagal afferent neurons convey information about the availability of nutrients in the gut to the brain. in the post - prandial period, the vagal afferent pathway plays a pivotal role in regulation of gastrointestinal and pancreatic function and also plays a role in the control of meal size and duration. here we demonstrate that knocking out leptin receptor expression in vagal afferent neurons prevents appropriate post - prandial gut - brain signaling, resulting in increased food intake, weight gain, and adiposity. taken together with our previous data showing that leptin resistance is an early event in high - fat diet induced hyperphagia and weight gain, the current data strongly suggest that defects in leptin signaling in the vagal afferent pathway is a novel mechanism for the initiation of obesity. this novel approach may provide insight into the role of other factors (i.e. hormones, cytokines, microbial products, mechanosensitivity, and nutrients) involved in gut - brain signaling as they relate to food intake, inflammation, microbiota - brain signaling, and neurodegenerative diseases. all experiments were approved by the uc davis institutional animal care and use committee (protocol # 16793) and phs animal welfare assurance to uc davis (# a3433 - 01). cre - negative, lepr, and lepr littermates (wt) were used as controls in all studies. mice were individually housed after weaning under a 12 h light:12 h dark schedule and allowed ad libitum access to food (purina5008) and water unless specified otherwise. all experiments were performed in 12-week - old mice except for the high fat diet experiment in which 9-week - old chow fed mice were either kept on chow or given ad libitum access to high fat diet (45% kcal / g fat ; research diets d12451) for 12 weeks. mice were fasted overnight, or fasted and refed 2 h. tissue and cardiac blood were collected immediately. the quantity of food ingested during the last meal and the time of the last meal before blood collection are important confounding variables since leptin is known to be released from the gut in response to a meal and can account for as much as 20% of circulating leptin. therefore we minimized the variability in circulating leptin levels between animals by collecting blood, and measuring leptin levels, from fasted animals. leptin was measured by elisa according to the manufacturer 's protocol (alpco diagnostics, salem, nh). for qpcr tissue was snap frozen. for immunohistochemistry tissue was fixed in 4% paraformaldehyde and left in 25% sucrose overnight prior to sectioning. flox mice were genotyped according to prior reports. to quantify lepr knockdown in tissue we used real - time pcr. lepr was expression was quantified using validated taqman primer / probe sets (mm01262072_m1) and conditions for the real - time rt - pcr detection of mouse leptin receptor. specific pcr products were confirmed by demonstrating the presence on an agarose gel by electrophoresis. nodose ganglia and superior cervical ganglia samples from 2 mice were pooled together to get sufficient cdna, a total of 12 animals were used for these tissue. as previously described in ref.. primary antibodies raised against cart h-003 - 63 and mch h-070 - 47 (1:200 ; phoenix peptides, buringlame, ca), cb1 sc20754, y2 sc14736, and lepr sc8391 (1:100 ; santa cruz biotechnology inc., dallas, tx), pstat3 9145 (1:100 ; cell signaling technology, beverly, ma), and cre recombinase mms-106p (1:200, covance inc, emeryville, ca) were used. secondary antibodies were used as appropriate and included donkey anti - rabbit immunoglobulin and donkey anti - goat immunoglobulin conjugated with alexa fluor 488 or 555 (1:400 ; molecular probes, eugene, or). percentage of positive pixels and positive neurons were quantified using scion software as previously described. energy expenditure was evaluated in two separate cohorts of age matched (2 days) wt and nav1.8/lepr mice fed powdered chow diet (labdiet 5058) using a comprehensive lab animal monitoring system (clams, columbus instruments, comlubus, oh). data was combined since no statistical difference between runs was identified (data not shown). mice were fed powdered diet for one week and acclimated to monitoring chambers for 2 days prior to 48 h data collection, data is presented as an average of both days. activity levels were determined by counting laser breaks along a x, y, and z axis. the food bout was defined as an episode of uninterrupted feeding of at least 0.02 g, and meal termination was when a bout of feeding was followed by 10 min with no measurable intake. cholecystokinin (octapeptide, sulfated) was purchased from bachem (torrance, ca) and leptin (rat) from sigma aldrich (st. g / kg ; ip), leptin (120 g / kg ; ip), or saline (100 l ; ip) were administered and food was immediately returned to the cage ; food intake was recorded every 20 min over 2 h for cck studies and every hour for 7 h in leptin studies. statistical analysis was performed using prism software (prism 5.0 ; graphpad software, la jolla, ca). unpaired t - test was used to make direct comparisons between wt and nav1.8/lepr mice. in feeding experiments paired t - test was used to compare saline with either cck or leptin. two - way anova with bonferroni post hoc test was used to compare the effects of leptin and saline over time in the wt and nav1.8/lepr mice ; to compare circulating leptin concentrations in wt and nav1.8/lepr mice at 6 and 12 weeks ; to compare the expression of cart, mch, y2, and cb1 expression in nodose ganglia of wt and nav1.8/lepr mice in response to feeding or fasting ; and to compare weight gain over time in chow and high fat fed animals. one way anova with bonferroni post hoc test was used to compare adiposity between nav1.8/lepr mice fed high fat or chow diets, and their starting weights before going on their respective diets. represents p < 0.05 ; represents p < 0.01 ; and p < 0.001. for all experiments in which one way anova was performed different letters denote significant differences between groups. | the vagal afferent pathway senses hormones released from the gut in response to nutritional cues and relays these signals to the brain. we tested the hypothesis that leptin resistance in vagal afferent neurons (van) is responsible for the onset of hyperphagia by developing a novel conditional knockout mouse to delete leptin receptor selectively in sensory neurons (nav1.8/leprfl / fl mice). chow fed nav1.8/leprfl / fl mice weighed significantly more and had increased adiposity compared with wildtype mice. cumulative food intake, meal size, and meal duration in the dark phase were increased in nav1.8/leprfl / fl mice ; energy expenditure was unaltered. reduced satiation in nav1.8/leprfl / fl mice is in part due to reduced sensitivity of van to cck and the subsequent loss of van plasticity. crucially nav1.8/leprl / fl mice did not gain further weight in response to a high fat diet. we conclude that disruption of leptin signaling in van is sufficient and necessary to promote hyperphagia and obesity. |
transcutaneous electrical nerve stimulation (tens) is commonly used in the clinical management of chronic pain, such as for patients with low back pain and knee osteoarthritis1,2,3. typically for chronic pain management, tens is applied to the peripheral site of the lesion, with the stimulation administered for at least 15 min4,5,6. these general stimulation parameters produce a diffuse inhibition of pain, likely to be mediated by descending pain control pathways7, 8. as well, a dose - response relationship of tens on pain control has been reported, with stronger intensities of stimulation having a greater effect on pain relief9. these reported effects of tens on pain control could be of clinical benefit in the management of post - operative pain. yet, tens is seldom used after surgery as the required stimulation period is typically too long and the placement of electrodes on the surgical site difficult in the early period of post - operative rehabilitation. therefore, the purpose of our study was to investigate the effects of short - duration, high - intensity and low - intensity tens on pain thresholds in healthy adults, where pain thresholds were evaluated on the side contralateral to the tens application. twenty - five healthy adults (16 males and 9 females ; mean age, 24.5 4.8 years) were recruited for the study. participants were screened for relevant contraindications, injury or nerve damage to the upper and lower limbs, chronic illness, pregnancy, cardiac pacemaker, sensitivity to the tens electrodes, current use of pain medication, skin conditions or impairments in skin sensation in the region of electrode placement. the methods and procedures for this study were approved by the institutional ethics committee of kochi university. the study was conducted in compliance with the declaration of helsinki, and all participants provided written informed consent. tens was administered using a nihon kohden unit (nihon kohden corporation, japan), using the following parameters of stimulation : 10 hz frequency, 500 s pulse width, 30 s stimulation duration. this stimulation was delivered at two intensity levels, a low intensity (sensory) level and a high intensity (motor) level. for the motor - level, the intensity of the stimulation was set at 1.5 times the motor threshold of the muscle, while for the sensory - level, the intensity was set at the sensory threshold of the common peroneal nerve. the two intensity levels of stimulation were administered on separate days, with participants randomly selecting the order of stimulation by drawing random numbers. the tens stimulation was applied to the peroneal nerve of the dominant leg, through two circular, self - adhesive electrodes (2.5 cm diameter ; vitrode d, nihon kohden, japan), positioned on the head of the fibula and over the popliteal fossa. the effects of tens stimulation on pain thresholds was measured using a pressure pain threshold (ppt) technique previously described in the literature10, 11. to determine the ppt, pressure was applied perpendicularly to the skin, at a rate of 50 kpa / s, using a flat, circular, 1 cm probe tip. a 100-point visual analog scale (vas) was used to quantify the ppt, with anchors at 0, no pain, and 100, worst pain. as the pressure was applied, participants were asked to rate the associated pain on the vas. the vas was considered to be an appropriate measure of the ppt based on findings by kemp. who reported that even pressure stimulation below the pain threshold was consistently perceived and rated significantly higher than 0, no pain, on the vas12. ulrika. reported a vas score between 30 and 40 to be representative of the ppt in healthy adults13. based on these previous studies, the vas score of 30 was set as a reference point, a priori, in our study, and participants were asked to push a button when they perceived the applied pressure to exceed this pain threshold. ppts were assessed at two different sites on the non - dominant limbs : 1) the belly of the deltoid muscle of the upper limb, at a location 5 cm distal to the acromion, along the midline of the muscle, and 2) the tibialis anterior (ta) muscle of the lower limb, at a location 5 cm distal and 3 cm lateral to the tibial tuberosity, along the midline of the tibia. these two ppt sites provided information on the segmental (i.e., ppt of the ta) and non - segmental, or diffuse, (i.e., ppt of the deltoid muscle) effects of tens. ppt was recorded before, during, and 30 min after the application of tens (i.e., before - tens, during - tens, and after - tens, respectively). three ppt measurements, taken 30 s apart, were recorded before and after tens, with the average of the three ppt values used for analysis ; one ppt measurement was made during the 30 s application of tens (fig. three ppt values were obtained, at 30 s intervals, before- and after - tens application, with the average ppt of the three measures used in the analysis. a single ppt measurement was obtained during the 30 s duration of tens application.). the ppt values were highly consistent, with calculated intra - class correlations of 0.94, for the deltoid muscle site, and 0.82, for the ta. three ppt values were obtained, at 30 s intervals, before- and after - tens application, with the average ppt of the three measures used in the analysis. a single ppt measurement was obtained during the 30 s duration of tens application. all participants completed the study protocol and all data points were included in the analysis. the percent change in ppt (% ppt) was calculated (i.e., (during - tens / before - tens) 100) to compare the effects of the sensory - level and motor - level tens. all statistical analyses were performed using spss 21.0 software. the level of significance was set at p < 0.05 for all tests. ppt measures are reported in table 1table 1.pressure pain threshold (ppt) of each time period of tens application (unit : kpa)motor - levelsensory - level(a) ta muscle sitebefore248.8 21.6256.5 22.1during329.4 30.2 272.0 18.6after260.3 22.1244.5 20.7(b) deltoid muscle sitebefore161.4 21.7174.2 19.9during217.4 24.2 200.6 23.9after161.4 18.4171.6 21.2mean se ;, significant difference between during- and before - tens ;, significant difference between during- and after - tens (p<0.05). there were no differences in baseline ppt measures (i.e., before - tens) for the sensory - level and motor - level tens conditions, both at the deltoid and the ta muscle sites. motor - level tens produced significant increases in the ppt at both the deltoid and ta muscle sites (i.e., segmental and diffuse effect), whereas sensory - level tens increased the ppt only at the deltoid muscle site (i.e., diffuse effect only). the ppt at both muscle sites decreased immediately after stimulation for both levels of tens application. mean se ;, significant difference between during- and before - tens ;, significant difference between during- and after - tens (p<0.05) percent changes in the ppt (% ppt) during tens application are reported in table 2table 2.percentage of change in ppt during tens (unit : %) motor - levelsensory - levelta135.8 8.0 112.0 5.9deltoid147.2 10.0 116.3 4.0meanse ;, p<0.05. there were significant differences in % ppt for both the motor- and sensory - level tens at both muscle sites, deltoid (motor level : 147.2% 10.0%, sensory level : 116.3% 4.0%) and ta (motor - level : 135.8% 8.0%, sensory - level : 112.0% 5.9%). in this study, we investigated the immediate effect of two intensity levels of tens application, motor - level and sensory - level, on the modulation of the ppt of the contralateral lower limb (i.e., segmental effect) and upper limb (i.e., diffuse effect). the most important outcome of this study was a finding of the segmental and diffuse effects of motor - level tens, increasing the ppt at both deltoid and ta muscle sites. motor - level tens also produced a larger increase in the ppt (i.e., higher % ppt), compared to the sensory - level tens intensity. these results are in agreement with findings from claydon.14 who reported high - intensity tens stimulation to be of fundamental importance to effective dosage, regardless of the frequency of stimulation. to the best of our knowledge, no other studies have shown the immediate effects of high - intensity, motor - level, tens application on the ppt at contralateral sites. positive findings of the diffuse and segmental effects of motor - level tens on contralateral ppts provides evidence of the feasibility of including tens as one component of an effective pain management strategy in the early post - operative phase of rehabilitation, and laying a foundation for a novel approach to pain management in physical therapy. as motor - level tens produced an increase in the ppt at both segmental and diffuse contralateral sites, it is reasonable to suppose that motor - level tens activated a systemic pain modulating response, rather than only a local response. animal studies have demonstrated a positive correlation between nociception (i.e., application of painful stimuli) and activation of diffuse noxious inhibitory controls (dnic)15. in humans, pud.16 reported both a local (i.e., segmental) and diffuse effect of endogenous analgesia. for their noxious stimuli, pud. asked subjects to immerse themselves into noxious cold water (1 0.5 c) for 30 s. in our study, we demonstrated that a 30 s of motor - level tens was an effective stimulus to trigger a diffuse, pain analgesic, effect. the intensity of the motor - level tens stimulus was a very strong but subjectively still non - painful stimulation (i.e., no participants withdrew from the painful stimulus), as used in previous studies17. we hypothesize that the intensity of the motor - level tens was sufficient to activate a dnic - like mechanism which contributed to the generalized increase in ppts. exercise - induced hypoalgesia (eih), using a variety of exercise modalities, has been reported to be effective in humans. although many studies have used high - intensity exercise (e.g., aerobic exercise or exhaustive isometric exercise) to produce hypoalgesia18,19,20, positive analgesic effects of low intensity exercise (i.e., hand grip or raising leg straight) have also been reported21,22,23. in the present study, although it was not a voluntary contraction, this stimulus may have produced an eih effect. placebo analgesia is activated by expectation and distraction of attention. in our study, it is possible that the tens stimulation distracted attention away from the pressure point. however, analysis of this specific mechanism lies outside the scope of this study. reported that, during voluntary contraction of the quadriceps at an intensity of 20% to 25% of maximal voluntary contraction, ppts increased in both the contralateral quadriceps (i.e., segmental) and infraspinatus (i.e., diffuse) muscles, compared to baseline27. in our study, we demonstrated that these segmental and diffuse analgesic effects can be achieved even with a low intensity muscle contraction. the baseline ppt indicated a higher sensitivity of the deltoid to pressure, compared to the ta muscle. this result was consistent with previous studies28, 29, with the higher sensitivity of the deltoid to effects of tens contributing to the significance of ppt measures at this site only. the limitations of our study must be considered in the interpretation of the outcomes. foremost, participants in our study were healthy adults and, therefore, it is unclear whether similar results would be obtained in patients. as well, we did not measure ppts on the ipsilateral side of stimulation and, therefore, comparisons to measured contralateral effects are not possible. finally, the effects for only two levels of stimulation intensity were evaluated and, therefore, the most effective intensity to achieve an analgesic effect can not be determined. in conclusion, our study provided evidence of the beneficial effects of motor - level tens to produce an immediate and large increase in ppt, compared to sensory - level tens. while additional research is required, outcomes of this study provide a foundation for a novel approach for post - operative pain management in physical therapy. | [purpose ] this study aimed to investigate the differential effects of high - intensity and low - intensity transcutaneous electrical nerve stimulation on the contralateral side on the pain threshold in healthy subjects. [subjects and methods ] twenty - five healthy adults, volunteers received two intensity levels (motor - level, 1.5 times the muscle motor threshold ; sensory - level, sensory threshold of the common peroneal nerve), for 30 s on separate days. pressure pain threshold was recorded on the contralateral tibialis anterior and deltoid muscle before, during, and after stimulation. [results ] motor - level stimulation significantly increased the pressure pain threshold at both muscle sites, while effects of sensory - level stimulation on pressure pain thresholds were significant only at the deltoid site. the percent change in pressure pain thresholds at both sites was significantly higher during motor - level stimulation. [conclusion ] motor - level stimulation, applied unilaterally to one leg, produced immediate contralateral diffuse and segmental analgesic effects. this may be of therapeutic benefit in patients for whom transcutaneous electrical nerve stimulation can not be directly used at the painful site. |
b6 mice carrying myd88 and tlr mutations were backcrossed 10 - 12 times to nod / ltj males and intercrossed to produce ko and heterozygous animals. gf animals were re - derived from nod / ltj females impregnated by nod.myd88ko males and kept gf at taconic farms, germantown, ny, the university of chicago and washington university (st. asf was introduced to gf nod.myd88ko mice by adding cecal contents from donor mice to sterile drinking water. wild - type gf mice were colonized with microbiota from spf nod.myd88ko animals by co - housing gf nod females and newborn progeny with spf myd88.ko females. damage to the islets was scored in a blinded fashion, and graded as follows : 0 no visible infiltration ; i periinsulitis ; ii - insulitis with 50% islet infiltration (supplemental fig. at least 100 islets in each group of 5 to 12 animals were scored. in microbiota transfer experiments 20 sections per pancreas cut at 40m intervals (10 islets / section) were examined and scored (combining grades ii and iii). 610 splenocytes alone, or 210 lymph node cells mixed with 410 irradiated splenocytes from b6.nod-(d17mit21-d17mit10)/ltj (b6.g7) mice, per well of 96-well plates pretreated with anti - ifn- antibodies were incubated overnight with listed peptides. lymphocytes were stained with antibodies against cd4 and cd8 to calculate the frequency of peptide - specific cells per 10 cd4 or cd8 t cells. 16s rrna gene sequences were edited and assembled into consensus sequences using phred and phrap, aided by xplorseq23, and bases with a phrap quality score of < 20 were trimmed. sequences were aligned using the nast online alignment tool (http://greengenes.lbl.gov/cgi-bin/nph-index.cgi), and checked for chimeras using the online greengenes server (http://greengenes.lbl.gov/cgi-bin/nph-bel3_interface.cgi) with a window size of 300 and using the nast - aligned sequences31. b6 mice carrying myd88 and tlr mutations were backcrossed 10 - 12 times to nod / ltj males and intercrossed to produce ko and heterozygous animals. gf animals were re - derived from nod / ltj females impregnated by nod.myd88ko males and kept gf at taconic farms, germantown, ny, the university of chicago and washington university (st. asf was introduced to gf nod.myd88ko mice by adding cecal contents from donor mice to sterile drinking water. wild - type gf mice were colonized with microbiota from spf nod.myd88ko animals by co - housing gf nod females and newborn progeny with spf myd88.ko females. damage to the islets was scored in a blinded fashion, and graded as follows : 0 no visible infiltration ; i periinsulitis ; ii - insulitis with < 50% and iii at least 100 islets in each group of 5 to 12 animals were scored. in microbiota transfer experiments 20 sections per pancreas cut at 40m intervals (10 islets / section) were examined and scored (combining grades ii and iii). 610 splenocytes alone, or 210 lymph node cells mixed with 410 irradiated splenocytes from b6.nod-(d17mit21-d17mit10)/ltj (b6.g7) mice, per well of 96-well plates pretreated with anti - ifn- antibodies were incubated overnight with listed peptides. lymphocytes were stained with antibodies against cd4 and cd8 to calculate the frequency of peptide - specific cells per 10 cd4 or cd8 t cells. 16s rrna gene sequences were edited and assembled into consensus sequences using phred and phrap, aided by xplorseq23, and bases with a phrap quality score of < 20 were trimmed. sequences were aligned using the nast online alignment tool (http://greengenes.lbl.gov/cgi-bin/nph-index.cgi), and checked for chimeras using the online greengenes server (http://greengenes.lbl.gov/cgi-bin/nph-bel3_interface.cgi) with a window size of 300 and using the nast - aligned sequences31. | type 1 diabetes (t1d) is a debilitating autoimmune disease that results from t cell - mediated destruction of insulin - producing cells. its incidence has increased during the past several decades in developed countries 1, 2, suggesting that changes in the environment (including human microbial environment) may influence disease pathogenesis. the incidence of spontaneous t1d in non - obese diabetic (nod) mice can be affected by the microbial environment in the animal housing facility3 or by exposure to microbial stimuli, such as injection with mycobacteria or various microbial products 4,5. here we show that specific - pathogen free (spf) nod mice lacking myd88 protein (an adaptor for multiple innate immune receptors that recognize microbial stimuli) do not develop t1d. the effect is dependent on commensal microbes as germ - free (gf) myd88-negative nod mice develop robust diabetes, whereas colonization of these gf nod.myd88-negative mice with a defined microbial consortium (representing bacterial phyla normally present in human gut) attenuates t1d. we also find that myd88-deficiency changes the composition of the distal gut microbiota, and that exposure to the microbiota of spf nod.myd88-negative donors attenuates t1d in gf nod recipients. together, these findings indicate that interaction of the intestinal microbes with the innate immune system is a critical epigenetic factor modifying t1d predisposition. |
total knee arthroplasty (tka) is a successful treatment applied worldwide for end - stage osteoarthritis. a good postoperative alignment is the foundation of achieving satisfactory treatment results and longevity of the prostheses. in conventional tka, bone cutting is guided by an intramedullary (i m) system on the femoral side and an extramedullary (em) system on the tibial side. in general, i m guide is more precise compared to em guide placed on the same site. lower extremity coronal alignment after conventional tka is often considered to be affected by tibial side more than the femoral side. however, we conducted a retrospective analysis of imaging data of tka finished by the same surgical team, and compared the effect of im - guided resection for femoral side and em - guided resection for tibial side on postoperative coronal alignment. in addition, we examined the rate of satisfactory postoperative alignment for the two different types of cutting guide system. we excluded patients with external joint deformity, including prior trauma or osteotomy history of lower extremity, abnormal condition resulted from other diseases (such as blount s, paget s, tertiary syphilis, or rickets). moreover, patients with limited motion of hip or ankle before surgery or contraction of knee extension 2 weeks after surgery, which would affect the quality full - length leg x - ray imaging, were also excluded. especially, the full - length x - ray should be finished in neutral position without knee flexion or rotation of lower extremity, which indicates that the anterior edge of tibial plateau could cover the posterior edge, and the proximity of tibia and fibula head should be overlapped a little, and the patella is in the middle or slightly lateral position of trochlea groove in the film. a midline incision was made anterior to the knee in all patients, and the joint capsule was entered through an incision medial to the patella. a pneumatic tourniquet inflated at 300320 mmhg (1 mmhg = 0.133 kpa) was used in the standard manner for hemostasis before making the incision and maintained until the end of the surgery. the i m entry point was chosen to be on the whiteside 's line (femoral anteroposterior axis), 1 cm above the terminal end of the medial border of the posterior cruciate ligament. the proximal pin was placed on the connecting lines between the medial edge of the lateral intercondylar spine and the medial third of the tibial tuberosity. as reference, anatomical landmarks were used the medial third of the tibial tuberosity, the proximal tibial spine, and the anterior tibial tendon. we chose an open intercondylar designed prosthesis (genesis ii, smith and nephew, memphis, usa). no patient received patellar replacement, and all surgeries were finished by the same surgical team. the main attending surgeon had a caseload > 200 tkas per year for 10 consecutive years. the coronal mechanical axis alignment and femoral valgus angle were measured on preoperative, full - length radiographs [figure 1 ]. two weeks after surgery, the femoral prosthesis was measured on full - length radiographs. the alignment was defined by the angle formed by the connecting line between the medial and lateral condyle and the perpendicular line to the mechanical axis of the lower limb. likewise, the angle formed by the tibial prosthetic platform and the vertical mechanical axial line of the tibia was measured to define the tibial prosthetic alignment [figure 2 ]. a varus angle was defined as positive (+) and a valgus angle was defined as negative (). preoperative full - length leg x - ray, the femoral mechanical axis was showed as line a, and line b indicated the tibial mechanical axis, the angle between line a and b was the angle of lower extremity mechanical axis. postoperative full - length leg x - ray, the bottom line of the tibial prosthetic platform was presented as line c, and the vertical line of line c was named line d, which crossed line b forming the angle to evaluate the prosthetic coronal position. the femoral prosthetic alignment was defined by the angle formed by the connecting line between the medial and lateral condyle line e and the perpendicular line to the mechanical axis of the lower limb line a (line f). the statistical analysis was achieved by the spss 19.0 statistical software (ibm corp., we chose the mean and standard deviation (sd) to describe normally distributed measurement data, as well as median, along with minimum and maximum values for describing measurement data that were not normally distributed. multiple linear regression analysis was applied to analyze factors related to the overall alignment of the lower extremity. we excluded patients with external joint deformity, including prior trauma or osteotomy history of lower extremity, abnormal condition resulted from other diseases (such as blount s, paget s, tertiary syphilis, or rickets). moreover, patients with limited motion of hip or ankle before surgery or contraction of knee extension 2 weeks after surgery, which would affect the quality full - length leg x - ray imaging, were also excluded. especially, the full - length x - ray should be finished in neutral position without knee flexion or rotation of lower extremity, which indicates that the anterior edge of tibial plateau could cover the posterior edge, and the proximity of tibia and fibula head should be overlapped a little, and the patella is in the middle or slightly lateral position of trochlea groove in the film. a midline incision was made anterior to the knee in all patients, and the joint capsule was entered through an incision medial to the patella. a pneumatic tourniquet inflated at 300320 mmhg (1 mmhg = 0.133 kpa) was used in the standard manner for hemostasis before making the incision and maintained until the end of the surgery. the i m entry point was chosen to be on the whiteside 's line (femoral anteroposterior axis), 1 cm above the terminal end of the medial border of the posterior cruciate ligament. the proximal pin was placed on the connecting lines between the medial edge of the lateral intercondylar spine and the medial third of the tibial tuberosity. as reference, anatomical landmarks were used the medial third of the tibial tuberosity, the proximal tibial spine, and the anterior tibial tendon. we chose an open intercondylar designed prosthesis (genesis ii, smith and nephew, memphis, usa). no patient received patellar replacement, and all surgeries were finished by the same surgical team. the main attending surgeon had a caseload > 200 tkas per year for 10 consecutive years. the coronal mechanical axis alignment and femoral valgus angle were measured on preoperative, full - length radiographs [figure 1 ]. two weeks after surgery, the femoral prosthesis was measured on full - length radiographs. the alignment was defined by the angle formed by the connecting line between the medial and lateral condyle and the perpendicular line to the mechanical axis of the lower limb. likewise, the angle formed by the tibial prosthetic platform and the vertical mechanical axial line of the tibia was measured to define the tibial prosthetic alignment [figure 2 ]. a varus angle was defined as positive (+) and a valgus angle was defined as negative (). preoperative full - length leg x - ray, the femoral mechanical axis was showed as line a, and line b indicated the tibial mechanical axis, the angle between line a and b was the angle of lower extremity mechanical axis. postoperative full - length leg x - ray, the bottom line of the tibial prosthetic platform was presented as line c, and the vertical line of line c was named line d, which crossed line b forming the angle to evaluate the prosthetic coronal position. the femoral prosthetic alignment was defined by the angle formed by the connecting line between the medial and lateral condyle line e and the perpendicular line to the mechanical axis of the lower limb line a (line f). the statistical analysis was achieved by the spss 19.0 statistical software (ibm corp., armonk, ny, usa). we chose the mean and standard deviation (sd) to describe normally distributed measurement data, as well as median, along with minimum and maximum values for describing measurement data that were not normally distributed. multiple linear regression analysis was applied to analyze factors related to the overall alignment of the lower extremity. data were collected continuously between june 2014 and december 2014, from 195 patients who underwent primary tka at peking university third hospital. seven patients were excluded due to low quality full - length lower extremity radiographs 2 weeks after surgery ; therefore, 188 cases were included in the study. among 188 patients included in the study, left- and right - sided procedures were equally represented. in the study group, 196 cases had varus knee preoperatively, with the varus angle (mean sd) 11.78 5.54, and 16 cases had valgus knee, with the valgus angle 10.28 8.36 [table 1 ]. coronal alignment was within the range of 3 in 144 cases with femoral side prostheses alignment and 191 cases with tibial side prostheses alignment. the paired chi - square test on the satisfactory rate of the two groups showed that the difference was statistically significant (p = 0.02) [table 2 ]. satisfactory postoperative alignment of femoral and tibial prosthesis (number of cases) multiple linear regression analysis was applied, where dependent variables were determined by postoperative coronal alignment of the lower extremities, and independent variables were set as the coronal prosthetic alignment on the femoral and tibial sides. results showed that femoral side prosthesis alignment had greater effect on overall lower extremity alignment than the tibial side. the standardized regression coefficient for femoral side was 0.666 and for tibial side was 0.414 (p subgroup analysis was conducted based on the varus or valgus status of the prostheses. in case of both femoral and tibial side varus, 0.001) and tibial side = 0.377 (p 0.05, clearly no statistical significance). therefore, this conclusion will need to be confirmed by further studies with a larger sample size. the significance of this retrospective, observational study is that it analyzed, for the first time, the effect of femoral and tibial side bone cutting on postoperative alignment, and noted that postoperative coronal alignment is mainly affected by femoral side im - guided resection. in addition, our preliminary results suggest that the incidence of unsatisfactory postoperative femoral side alignment may be higher. different factors, such as the distal curve of the femur, wrong medullary entry point, and inappropriate i m rod diameter and length, may all contribute to unsatisfactory femoral side alignment. given that femoral side i m guide procedure is affected by so many factors, surgeons should pay closer attention. the x - ray films in this study were affected by the rotation of lower extremity and the preoperative contraction of knee extension. although we controlled the quality of our radiographs, as a retrospective study, we were unable to completely eliminate this effect on our results. therefore, this study has certain limitations. in future, the results with a larger sample size should be analyzed by the difference valgus angle of femoral cutting guide and the preoperative contraction of knee extension in subdivided groups, to avoid the improper conclusion caused by the deviation data. | background : a good postoperative alignment in total knee arthroplasty (tka) is the key to achieving satisfactory results. we assessed the effect of femoral and tibial resection on the overall alignment after conventional tka.methods:we conducted a retrospective analysis of 212 primary tkas in 188 patients. intramedullary (im)-guided resection was applied on the femoral side while extramedullary (em)-guided resection was used on the tibial side. using full - length x - ray, the preoperative femoral valgus angle and lower extremity alignment, as well as 2-week postoperative femoral and tibial prosthetic coronal alignment and overall lower extremity alignment, were measured.results:postoperatively, good prosthetic alignment was achieved in 191 cases (90.1%) on the tibial side and in 144 cases (67.9%) on the femoral side (2 = 5.441, p = 0.02). multiple linear regression analysis was used to assess the effect of different alignment sides on the overall alignment in the coronal plane. data were divided into five subgroups based on the valgus or varus status of the prostheses. the standardized regression coefficients of the femoral and tibial prosthetic alignment on the overall alignment were 0.666 and 0.414, respectively ; in varus on both sides were 0.658 and 0.377, respectively ; in valgus, 0.555 and 0.030 ; femoral side varus and tibial side valgus, 0.702 and 0.211 ; femoral side valgus and tibial side varus, 0.416 and 0.287. the study showed that the overall low extremity alignment was statistically influenced by the prosthetic alignment, except for the tibial prosthetic alignment when femoral prosthesis was in valgus (p = 0.153).conclusions : in conventional tka, tibial side em - guided resection may offer satisfactory postoperative alignment, and femoral resection relying on i m guide may lead to more undesirable results. postoperative coronal alignment is mainly affected by the femoral resection. therefore, femoral side operation should receive adequate attention from the surgeons. |
it is commensal microorganism that constitutes a major component of the normal skin and mucosal microflora of humans. however, in recent years these bacteria have emerged as an opportunistic pathogen, important causative agents of bacteremia, and the leading cause of nosocomial infections particularly associated with indwelling medical devices (e.g., prosthetic joints and heart valves) and in individuals with a compromised immune system (e.g., cancer patients and neonates) [2, 3 ]. staphylococcus epidermidis pathogenesis relies on the ability to adhere and form biofilms on the surfaces of the medical devices mentioned earlier. staphylococci are considered as naturally susceptible to almost all antimicrobial agents developed but at the same time have a reputation of rapidly developing resistance. resistance to methicillin is at 7590% among hospital isolates of s. epidermidis, which is even higher than the corresponding rate for s. aureus (4060%). in addition to methicillin resistance, s. epidermidis strains have acquired resistance to several other antibiotics. most antibiotic resistance genes are plasmid - encoded and are more often found in methicillin - resistant than methicillin - susceptible strains. these facts together with the ubiquity of s. epidermidis as a human commensal microorganism render this bacterium an optimal carrier and reservoir for antibiotic resistance genes and for the transfer of genetic elements to pathogenic bacteria. the increasingly growing rate of antibiotic resistance of microorganisms necessitates the development and research of new antimicrobial agents or resistance modifiers. medicinal plant - derived compounds have increased widespread interest in the search of alternative antibacterial agents because of the perception that they are safe and have a long history of use in folk medicine for the treatment of infectious diseases. natural products of higher plants may possess a new source of antimicrobial agents with possibly novel mechanisms of action [9, 10 ]. they are effective in the treatment of infectious diseases while simultaneously mitigating many of the side effects that are often associated with conventional antimicrobials. systematic and methodical screening of them may result in the discovery of novel active compounds. one of the promising methods in coping with bacterial resistance is, along the use of alternative classes of antimicrobial agents, also the application of synergistic activity between antibiotics and nonantibiotics. many plants have direct antimicrobial activity but also resistance modifying / modulating activities. resistance modifying agents may inhibit multidrug resistance mechanism. it is well documented that some plants belonging to asteraceae and lamiaceae families possess suitable medicinal properties, which are based mainly on the presence of essential oils. antimicrobial activity of many species of salvia plants against several microorganisms has been recognized for decades and has been attributed to the presence of 1,8-cineole, -thujone, camphor, borneol, and p - cymene, among others. terpenes of essential oils extracted from different herbs are proved to have antimicrobial activity and some of them may act as resistance modifiers. researchers studied antibacterial properties of various plants against gram - negative as well as gram - positive bacterial strains including staphylococcus aureus [1618 ], but there are only few reports on antibacterial activity against staphylococcus epidermidis. moreover, the interactions between plant extracts or essential oils and antibiotic in methicillin - resistant s. epidermidis have not been documented earlier. the principal objective of the present study was to evaluate in vitro antibacterial activities of selected plant extracts from asteraceae and lamiaceae family against clinical isolates of s. epidermidis and to evaluate interactions between antibiotic and plant extracts or essential oils in coping with methicillin resistance. in this work crude extracts of plants from asteraceae (anthemis tinctorium, chamaemelum nobile, matricaria recutita, tanacetum argyrophyllum, and tanacetum parthenium) and lamiaceae (salvia fruticosa, salvia officinalis, and salvia sclarea) family were used. essential oils from salvia officinalis and salvia sclarea were prepared in accordance with the european pharmacopoeia. air - dried aerial parts were subject to hydrodistillation for 4 h, and isolated oil was diluted in n - hexane and dried over anhydrous sodium sulphate. methicillin - resistant and methicillin - susceptible staphylococcus epidermidis strains were isolated from patients with positive haemocultures from university teaching hospital old town, bratislava, slovak republic, and were kindly provided by dr. slobodnkov from institute of microbiology, faculty of medicine, comenius university in bratislava, slovak republic. all strains were routinely grown aerobically in brain - heart infusion medium (biomark, india) with shaking for 24 h at 37c. the plant extracts were tested for antimicrobial activity by disc diffusion assay on mueller - hinton agar (himedia, india). suspension of the tested bacteria (0.1 ml of 10 cells / ml) was spread onto solid media plates. the sterile paper discs (6 mm in diameter), which were impregnated with 10 l of individual extract, were placed on the incubated plates. these plates after 2 h of maintenance at 4c were incubated for 24 h at 37c and the diameters of the resulting zones of inhibition were measured in millimeters. the mic values of plant extracts were determined by broth microdilution method using 96-well microtiter plates in accordance with clsi (2011) guidelines. serial twofold dilutions of the plant extracts were prepared by vortexing the extracts in millipore water. inoculum of microorganism was prepared in mueller - hinton broth (himedia, india), and the turbidity was adjusted to 0.5 mcfarland and diluted to obtain a final turbidity in wells approximately 1 10 cfu / ml. twenty l of solution of plant extract and 180 l of bacterial inoculum were placed into wells of microtiter plate and incubated at 37c for 24 h. growth of the bacteria was examined as a function of turbidity (optical density (od) at 600 nm) using varioskan flash (thermo fisher scientific, finland). the mic is defined as the lowest concentration of antimicrobial agent that completely inhibits growth of the organism. detection of the meca gene in staphylococcus epidermidis strains was accomplished using polymerase chain reaction (pcr) amplification. cells were suspended in a lysis buffer containing 1 m tris hcl, 5 m nacl, and 0.1 m edta, which was incubated at 95c for 10 minutes. after incubation, the suspension was centrifugated at 23 000 g for 5 min. the final pcr products were visualized using uv - transilluminator after electrophoresis on 1.5% agarose gel containing 50 mg / ml etbr. the 96-well microtiter plates were inoculated with test organism and serial dilutions of two antimicrobial agents antibiotic and plant extract. each well contained unique combination of plant extract / antibiotic concentrations. the absorbance of the plates was recorded at 600 nm using varioskan flash (thermo fisher scientific, finland). interactions between antimicrobial agents were determined by calculating the fractional inhibitory concentration (fic) indices. the fic is defined as follows : mic of substance a tested in combination / mic of substance a tested alone + mic of substance b tested in combination / mic of substance b tested alone. the fic index is interpreted as fic 4.0antagonistic effect. the effect of combinations of plant extract and oxacillin against methicillin - resistant s. epidermidis was evaluated using the time - kill assay method. all antimicrobial agents alone and in combination were tested against six strains of methicillin - resistant s. epidermidis. time - kill curves were performed in tube containing nutrient broth, using inoculum density of approximately (10 cfu / ml) in the presence of a single agent or a combination of antimicrobial agents. samples were obtained at 0, 6, 10, and 24 h. at each sample time, aliquots were taken and serially diluted. the plates were incubated at 37c for 24 h. after incubation, the numbers of colonies were enumerated and the mean counts (cfu / ml) for each test and controls were determined and expressed as log10. the effect of the antimicrobial combinations was interpreted as follows : synergy was defined as a decrease of 2 log10 cfu / ml in colony counts after 24 h by the combination compared to the most active single agent. additivity or indifference was defined as a < 2 log10 cfu / ml change in the average viable counts after 24 h for the combination, compared with the most active single agent. antagonism was described as a 2 log10 cfu / ml increase in colony counts after 24 h by the combination compared to that by the most active agent alone. loss of 260 nm absorbing material released from bacteria was measured by the technique of devi.. cells were separated from medium by centrifugation at 400 g, for 15 min, washed twice in phosphate - buffered - saline (ph 7.4), and resuspended in the same buffer. different concentrations of plant extracts 10%0.03% (v / v) were added to the cell suspension. ciprofloxacin (500 mg / l) and cell suspension without plant extract were used as controls. samples from 0 and 60 min of the experiment were centrifuged at 13 400 g, for 15 min. for each time point and treatment agent optical density was measured at 260 nm with spectrophotometer (nanodrop, thermoscientific, usa). six clinical isolates of methicillin - resistant staphylococcus epidermidis (sep1sep6) and one methicillin - susceptible strain (sep7) were used to evaluate the possible antistaphylococcal activity of selected plant extracts. our results, determined using disc diffusion and broth microdilution methods, are presented as average values in tables 1 and 2. according to inhibition zone diameters, most effective were extracts from three species of salvia (inhibition zone from 12.4 mm to 12.7 mm) followed by extract from matricaria recutita. extracts from the rest of asteraceae family plants showed smaller inhibition zones, from 7.0 mm to 10.4 mm. there was no difference between inhibition zones of methicillin - resistant and methicillin - susceptible strains. more precise data on the antimicrobial properties were obtained through the determination of minimum inhibitory concentration and the results from microdilution method confirmed the previous results. extracts from s. officinalis and s. sclarea showed higher inhibitory properties with mic values in the range of 1.25% to 10% (v / v) than those of asteraceae extracts, most of which had mic 10% or more than 10% (v / v). antibacterial effects of crude extracts and, namely, of essential oils from different species of salvia on the growth of gram - positive and gram - negative bacteria were evaluated by many authors. some of them confirmed antibacterial effect of different salvia species also on staphylococci [25, 26 ], while other stated that extracts from salvia officinalis, s. divinorum, and others had no effect on the growth of staphylococcus aureus and s. epidermidis. we extended our investigation of antibacterial effect of plant extracts to evaluation of its possible mechanism. the mechanism of action of terpenes from essential oils, which are main parts of tested plants, is not fully understood, but it is assumed that membrane perturbation by these lipophilic components is involved in the antibacterial action. marked leakage of cytoplasmic material is considered indicative of gross and irreversible damage to the cytoplasmic membrane [30, 31 ] and is commonly quantified by the loss of intracellular material that absorb at wavelengths of 260 nm (nucleic acids). after treatment with plant extracts at increased concentrations from 0.3% to 5% (v / v) the od260 of filtrates of all tested strains increased and the most remarkable increases occurred after 60 min treatment with 5% salvia sclarea and 5% salvia officinalis (figure 1). at the same time these results suggest that crude extracts from tested plants damage the cytoplasmic membrane and cause loss of intracellular components. in order to confirm the assumptions that this effect have been based on the nature of essential oils, in figure 2 we compared the leakage of intracellular compounds in the presence of crude plant extract and of essential oil from the same plant salvia sclarea. it has been reported that some antimicrobial agents cause gross membrane damage and provoke whole cell lysis. among these compounds can be found also essential oils from oregano, rosewood, and thyme, -pinene, lemongrass oil, and tea tree oil from melaleuca alternifolia. the resistance of the six clinical strains s. epidermidis to oxacillin is obvious from the table 2. mg / l to 256 mg / l, while mic of methicillin - susceptible strain sep7 was 0.125 methicillin resistance of sep1sep6 strains was confirmed by determination of gene meca (figure 3). pcr for the meca gene coding for methicillin resistance via penicillin binding protein 2a (pbp2a) is well established and is considered as gold standard for detection of methicillin resistance in comparison with phenotypic methods [3840 ]. the evaluation of in vitro interactions between plant extracts and oxacillin are described in terms of fractional inhibitory concentration (fic) indices. the fics for all used plant extracts and oxacillin against bacterial strain of methicillin - resistant s. epidermidis sep6 are shown in table 3.. from all asteraceae only extract from matricaria recutita had synergistic effect with oxacillin, and all rest extracts had additive effect. it was found that the crude extract of salvia officinalis reduced the minimum inhibitory concentration of aminoglycosides in vancomycin - resistant enterococci and then the effective compound was isolated. carnosol, the active compound showed weak antimicrobial activity and greatly reduced the mics of various aminoglycosides. the exact mechanism for the reduction of -lactam (methicillin) resistance by the natural antimicrobials is unknown but is likely due to some structural change in the resistant bacteria. epigallocatechin gallate from green tea inhibited the activity of penicillinase produced by s. aureus and synergistically enhanced the activity of carbapenems against methicillin - resistant s. aureus (mrsa). tellimagrandin i from red rose (rosa canina l.) petal extract greatly reduced the mic of -lactam antibiotic against mrsa. similarly, corilagin, an active compound extracted from arctostaphylos uva - ursi, was found to reduce the mics of oxacillin and cefmetazole against mrsa. the synergistic effects arising from the combination of oxacillin and plant extract from salvia species in checkerboard assays were explored in greater detail by using time - kill assays. as is shown in figure 4, the synergistic effects (difference 2 log10) could be observed starting from 10 hours of incubation and continued up to 24 hours. the results of the checkerboard and time - kill assays agreed in all cases. regarding the synergistic effect of extracts from salvia species, in this part of work we were concerned with evaluation of interactions between essential oils from salvia officinalis and oxacillin. the results of the checkerboard method indicated synergism in all six strains of methicillin - resistant s. epidermidis for which the combination of essential oil and oxacillin were tested (table 4). this is in accordance with the fact that the combination of two agents exhibit significant synergism only if the test organism is resistant to at least one of the agents. the observed synergistic effects of plant extracts (essential oils) and oxacillin could be theoretically the results of the perturbation of the cell membrane coupled with the action of oxacillin. -lactam antibiotic oxacillin inhibits the final stage involved in the synthesis of peptidoglycan of cell wall (transpeptidation reaction), which occurs outside the cell membrane and is mediated by alternative protein binding protein pbp2a encoded by meca gene. compounds having synergic effect with oxacillin may inhibit the pbp2a activity or inhibit its production. first mechanism can be connected with the perturbation of cell membrane, which we have confirmed in this work ; the second mechanism : the inhibition of the production pbp2 by inhibition of meca gene expression is the content of our current research and its result will be published in later stage. the antibacterial activity of plant extracts from asteraceae and lamiaceae family was confirmed and contributed to the ability of contained essential oils to disturb biological membranes. synergistic activity of extracts as well as essential oil from s. officinalis and oxacillin could suggest an alternative manner to overcome a problem of bacterial infections caused by methicillin resistant staphylococcus epidermidis. further research is necessary to identify active compounds and research mechanism of interaction with antibiotics. | the crude extracts of plants from asteraceae and lamiaceae family and essential oils from salvia officinalis and salvia sclarea were studied for their antibacterial as well as antibiotic resistance modifying activity. using disc diffusion and broth microdilution assays we determined higher antibacterial effect of three salvia spp. and by evaluating the leakage of 260 nm absorbing material we detected effect of extracts and, namely, of essential oils on the disruption of cytoplasmic membrane. the evaluation of in vitro interactions between plant extracts and oxacillin described in terms of fractional inhibitory concentration (fic) indices revealed synergistic or additive effects of plant extracts and clearly synergistic effects of essential oil from salvia officinalis with oxacillin in methicillin - resistant staphylococcus epidermidis. |
the emergence and spread of carbapenemase - producing enterobacteriaceae is an increasing problem of global dimensions. originally, metallo--lactamases (mbls) were associated with resistance in gram - negative non - fermenters, but they have become increasingly important regarding carbapenem resistance in enterobacteriaceae. mbls confer resistance to almost all -lactam antibiotics and are not inactivated by -lactamase inhibitors, hence limiting treatment options in the individual patient and presenting a major challenge for infection control within the hospital setting 1. to date, the occurrence of carbapenem - resistant enterobacteriaceae in germany is still a rare event ; however, outbreaks involving kpc and vim-1 carrying klebsiella pneumoniae isolates have been described 2,3. in the year 2012, vim-1 was the most prevalent mbl detected in enterobacteriaceae in germany, followed by ndm-1 and gim-1 2. imp type mbls were first identified in pseudomonas aeruginosa in japan 4 and have since been reported predominantly from asia 5. with the exception of italy, imp - type enzymes have rarely been reported from other countries in europe 1,5,6. here, we report the isolation of citrobacter freundii harbouring mbl imp-8 from three patients between march 2012 and march 2013. all three patients had underlying haematological conditions (acute myeloid leukaemia n = 2 and myelodysplastic syndrome they were screened from rectal swabs for colonization with multidrug resistant gram - negative bacteria on a weekly routine schedule. identification of the isolates was performed using matrix - assisted laser desorption / ionization time of flight mass spectrometry (maldi tof - ms) (axima assurance, biomrieux sa, marcy letoile, france ; saramis database version 4.09) and the vitek 2 identification system (biomrieux sa). antimicrobial susceptibility testing was initially performed with the vitek 2 system (biomrieux sa) and confirmed by etest (biomrieux sa). results were interpreted according to the european committee on antimicrobial susceptibility testing (eucast) guidelines (http://www.eucast.org/fileadmin/src/media/pdfs/eucast_files/breakpoint_tables/breakpoint_table_v_3.1.pdf). the detection of extended - spectrum -lactamase genes blactx - m, blatem, blashv, 7 and carbapenemase genes blaoxa-48 8, blakpc 9, blandm 10, blaimp and blavim 11 was performed as described previously. sequencing of the imp genes was performed using class 1 integron primer 3cs (5-aag cag act tga cct ga-3) in combination with primer imp - a, and 5cs (5-ggc atc caa gca gca ag-3) in combination with primer imp - b 11. sequence identification was determined by comparison with the sequences available in genbank (http://www.ncbi.nih.gov/blast) and with the reference sequences of the lahey database (http://www.lahey.org/studies). alignment was performed using bioedit version 7.1.11 (ibis biosciences, carlsbad, ca, usa). genomic fingerprinting of the isolates was done by the enterobacterial repetitive intergenic consensus (eric) method using the eric2 primer as described previously 12. plasmids were extracted using the qiagen large construct kit (qiagen, hilden, germany) and digested using ecori and bamhi to allow for size estimation of the plasmids. briefly, dna was blotted onto positively charged nylon membranes (roche biochemicals, basel, switzerland) in denaturation solution (3 m nacl, 0.4 m naoh) by capillary transfer. high stringency hybridization was performed in accordance with the instructions given by the manufacturer of the digoxigenin labelling and detection kit (roche biochemicals). digoxigenin - labelled dna probes were generated with a digoxigenin - labelling pcr kit as described in the manufacturer s instructions (roche biochemicals) using the oligonucleotides imp - a and imp - b. l for the isolate of patients two and three, whereas meropenem mic for patient one was intermediate with an mic of 8 molecular detection of esbl genes blactx - m, blatem, blashv and carbapenemase genes blaoxa-48, blakpc, blandm and blavim was negative in all three isolates. the imp pcr gave a positive result and subsequent determination of the nucleotide sequence revealed an mbl of the imp-8 type. the sequence was compared with the reference sequence (genbank accession number af322577) 13. the blaimp-8 gene detected in the three isolates harboured a non - coding point mutation at position 18 (tc) as shown in fig.1. additionally, an imp-8 carrying plasmid of approximately 28 500 bp could be isolated from all c. freundii strains. genomic fingerprinting by the eric method revealed indistinguishable pcr patterns in all isolates (data not shown). characteristics of imp-8 carrying citrobacter freundii isolates from rectal swabs of three hospitalized patients in germanya i, intermediate ; r, resistant ; s, susceptible ; an, amikacin ; atm, aztreonam ; caz, ceftazidime ; cip, ciprofloxacin ; col, colistin ; ctx, cefotaxime ; cxm, cefuroxime ; ert, ertapenem ; mem, meropenem ; tig, tigecycline ; tzp, piperacillin - tazobactam. interpretation according to the eucast clinical breakpoints (http://www.eucast.org/fileadmin/src/media/pdfs/eucast_files/breakpoint_tables/breakpoint_table_v_3.1.pdf). sequence of the blaimp-8 gene of three citrobacter freundii strains isolated from hospitalized patients in germany. compared with the sequence of the imp-8 reference strain (genbank accession number af322577 13), a non - coding point mutation at position 18 was identified in the imp-8 from the three citrobacter freundii strains (grey shaded). imp-8 is very uncommon in europe, only once reported from portugal in a pseudomonas mendocina strain 6,14. in contrast, imp-8 is frequently encountered in asia, especially in taiwan, where imp-8-producing enterobacteriaceae are involved in serious infections.. reported on a case series of 37 patients with bloodstream infections caused by a large variety of imp-8-producing enterobacterial species including escherichia coli, k. pneumoniae, enterobacter cloacae and c. freundii 15. furthermore, aggravating the issue, phenotypic screening for imp-8-positive enterobacteriaceae is extremely difficult because of the lack of distinctive phenotypes. investigation of 95 imp-8-positive enterobacteriaceae revealed susceptibility to ertapenem in 21% and to meropenem in 45%, whereas phenotypic combined disk tests using edta and phenylboronic acid were positive in only 40% of the isolates 16. these observations from taiwan suggest that imp-8 is capable of spreading between enterobacterial species, causing serious problems in terms of infection control measures and limiting therapeutic options in critically ill patients. the role of faecal carriage of mbl - producing enterobacteriaceae remains unclear and has not been investigated in a large - scale epidemiological study. faecal carriage of one c. freundii vim-1 has been reported from spain in an outpatient, who was also colonized with two different vim-1-carrying k. pneumoniae isolates 17. in an italian hospital, eight vim-1-carrying c. freundii strains were isolated from rectal swab samples during active screening following the detection of a k. pneumoniae carbapenemase (kpc)-positive patient 18. none of our three patients became infected by the imp-8 c. freundii strains, but nevertheless it is alarming that imp-8 mbl circulates in the gut flora of patients at high risk of nosocomial infections. even more worrisome is the fact, that the imp-8 gene is located on a plasmid, which might facilitate the transfer of the resistance gene within enterobacterial species. our findings emphasize the importance of establishing screening schemes and laboratory diagnostic algorithms to ensure the implementation of efficient infection control measures and therapeutic strategies, not only in high - prevalence countries, but also in countries with a low incidence of mbl producing enterobacteriaceae. | metallo--lactamases (mbls) in enterobacteriaceae are an increasing problem worldwide. this report describes the isolation of citrobacter freundii carrying imp-8 mbl from three patients during the period from march 2012 until march 2013 in germany. the blaimp-8 enzyme is predominantly found in asia, where imp-8 has spread to various enterobacterial species causing serious infections. to our best knowledge, this is the first report of blaimp-8 habouring enterobacteriaceae in europe. |
type and screen (t&s), done pre - operatively to prevent complications from blood transfusion incompatibility between donor and recipient for existence of alloantibody. alloantibody screening is a laboratory method for detection of broad range unexpected (usually clinically significant, igg) an antibody(s) against an antigen(s) (usually the minor blood group) in sample sera that blood recipient lacks. indeed if the blood recipient recognizes the donor rbc surface antigens as foreign, the host will mount an immune response to the donor rbc s. those unexpected antibodies are usually results of rbcs stimulation including history of blood transfusion, pregnancy or history of miscarriage. although, naturally occurring alloantibody(s) may be present in some patient sample. clinically significant alloantibody(s) affected by igg immunoglobulin could be associated with hemolytic disease of new born (hdn), hemolytic transfusion reaction (htr) and notable decreases in the survival of transfused rbcs. according to similar studies, the prevalence of clinically significant alloantibodies has been reported from less than 0.5% to up to 60% of samples depending on the population study and the test method sensitivity, while in those patients receiving blood products regularly consisting of patients suffering from different type of malignancy, hemoglobinopathies or hematological disorders the rate of unexpected antibodies is significantly higher compared to those patients underwent elective surgery or those women had a history of pregnancy or miscarriage. this procedure (t&s) is much reliable and even less expensive than full cross matching and gives the same immunohematological safety. in the t&s test, each recipient s blood sample is typed for its abo and rh d blood groups and screened for unexpected but clinically significant antibody(s) that could to lead to red blood cell (rbcs) alloimmunization. the recipient s serum is incubated with two or three different group - o screening red cells (not pooled) in 2 - 5% rbcs suspension, which carry important and representative blood group antigens as homozygote including d, c, e, c, e, m, n, s, s, p1, lea, leb, k, k, fya, fyb, jka, and jkb antigens. it is one of the most important complications of blood transfusions. in the presence of alloantibody, the life span of red blood cells is shortened and the patient s need for blood increases. identification of the types of antigens present and transfusion of fully compatible blood may prevent alloimmunization. so, in this study, we evaluated relative frequencies of alloantibody against rbc in a general population of ardabil, (ardabil province in north west of iran with azeri race) whose referred to imam khomeini hospital for elective surgery and may be candidate to receive blood transfusion. in this cross - sectional study, type and screen test done as a pre - transfusion test for 1420 patients of imam khomeini hospital in ardabil that underwent elective surgery between march 2012 and february 2013. blood group typing done by anti - a, anti - b and anti - d reagent (iranian blood research and fractionation holding company, tehran - iran) in tube test procedure for each patient. screening test, also done by antibody screening kit contains three vials of rbc with known surface antigen (iran blood transfusion organization, iran, lot no : 11ip3c40 - 13ip3c65) according to manufactory method in room temperature and 37c. briefly, add 2 drops of patient plasma / serum to be tested to each labeled tube. then, add one drop of thoroughly mixed reagent red cells to the appropriate labeled tube. afterwards, 2 drops of albumin 22% added to each tube. after mixing the contents of each tube, incubate all tubes at room temperature and 37c (1c) for 10 - 30 minutes. then proceed directly with the antiglobulin test phase following incubation. immediately re - suspend the cells by gentle agitation ; examine the tubes macroscopically for agglutination. patients with positive results (presence of antibody in their serum) continued by antibody identification test (iran blood organization, iran, lot no : 11ip11c40 - 13ip11c65). this kit contains 11 cell panels and had a similar method to antibody screening test (described above). dat (direct anti - globulin test) done for patients with positive auto - control. in this study, of 1420 patients, 842 (59.3%) were male and 578 (40.7%) were female. patient s age ranged between 5 and 82 year and had mean age of 43.2 24.7 year. prevalence of alloantibody in this population was 0.92% (13 patients) and 99.2% (1407 patients) showed no alloantibody in their serum. of 13 alloimmunized patients, 7 patients (8% of male population) were male and 6 patients (1% of female population) were female. there was not any significant relationship between sex and prevalence of alloantibody (p > 0.05). the identified antibodies contains : k, e, c, lea, c, fya and d (table 1). anti - k and anti- lea, was the most prevalent clinical significant, and non - significant antibodies, respectively. the mean age of alloimmunized group was 45.2 year and in non - alloimmunized group had mean age of 43.8 year. no significant correlation found between age and prevalence of alloantibody among patients (p > 0.05). prevalence of alloantibody among patients ready to elective surgery of total 1420 under study patients, 1318 patients (93%) had no history of blood transfusion and 7% of patients had blood transfusion in past. among patients with positive antibody screening test, 10 patients had history of blood transfusion and a significant relationship found between history of transfusion and alloimmunization (p < 0.05). during the study, observed that demands for reservation of blood reduces significantly than before establishment of ts policy (p < 0.05). a reason for this assurance is the successful use of maximum surgical blood order schedules, which guide clinicians and blood banks alike in predicting the number of units that will need to be prepare for commonly performed surgical procedures. after the introduction of the indirect anti - globulin test by coombs in 1945, which added a new dimension to the safety of blood transfusion, there was a rapid increase in the identification of alloantibody that caused transfusion reactions or hemolytic disease of the newborn. this includes rbc antigenic difference between the blood donor and the recipient, the recipient s immune status and immunomodulatory effect of the allogenic blood transfusions on the recipient s immune system. in our study, prevalence of alloantibody was 0.9%. in a study conducted in minnesota region on normal population between 1975 and 1995, the rate of prevalence of alloantibody against red cell antigens was less than one percent. the most prevalent antibody was against e, le and k antigens, respectively. in another study in tehran (central part of iran) between 1997 and 1998, these efforts beside our study data and more similar reports in worldwide indicated that in normal population, incidence of occurrence of alloimmunization is less than one percent. history of blood transfusion and pregnancy can lead to formation of alloantibody. beside alloantibody, also autoantibody can be a source of potential risk in blood transfusion and must be regarded in antibody screening tests. in our study, we did not find any correlation between formation of alloantibody and gender. like our study in similar study in tehran, also no relationship found between gender and prevalence of alloantibody. while, in a study in kuwait occurrence of alloantibody was higher in females., in our study, antibody against k, e and c antigens with 30%, 15% and 15% prevalence, respectively, had higher rate among the other rbc antigens. in western europe and the united states, the most frequently reported alloantibody in alloimmunized patients were alloantibody against rh and kell antigens. in a study in minnesota, also anti - e and anti - k had higher rate than the other. prevalence of a specific antigen in region under study and also application of different methods and reagents can be considered as an explanation for this difference. alloantibody against these antigens has ability to mediate an intermediate to strong hemolytic reaction in recipients. thus, identification of these antigens in donors associated with precise performance of antibody screening and antibody identification tests have a significant role in reducing the risk of a hemolytic reaction. in our study, as expected, history of blood transfusion has a direct correlation with production of alloantibody especially in males, because women due to pregnancy have higher ability to produce alloantibody than men. the t&s test provides many benefits to patients and the main one is that, blood issued is safe and compatible. in cross - match test, blood units are randomly tested without information about the patient s antibody status. for cross - match test, blood units must reserve for a designated patient and this led to overestimate the number of units that would be required. in addition, as a repeat cross - match required at least another 1 to 2 hours and increase the workload of the blood bank staff. by t&s test, blood units are no longer reserved for a patient if the results from the antibody screen are negative and instead, a validity period is given to an individual for their negative antibody screen status. therefore, alloantibody screening must to preformed for patients needing blood transfusion, pregnant women, cases of blood cells transfusion reactions by blood bank laboratory in hospitals and finally for blood and plasma donors by blood transfusion establishments. | introductionalloimmunization is a reaction of the immune system to foreign antigens. for prevention of alloantibody formation, performing of type and screen test is necessary on a patient s blood specimen as part of pre - transfusion testing.materials and methodin this cross - sectional study, type and screen test done for 1420 patients with elective surgery for detection of alloantibody in imam khomeini hospital in ardabil.resultsprevalence of alloantibody in this population was 0.92% (13 patients) and 99.2% (1407 patients) showed no alloantibody in their serum. the most prevalent alloantibody was anti - k, anti - e and anti - c. no significant relationship observed between sex and alloimmunization rate.conclusionperformance of type and screen test play an important role in reducing the rate of alloimmunization, and also, could reduce the demands for blood reservation in hospital blood banks. |
microorganisms and their by - products are considered to be the major cause of pulp and periradicular pathosis. anaerobic bacteria especially black - pigmented gram - negative species have been linked to the signs and symptoms of these diseases. facultative bacteria such as enterococcus faecalis have also been isolated from infected root canal treatment. e. faecalis has been frequently found in root canal - treated teeth in prevalence values ranging from 30% to 90% of the cases. root canal - treated teeth are about nine times more likely to harbor e. faecalis than cases of primary infections. this finding can be explained by various survival and virulence factors possessed by e. faecalis, including its ability to compete with other microorganisms, invade dentinal tubules, and resist nutritional deprivation. various nanoparticles have gained popularity as antimicrobial agents as a result of their broad spectrum of activity and biocompatibility ; recent studies have focused on using nanoparticulate materials to disinfect root canals. nanosilver (ns) shows antibacterial effect ; it also exhibits novel physicochemical and biological activities. chlorhexidine is a synthetic cationic bis - guanide that consists of two symmetric 4-chlorophenyl rings and two biguanide groups, connected by a central hexamethylene chain. at higher concentration (2%), chx is bactericidal as precipitation of the cytoplasmic contents occurs, which results in cell death. also canal dressing for 1 week with 2% chx may provide residual antimicrobial activity against e. faecalis. camphorated phenol is among phenolic group of medicaments which have been applied either on a cotton wool pellet placed in the pulp chamber or on a paper point placed in the root canal, with the rationale being that the antimicrobial effect is delivered through vaporization of the medicament. hence, some bacteria may survive and have opportunity to multiply and persist in the root canal system. the aim of this study was to compare the effectiveness of various concentrations of nanosilver gel with 2% chlorhexidine gluconate and camphorated phenol on enterococcus faecalis biofilm using methyl thiazolyl tetrazolium (mtt) assay and confocal laser scanning microscopy (clsm) analysis. the hypothesis tested was that nanosilver gel, chlorhexidine gluconate, and camphorated phenol are equally effective on enterococcus faecalis biofilm. two tests were done (mtt assay and clsm analysis) to determine the effectiveness of nanosilver gel, chlorhexidine gluconate, and camphorated phenol on e. faecalis biofilm. pure strain of e. faecalis (atcc 29212) from nanjing biotechnology co. ltd. was used, to create the bacterial inoculum ; isolated colonies (24 hours) of pure cultures of e.f grown aerobically on brain heart infusion (bhi) agar plates were suspended in 5.0 ml bhi. the cell suspension was spectrophotometrically adjusted to match the turbidity equivalent to 0.5 mcfarland standards. polystyrene microtiter 96-well plates were used to evaluate the effect of medicaments on e. faecalis biofilm. 50 l of the 0.5 mcfarland standards inoculum e. faecalis prepared was added to 17 columns and a g rows and then 150 l of sterile bhi liquid medium was added to the microtiter plate where bacterial cells were seeded plus one column more (18 columns) ; the plate was then covered with the lid and sealed with parafilm and incubated at 37c for 24 hours afterwards. the biofilm was formed on the base of the plate after 24 hours ; 50 l of the experimental medicaments was added to different columns to test their effectiveness. medicaments were as follows : normal saline (shandong qidu pharmaceutical co. ltd.) as positive control;0.05%, 0.1%, and 0.2% nanosilver gel (shenyang dekang medicine technology co. ltd);2% chlorhexidine gluconate (shantou makat hi - tech co. ltd);camphorated phenol (hubei taichen janrui pharmaceutical co. ltd). normal saline (shandong qidu pharmaceutical co. ltd.) as positive control ; 0.05%, 0.1%, and 0.2% nanosilver gel (shenyang dekang medicine technology co. ltd) ; 2% chlorhexidine gluconate (shantou makat hi - tech co. ltd) ; camphorated phenol (hubei taichen janrui pharmaceutical co. ltd). the medium was carefully aspirated ; then each well was rinsed with sterile phosphate buffered solution (pbs) 2 - 3 times for about 5 minutes during each wash. 10 l of mtt solution was then added to the experimental wells, the plate was covered with aluminium foil to attain dark environment, and it was there after incubation at 37c for 46 hours. after 46 hours the medium was aspirated and 150 l of dimethyl sulfoxide (dmso) was added to each experimental well. the plate was kept in a microplate reader where it was shaken first for 10 minutes and then the absorbance value was measured at 630 nm wavelength. sterile 22 22 coverslip was placed in a 6-well culture plate and left there for five minutes. 500 l of the 0.5 mcfarland standards inoculum e. faecalis prepared was added on the surface of the coverslip and left there for 5 minutes. then carefully 5 mls of sterile bhi liquid medium was added along the side walls of the plate ; the plate was sealed with the parafilm and then incubated at 37c for 24 hours for formation of e. faecalis biofilm on glass coverslips. 200 l of the experimental medicaments was added ; the plates were sealed and then incubated at 37c for 24 hours. the culture medium and medicine were carefully aspirated without touching the coverslip. the cells were then washed twice with sterile pbs solution for 2 minutes each time. 300 l of the prepared ao / eb dye solution was added, and the plates were left in the dark for 15 minutes. then the specimens were observed under clsm with absorbance wavelength of 543 nm for ao (green) and 488 nm for eb (red) dyes under 200 magnification. mean and standard deviation values for each experimental group and control groups were calculated using spss 20.0 software and the percentage inhibition for each group of experimental drug was calculated using the following formula below and results are shown in table 2 : (1)% inhibition = control od valueexperimental group od valuecontrol od value blank control od value 100%. statistical analysis using one - way anova showed significant differences among groups (0.05% nsg and cp, 0.1% nsg and cp, 0.2% nsg and cp, 0.1% nsg and 2% chx, and 0.2% nsg and 2% chx) (p 0.05). the live and dead cells of e.f in the biofilm formed on the coverslip were observed according to the uptake of green and red / orange dye by the bacterial cells. figures 1, 2, 3, 4, 5, and 6 show clsm images of bacteria biofilm treated with different groups of medicaments. the colour of the biofilm changes from green to yellowish red, nanosilver gel being more yellowish red (in all 3 concentrations) compared to 2% chlorhexidine gluconate and camphorated phenol and more green for the control group indicating that almost all the cells are alive (figures 16). the term biofilm was introduced to designate the thin layered condensations of microbes that may occur in various surface structures in nature. free floating bacteria existing in an aqueous environment, the so - called planktonic form of microorganisms, are prerequisite for biofilm formation. biofilms may thus become established on any organic or inorganic surface substrate where planktonic microorganisms prevail in a water based solution. in dental context, a well - known and extensively studied biofilm structure is dental plaque. here, bacteria free in saliva (planktonic organisms) serve as primary source of organisms for the organization of this specific biofilm. in endodontics, the biofilm concept was initially discussed mainly within the framework of bacteria on the root tips of the teeth with necrotic and infected pulps and infected root canals. such bacteria aggregations have been thought to be the cause of therapy - resistant apical periodontitis. although not described in as much detail, bacterial condensations (biofilm) on the wall of infected root canals have been observed. antimicrobial agents have often been developed and optimized for their activity against fast growing, dispersed populations containing a single microorganism. however, microbial communities grown in biofilms are remarkably difficult to eradicate with antimicrobial agents and microorganisms in mature biofilms can be notoriously resistant for the reasons that have yet to be adequately explained. there are reports showing that microorganisms grown in biofilms could be twofold to 1000-fold more resistant than the corresponding planktonic form of the same organisms. e. faecalis has a strong tendency to form biofilm in the root canals and hence has a tendency of having antibiotic resistance to conventional therapy and is also proved to be resistant to the most widely used medicaments to disinfect the canals. therefore, an endodontic irrigant / medicament should ideally exhibit powerful antimicrobial activity, disinfect the root canal space, and have no cytotoxic effect on periradicular tissues, among various other properties required. confocal laser scanning microscope (clsm) has provided the ability to examine biofilms in situ without the limitations encountered with the sem, albeit at lower magnifications. clsm is now being used to determine the true architecture of plaque and the location of selected bacteria within the biofilm. the use of clsm requires that the organisms in the biofilms be stained with fluorescent stains. these stains are designed to emit light at specific wavelengths and can be used to probe specific cellular functions. using a suite of such stains allows the biofilm researcher to quantify all the cells and determine which ones are viable. nanosilver gel was found to be more effective than chlorhexidine gluconate and camphor phenol against e. faecalis biofilm. the results of this study demonstrated antibacterial activity against bacterial strains by all the intracanal medicaments tested. the bacterial strains chosen for this study belong to bacterial species that are clinically relevant, since they are part of the endodontic pathogens and are also new emerging pathogens causing infections in other clinical fields [1618 ]. the microorganism in the current study is a common isolated pathogen in both primary and secondary endodontic infections. the silver nanoparticles showed efficient antimicrobial property compared to other medicaments due to their extremely large surface area, which provides better contact with microorganisms. the bacterial membrane contains sulphur - containing proteins and the silver nanoparticles interact with these proteins in the cell as well as with the phosphorus containing compounds like dna. when silver nanoparticles enter the bacterial cell, it forms a low molecular weight region in the centre of the bacteria to which the bacteria conglomerate, thus protecting the dna from the silver ions. the nanoparticles preferably attack the respiratory chain, cell division finally leading to cell death. the nanoparticles release silver ions in the bacterial cells, which enhance their bactericidal activity [1922 ]. results showed that silver nanoparticles are cytotoxic in the case of exposure at high concentrations. there are contradictory studies on silver nanoparticles and ion cytotoxicity from laboratories around the world. silver is known to have a lethal effect on bacteria, but the same property that makes it antibacterial may render it toxic to human cells. concentrations of silver that are lethal for bacteria are also lethal for both keratinocytes and fibroblasts. in vitro studies have demonstrated that nanosilver has effects on reproduction and development and has an effect on dna among others. in contrast, it was found out that adding 1.0% silver nanoparticles (550 nm) to bone cement, a dose at which bactericidal activity was seen, did not result in (additional) cytotoxicity towards mouse fibroblasts (l929) or on growth of human osteoblast cell line (hfob 1.19). results of the current study indicate a dose dependent antimicrobial activity of nanosilver especially when compared with chlorhexidine gluconate ; nanosilver gel showed the ability to inhibit enterococcus faecalis biofilm formation at different concentrations, even at much lower concentration than chlorhexidine gluconate. the current study shows that camphorated phenol has lower inhibition effect compared to nanosilver gel and 2% chlorhexidine gluconate which coincides with the finding when camphorated phenol was compared to calcium hydroxide. it was found out that chx was less effective in eliminating e.f biofilm which concurs with the present study. on the contrary, it was found out that chx was more effective in eliminating e.f biofilm compared to other medicaments. according to the results of the current study, at a very low concentration nanosilver gel can be used to reduce bacteria load especially the most resistant e. faecalis as intracanal medicament. more studies using animal models and clinical studies are to be done to get a better understanding of the effects of nanoparticles on periodontal tissues ; also more data on the cytotoxicity of silver nanoparticles are needed on appropriate model organisms. | aim. to assess the effectiveness of nanosilver gel (nsg) in comparison to chlorhexidine gluconate (chx) and camphorated phenol (cp) against enterococcus faecalis (e.f) biofilm. methods and materials. two tests were done, methyl thiazolyl tetrazolium (mtt) assay and confocal laser scanning microscopy (clsm) analysis, to determine the effectiveness of nsg, chx, and cp on e.f biofilm. polystyrene microtiter 96- and 6-well plates were used for mtt and clsm, respectively. nanosilver gel was in three concentrations (0.05%, 0.1%, and 0.2%), chlorhexidine gluconate used was 2%, and camphorated phenol and normal saline were as control. analysis was done using one - way anova ; the post hoc test was run for multiple comparisons. the level of statistical significance was set at p 0.05). conclusions. 0.1% and 0.2% nanosilver gel is more effective on enterococcus faecalis biofilm as compared to chlorhexidine gluconate and camphorated phenol. |
many receptors for hormones, neurotransmitters, neuropeptides, chemokines, and autocrine and paracrine signaling molecules interact with heterotrimeric g proteins to exert their actions on target cells ; these receptors are considered g protein coupled receptors (gpcrs). it is estimated that more than 800 gpcrs are encoded in the mammalian genome, supporting that gpcrs are common membrane receptors in cells. heterotrimeric g proteins consist of an -subunit, which binds to and hydrolyzes guanosine-5-triphosphate (gtp), and - and -subunits, which form an indissociable complex. gpcrs transmit extracellular signals into the cell by binding to and activating different intracellular signaling proteins, termed g proteins (g, families gi, gs, gq/11, g12/13) or arrestins. the gq proteins, like all heterotrimeric g proteins, are composed of three subunits : gq, g, and g. gq - gtp and the g dimer then transmit receptor - generated signals to downstream effector molecules and protein binding partners until the intrinsic gtpase activity of g hydrolyzes gtp to gdp and the inactive subunits reassociate ; this is called the active and inactive cycle. each of the four major subfamilies of g proteins is associated with different signaling pathways : gq/11 activates the phospholipase c (plc) family ; gs stimulates the adenylyl cyclase (ac) pathway ; gi / o inhibits ac ; and g12/13 activates small gtpases. the gq/11 subfamily, including gq, g11, g14, and g15/16, shares structural similarity, and activation of the subunit within each protein complex can activate plc- [47 ]. furthermore, all of these four subunits regulate both overlapping and distinct signaling pathways, thereby stimulating inositol lipid (i.e., calcium / protein kinase c (pkc)) signaling through plc- isoforms [1, 49 ]. genetic studies using whole animal models have demonstrated the importance of gq in cardiac, lung, brain, and platelet functions, helping to define the physiological and pathological processes mediated by the gq [5, 10 ]. recent studies have described all four subtypes of gq/11 coupled gpcrs, including the muscarinic 1, 3, and 5 (m1, m3, and m5) receptors ; bombesin receptor, vasopressin receptor, endothelin receptor, thyrotropin - releasing hormone receptor (trhr), gonadotropin - releasing hormone receptor (gnrhr), membrane estrogen receptor (mer), chemokine receptors, adrenergic receptors (1ar), and angiotensin ii type 1 receptor (at(1)r) [1113 ]. in the field of immunology, chemokine and these gqpcrs are expressed on lymphocytes and are regulated by their ligands in the immune system [1418 ]. abnormal regulation of these receptors may be associated with the pathogenesis of autoimmunity and a variety of autoimmune diseases induced by autoreactive lymphocytes, leading to morbidity and mortality in individuals with autoimmune disorders [1924 ]. gq is the most commonly studied subclass of the gq/11 subfamily in the field of immunology and is mainly coupled to sex hormone receptors and some chemokine receptors, which are differentially expressed in certain types of lymphocytes [6, 18 ] (table 1). gonadotropin - releasing hormone (gnrh) is the primary hormone associated with reproduction ; gnrh is known to exert its actions largely through two related gq/11 protein receptors. the interaction between gnrh and its cognate type i receptor (gnrhr) in the pituitary results in the activation of gq, plc-, phospholipase a2 (pla2), and phospholipase d (pld). sequential activation of phospholipases generates the second messengers inositol 1, 4,5-tris - phosphate (ip3), diacylglycerol (dag), and arachidonic acid (aa), which are required for ca mobilization. further activation of various protein kinase c isoforms (pkcs) induces sequential activation of mitogen - activated protein kinases (mapks) and promotes nuclear transcription. gnrhr mrna and protein have been found in the pituitary, lymphocytes, mononuclear cells, and various types of cancer cells. many autoimmune diseases, particularly systemic lupus erythematosus (sle), exhibit gender - specific differences, and gnrhrs have been shown to function as immunostimulatory hormone receptors, playing pivotal roles in the observed gender - specific differences in immunity and/or autoimmunity. studies in mice and rats have shown that gnrh stimulates the expression of hormone - gqpcr and the interleukin- (il-) 2 receptor, the proliferation of b and t lymphocytes, and the elevation of serum igg levels. jacobson measured gnrhr mrna and gnrh binding in lupus - prone mice after in vivo exposure to gnrh or vehicle. their results showed that even vehicle - treated females expressed more gnrhr in immune cells than did vehicle - treated males ; gender differences were confirmed, with females expressing gq - coupled hormone receptor mrna and protein more than males. in mice given additional studies have shown that gnrh (through gqpcr) stimulates t / b lymphocyte proliferation in vitro in females only. these differences in expression and activation of gnrhr through gqpcr on lymphocytes contribute to the observed gender differences in immunity and/or autoimmunity. in addition to the pivotal function of intracellular estrogen receptors in autoimmunity, researchers have also shown that mers can stimulate gq - coupled gpcrs through pkc and calcium pathways. rider and abdou suggested that estrogen acting through gq - mers enhances t - cell activation in women with lupus, resulting in amplified t / b - cell interactions, b - cell activation, and autoantibody production. thus, the gender differences in gnrh and estrogen production and function can be directly associated with gq protein receptor expression, which plays a critical role in maintaining the balance of t / b lymphocytes and affects the morbidity of autoimmune diseases that predominantly affect women. chemokine receptors are expressed on t cells, b cells, monocytes, macrophages, and dendritic cells. chemokine receptors and their ligand axis play pivotal roles in leukocyte migration, differentiation, adhesion, and activation [32, 33 ]. many chemokine receptors have been implicated in the pathogenesis of autoimmune connective tissue diseases such as sle, rheumatoid arthritis (ra), and systemic sclerosis (ss) [1921, 32, 3437 ]. however, previous studies have demonstrated the indispensable role of chemokine receptors in autoimmune diseases, highlighting the role of gi protein - coupled chemokine receptors (rather than gq - coupled receptors) in directing the migration of immune cells, which mostly signal through the canonical ac pathway [19, 36 ]. in a cell - based study, arai and charo showed that monocyte chemotactic protein- (mcp-) 1-related chemokine receptors (mainly cc family receptors) interact with multiple subtypes of g proteins in a cell type - specific manner and that the third intracellular loop of cc type receptors mediates gq coupling. moreover, many studies have shown that chemokine receptors can interact with multiple g - protein subtypes ; the coupling is cell type - specific. shi and colleagues showed that chemokine receptors can be divided into cd38-dependent and -independent subclasses, depending on whether cd38 is needed for the chemotaxis of the ligand. cd38-dependent chemokine receptors couple to gq, indicating that there is indeed a novel gq protein - coupled alternative signaling pathway separate from the canonical gi - coupled classic pathway. autoimmunity - associated chemokine receptors mainly include the cc family (ccr5 and ccr7) and the cxc family (cxcr3, cxcr4, cxcr5, and cxcr7) [19, 20, 34, 3641 ]. to date, most chemokine receptors, such as cxcr4 and cxcr5 on t cells and b cells, have been shown to be induced by gi in the classic pathway, while ccr7 and cxcr4 have been shown to be dependent on gq pathways only on dendritic cells (dcs). hence, the dependence of autoimmune diseases on the specific gq coupled chemokine receptor alone is still unclear. since this chemokine receptor is engaged and activated in lymphocytes by gpcrs, it is possible that these gq - coupled receptors may interact functionally with gi to regulate chemotaxis in lymphocytes during the effector stage. at(1)r, one of the best - studied gpcrs, signals through gq to transduce signals on lymphocytes in autoimmune - regulated cardiomyopathy and hypertension. experimental findings support the concept that the balance between t cell - induced inflammation and t cell suppressor responses is critical for the regulation of blood pressure levels ; autoantibodies to these receptors can exacerbate the pathological process. high levels of autoantibodies against the second extracellular loop of 1-adrenoceptor (1-ar) are also found in patients with hypertension, suggesting an important role of 1-ar and at(1)r autoimmunity in the pathogenesis and management of hypertension, particularly in patients having high levels of receptor - associated autoantibodies [11, 45, 46 ] autoimmunity comprises a variety of autoreactive lymphocytes characterized by the loss of tolerance to a variety of autoantigens and imbalanced humoral and cellular immunity in biological systems [19, 23, 47 ]. gq - coupled gpcrs on different lymphocytes can transduce a series of extracellular signals into the nucleus to regulate immune function. immune responses are coordinated by the extracellular ligand to gpcr on lymphocytes, and activated intracellular gq protein then activates enzymes, second messengers, protein kinases, and nuclear translocation, consequently inducing the migration, activation, and apoptosis of lymphocytes. this well - orchestrated function of hematopoietic cells is complex ; however, it is clear that gq - coupled receptors and the signaling molecules that reside downstream of these receptors are critical to these functions. women are more likely to actively express gnrh and gnrhr than men, as described above, particularly during the reproductive period, at which time immune responses are different [26, 30 ]. furthermore, in the spleen and thymus, the expression of g proteins on mononuclear cells differs in a gender - dependent manner. gnrhr exhibits direct immunostimulatory properties, and lymphocytes produce gnrh and express gnrhr [23, 26, 49 ]. the g protein gq/11 regulates the transduction of signals from multiple hormones from specific cell surface receptors to a variety of intracellular effectors, including the ac pathway, plc-, and the ion channel pathway. jacobson. demonstrated that antisense nucleotides to gq/11 inhibit hormone and gnrhr signaling, suppressing the proliferation of t cells from female mice after in vitro culture. additional studies have shown that antisense oligonucleotides to gq - coupled gpcrs can also be effective in vivo for ameliorating murine lupus ; specifically, antisense oligonucleotides directed against gq in female lupus - prone mice effectively reduce serum igg levels, anti - dna antibody levels, hematuria, and proteinuria, even in terms of the histopathology of renal biopsies. thus, these studies demonstrate the utility of gnrh inhibitors to modulate gqpcr activation in mice and suggest a novel potential target for the treatment of lupus. have shown that signals mediated by chemokine receptors may compete with t - cell receptor stop signals and determine the duration of t - cell antigen - presenting cell interactions. during t - cell stimulation by antigen - presenting cells, t - cell chemokine receptors coupled to gq and/or g11 protein when chemokine receptors are sequestered at the immunological synapse, t cells become insensitive to chemotactic gradients, form more stable conjugates, and enhance proliferation and cytokine production. thus, chemokine receptor trapping at the immunological synapse enhances t - cell activation by improving t - cell antigen - presenting cell attractions and impeding the distraction of successfully engaged t cells by other chemokine sources.. suggested that optimal activation of the t - cell receptor requires signaling through chemokine receptor - gq and that removal of gq locks cells into a migratory phenotype, making the cell less responsive to t - cell receptor signaling. previous studies have shown that activation of gq inhibits migration through an lck - shp-1 pathway, priming cells for activation through the t - cell receptor - cd3 complex. thus, these novel gqpcr signaling pathways are involved in mediating the threshold of chemotaxis and t - cell receptor activation, playing an irreplaceable role in immunity and autoimmunity. a gi - coupled classic pathway activates t lymphocytes and alternative gq - dependent chemokine receptors, promoting the migration of dcs and monocytes. furthermore, gq, similar to cd38, regulates extracellular calcium entry in chemokine - stimulated cells. gq - deficient (gnaq/) dcs and monocytes are unable to migrate to inflammatory sites and lymph nodes in vivo, demonstrating that this alternative gq - coupled chemokine receptor signaling pathway is critical for the initiation of immune responses [32, 38 ]. binding partners to gqpcr distinct from plc- include novel activators (ric-8a and tubulin), candidate effectors (rhogefs, pi3k, gpcr kinases (grks), btk, and complex regulator of g - protein signaling (rgs) proteins), regulators (rgs proteins and grks), and scaffold / adaptor proteins (ebp50/nherf1, cdp / cd81, caveolin-1, and tpr1) [1, 4, 6, 50 ]. downstream of these signaling proteins, signals through gpcr to gq family members exhibit unexpected differences in signaling pathways and the regulation of gene expression profiles [8, 50 ]. plc- is the most well - known downstream effector molecule of gqpcr (figure 1). the canonical pathway for the gq/11 family is the activation of plc- enzymes, which catalyze the hydrolysis of the minor membrane phospholipid phosphatidylinositol bisphosphate (pip2) to release ip3 and dag [47, 13, 14 ]. these second messengers serve to propagate and amplify the gqpcr - mediated signal with calcium mobilization following release from ip3-regulated intracellular stores and dag - mediated stimulation of pkc activity [4, 5 ]. inositol lipids, dag, pkc, and calcium each participate in multiple signaling networks, linking gq family members through a host of different cellular events. this pathway has been widely studied as a marker of gqpcr signaling. as the aforementioned chemokine receptors, there are classic (gi) and alternative (gq) coupled gpcr pathways depending on the specific type of the chemokines and chemokine - stimulated cells. the gq activates the plc family that can regulate the extracellular calcium entry in chemokine - stimulated cell and also subsequently influence the downstream effectors such as pi3k / akt for survival of the cell. multiple reports have documented the negative influence of gq - coupled receptors on the growth factor - directed activation of pi3k and akt isoforms [1, 47, 13 ]. in addition, a previous study also showed that gq represses akt activation in fibroblast cell lines [5254 ] and cardiomyocytes [55, 56 ] ; however, overexpression of gq in cardiomyocytes leads to cardiac hypertrophy and cardiomyocyte apoptosis [10, 57 ]. in contrast, gq normally inhibits pi3k activation and prevents activation of akt [6, 7, 10, 14, 38 ]. furthermore, gq inhibits the activation of the pi3k - akt pathway, as has been demonstrated in gnq/ mice. indeed, by measurement of the phosphorylation of akt at ser473 (phospho - akt), a phosphorylation site under the control of pi3k demonstrated that the level of phospho - akt was higher in gnq/ mice than in wt b cells. furthermore, deletion of phosphatase and tensin homolog (pten), an inhibitor of pi3k, also promotes mature b - cell survival and can rescue autoreactive b cells from anergy. interestingly, the autoreactive prone marine zone - like b (mzb) cell compartment is also expanded in mice expressing activated p110 or lacking pten. in the absence of gq, b cells constitutively express higher levels of activated akt and preferentially survive bcr - induced cell death signals and baff (b - cell - activating factor of the tnf family, also known as blys, for b lymphocyte stimulator) withdrawal in vitro and in vivo [10, 58, 62 ]. the b cells isolated from multiple models of autoimmunity have been reported to express elevated levels of phospho - akt, and perturbations in the pi3k / akt axis can lead to the development of autoimmunity [51, 62 ]. in addition to plc- and pi3k, many studies have demonstrated that gq - coupled receptors can also regulate other intracellular signaling molecules, such as members of the mapk family [6, 7, 50, 57 ]. the mapk signaling cascade is one of the most ancient and evolutionarily conserved signaling pathways and responds to a broad range of extracellular and intracellular changes [6367 ]. among the mapks, p38 mapk regulates the expression of tumor necrosis factor- (tnf-), interferon- (ifn-), and other cytokines via transcriptional and posttranscriptional mechanisms. therefore, inhibiting p38 mapk may abrogate tnf-, providing potential anti - inflammatory effects [65, 68, 69 ]. predominant th1 and th17 cytokine production are characteristic of many organ - specific autoimmune diseases, and the dysregulation of p38 mapk activity specifically in autoreactive lymphocytes appears to enhance il-17 and ifn- expression [66, 7072 ]. additionally, the erk pathway can be activated by the small g protein ras via the raf group of map kinase kinase kinases (mkkks). solid evidence has supported that endothelin - dependent erk / mapk activation depends on the gqpcr / plc-/ca / src signaling cascade. taken together, these studies have shown that gqpcr and gq are involved in the activation of erk. thus, complex gpcr signaling should be studied as a concerted network at the systems level. the detailed cross - talk mechanism between these gqpcr pathways still needs to be explored in the future. in this review, we have outlined current evidence supporting the biological, pathological, and cell signaling functions of gq - coupled gpcrs in autoimmunity. this discussion reinforced the idea of cell signaling diversity and challenged the established paradigm that gq - coupled gpcr signals in immunology are functionally redundant. moreover, studies of traditional pathways alone do not account for many gq - mediated responses ; gq - linked signaling suggests that gqpcrs have complex roles in signal transduction that are not yet fully understood. our previous studies have shown that gq is associated with immune diseases and has pivotal roles in autoimmunity [17, 18, 24, 33, 38, 72, 74 ]. gq is downregulated in ra 's patients and relates to the disease 's activity ; it can control the ra 's progress via th17 differentiation. while gq - containing g proteins can regulate b - cell selection and survival and are required to prevent b - cell - dependent autoimmunity, the deficiency of gq can enhance the t - cell 's survival and influence the migration of dcs and neutrophils. therefore, we are interested in clarifying the role of gq - coupled gpcrs in immune tolerance and autoimmunity, with the aim of improving therapeutic approached. nonetheless, there are still some limitations to the available data describing the role of gq - coupled gpcrs in autoreactive lymphocytes. further studies using in vitro - derived lymphocytes may not accurately reflect the situations occurring in vivo. moreover, the aforementioned studies involved different races and small patient populations, which may also have influenced the final results. to date, many studies have focused on gq - coupled membrane receptors and, to a lesser extent, g11. however, future studies of autoimmunity may improve our understanding of the unique cell signaling roles and properties of other gq/11 family proteins, including g14 and g15/16. taken together, our discussion herein summarizes our current understanding of the complexity of gq - coupled membrane receptor signaling and highlights many exciting new areas for future investigations in autoimmunity. | heterotrimeric g proteins can be divided into gi, gs, gq/11, and g12/13 subfamilies according to their subunits. the main function of g proteins is transducing signals from g protein coupled receptors (gpcrs), a family of seven transmembrane receptors. in recent years, studies have demonstrated that gpcrs interact with gq, a member of the gq/11 subfamily of g proteins. this interaction facilitates the vital role of this family of proteins in immune regulation and autoimmunity, particularly for gq, which is considered the functional subunit of gq protein. therefore, understanding the mechanisms through which gq - coupled receptors control autoreactive lymphocytes is critical and may provide insights into the treatment of autoimmune disorders. in this review, we summarize recent advances in studies of the role of gq - coupled receptors in autoimmunity, with a focus on their pathologic role and downstream signaling. |
in cancer diagnosis, cancer patients with the same diagnostic profile may have different clinical outcomes. the difference probably lies in the limitation of the traditional classification of tumor types, based mainly on morphology. a reliable and precise classification of tumors is essential for successful diagnosis.1 modern sequencing and microarray technology have enabled more detailed molecular characterization of cancer samples, leading to the discovery of many cancer subtypes. in fact, cancer subtype identification has become an integral part of personalized medicine.1 traditionally, the problem of cancer subtype classification has been approached in many ways such as multinomial logit models,2 bayesian probit models,3,4 random forest,5 and support vector machine (svm).69 other discriminatory methods, including linear discriminant analysis (lda), k - nearest - neighbor (knn) classifier, and classification trees, were also investigated.10 among these, svm is a successful procedure applied to microarray - based cancer diagnosis problems.2,11 however, it suffers from the difficulty to interpret the resulted model as well as no direct outcome probability estimates produced.1214 multinomial logit model, on the other hand, is endowed with feature interpretability and probabilistic nature.15 classic multinomial logit model works well when the number of predictors is small. as the number of predictors increases, the generalization power of the model deteriorates because of model overfitting. when the number of predictors exceeds the number of observations as is commonly seen in genomic studies, the method breaks down. to deal with the curse of high dimensionality as well as to increase the model interpretability, regularized procedures that incorporate a sparsity penalty have been proposed.1620 among these methods, group lasso is particularly appropriate for models with multiclass responses, in which all the coefficients linked to a common predictor form a group and are required to shrink to zero simultaneously in order to achieve the selection of predictors.16 although the sparse multinomial logit models are capable of selecting variables, they can not efficiently use underlying structure information such as a network of regulatory relationships between genes or gene - products. such structure information could be obtained by a data - driven approach via clustering21 or other similarity - based methods,22,23 or be extracted from the external databases accumulated through years of biomedical research. databases such as kegg, reactome, and mips have been developed to organize different types of biological network information. cancer is a complex disease caused by dysregulation of pathways instead of a single gene.2426 thus, the incorporation of the network information can potentially increase the power of identifying cancer subtypes. networks are often represented as graphs, with each vertex indicating a gene or a gene - product and each edge indexed by a relationship between two vertices. a constraint, induced by the laplacian matrix of the graph,27 has been proposed to facilitate the selection of predictors in ordinary regression settings, enhancing both the global smoothness over the network and the interpretability of the association between selected genes and responses in the context of known biology. in this paper, we propose a network - constrained sparse multinomial logit model for high - dimensional multinomial classification, aiming at improving prediction performance by utilizing the underlying network prior. the remainder of this article is organized as follows. the model is first presented, followed by the algorithm to fit the model. finally, the model is applied to a real data set of predicting the subtypes of glioblastoma multiforme (gbm). n with n observations and p predictors, yi denotes an observation of the categorical response variable y { 1,, k } and xi = (xi1, xi2,, xip) r indicates an observation of a p - dimensional vector of predictors. assuming that yi follows a multinomial distribution, a multinomial logit model is built with logit link, which is ir = p(y = r|xi)=exp(r0+xir.t)s=1kexp(s0+xis.t)=exp(ir)s=1kexp(is)(1)where r.=(r1,r2, we choose category k as the reference category by setting k0 = 0 and k. k1, correspond to the log odds ratio between category r and the reference category k. we regularize the multinomial logit model using a penalized likelihood approach, in which one maximizes the penalized log - likelihood lp()=l()j(),(2)over a (k 1) (p + 1)-dimensional parameter vector = (10,, (k1)0, 1.,, k1,.). in equation (2), l()=i=1nr=1kyirlogir=i=1n(r=1k1yirirlog(s=1kexp(is)))is the ordinary log - likelihood of a multinomial logit model, and j () is a function penalizing the magnitude of the parameters and regularizing the structure of features., the regulatory parameter, controls the strength of the regularization. assuming that all predictors are metric and standardized, that is, each predictor has one degree of freedom, and the differences in scale will not influence the penalty and, thus, the variable selection. in the multinomial logit model, we use a vector.j = (1j, 2j,, k1j) of parameters to capture the effect of variable xj, so that variable selection is achieved only when the k 1 parameters shrink to zero simultaneously. since the ordinary lasso facilitates only parameter selection rather than predictor selection, a group lasso penalty is utilized to penalize the parameters at a group level, defined as j1()=j=1pj.j=j=1pj(1j2+2j2++k1j2)1/2,(3)where j is a penalty weight, set as 1 by default. in group lasso, all the parameters in a group.j would shrink to zero simultaneously. in an association study, the graphs or networks depicting relationships among predictors are informative, supplementary to numerical data. consider a network represented by a weighted graph g = (v, e, w) with the set of vertices v = 1, p corresponding to p predictors, the set of edges e = { (j, k) : j and k are linked }, and the set of weights w = { wjk : (j, k) e}. wjk measures the level of concordance of predictors j and k, with 1 for identity and 0 for complete difference, if normalized to the scale of. we then construct an adjacency matrix a by ajk={wjk(j, k)e,0(j, k)e.and a degree matrix d = diag(d1, d2,,dp), where dj = (j, k)e jk is defined as the degree of vertex j. the laplacian matrix associated with graph g is l = d a, which is always non - negative definite and can be factorized as l = ss. by simple algebra, r.lrt can be written as r.lr.t=(j,k)e(rjrk)2wjk. thus, the network - constrained penalty,22,27,28 defined as j2()=r=1k1r.lr.t,(4)induces a smooth solution of the vector r. with respect to the labeled weighted graph g. typically, the adjacency matrix can be constructed from external information using the abovestated method. however, several data - driven methods are also applicable.22,23 adjacency coefficients can be defined based on similarity measures such as pearson correlation coefficient, which are transformed into adjacency by a monotonically increasing function. the most widely used transformation functions include the signum function, the sigmoid function, and the power function. detailed examples of adjacency measures are provided by huang and ma.22 to sum up, in our regularized model, the penalized log - likelihood function is given by lp()=l()j()(5) = i=1n(r=1k1yirirlogs=1keis)1j=0pj.j2r=1k1r.lr.t,(6)of which the second term, the sparse penalty, induces model sparsity and the third term, the network penalty, imposes smoothness over the network. when 2 = 0, the model reduces to the group lasso multinomial logit model. the incorporation of this extra tuning parameter expands the parameter search space and directs the search to more biological meaningful regions. like ordinary lasso, group lasso also suffers from an issue of estimation bias, which is resulted from the fact that all predictors are penalized to the same degree. in order to reduce the bias, we use adaptive group lasso, which penalizes predictors to different degrees by assigning a weight to each predictor. in our model, the weight is set to be the reciprocal of the l2 norm of the fitted coefficients in univariate analysis, where we fit the model with each individual predictor only. denoting.j as the univariate estimate, the group lasso penalty term (3) becomes j1()=j=1p1.j.j. we use the proximal gradient - based fista (fast iterative shrinkage - thresholding algorithm) to fit the model.29,30 consider the optimization of the general penalized log- likelihood lp () = l() 1j1(), composed of a concave and continuously differentiable term l(), and a convex penalty term j1(). the penalized maximum likelihood (ml) estimator is defined by ^=argmaxrdlp()=argmaxrd(l()+1j1()),(7)where l()=i=1n(r=1k1yirirlogs=1keis)2r=1k1r.lr.tis a smooth function with respect to parameter. with a positive step size v, the quadratic approximation29 of lp() at a given point 0 is qv(,0)=l(0)l(0)t(0)+12v02+1j1(). l(), the first - order derivative of l(), is a (k 1) (p + 1)-dimensional vector, whose element corresponding to rj is [l()]rj=lrj=i=1n(yirir)xij+2lj.r.t. the iterations of proximal gradient methods are defined by ^(t+1)=argminrd{l(^(t))l(^(t))t(^(t))+12v^(t)2+1j1()}(8)which consists of a linear approximation of the negative modified log - likelihood at the current value ^(t), a proximity term, and the penalty term. first, we set 1 = 0, and based on the standard formula for the iterates of gradient methods for smooth optimization, the unpenalized estimator (t+1) has an explicit form (t+1)=(t)+vl((t)). then we move back to the optimization problem with an active penalty. via lagrange duality, equation (7) can be equivalently expressed by ^=argminc(l()),where c={rd|j1()(1) } is the constraint region corresponding to j1() and (1) is a tuning parameter that is linked to 1 by a one - to - one mapping. given a search point u r, the so - called proximal operator associated with j1() is defined as p1(u)=argminrd(12u2+1j1(.(9)which is the projection of u onto region c. then the proximal gradient iterates defined in equation (8) can be equally expressed by the projection ^(t+1)=p1v(^(t)+vl(^(t))). next, consider the proximal operator (9). owing to the block - separability of this specific penalty, the proximal operator can be written as p1()=(p1(^.0),p1(.1),,p.(10) with (u)+ = max (u, 0), the explicit solution to the proximal operator (10) can be derived from the karush set (t+1)=^(t)+vl(^(t)), then the solution to the optimization problem (8) can be expressed as ^(t+1)=p1v((t+1))=(p1v(.0(t+1)),p1v(.1(t+1)),,p1v(.p(t+1)))=(.0(t+1),(11j.1(t+1))+.1(t+1),,(11j.p(t+1))+.p(t+1)) to summarize, the basic idea of proximal gradient methods is as follows : first, remove the l1 penalty of the objective function (6) and then optimize the smooth part by taking a step toward its ml estimator via first - order methods, which creates a search point. second, project this search point onto the constraint region c in order to account for the non - smooth penalty term. to accelerate the convergence rate, we extrapolate the current and the previous iterations with the help of deliberately chosen acceleration factors at,20 ^(t)=^(t)+at11at(^(t)^(t1)). the extrapolate point ^(t), instead of the current iterate ^(t) is used as a starting point to generate a search point, which is then projected on the penalty region. to select the tuning parameters 1 and 2, we use cross - validation, where we divide the data set into training and test data sets. the model is trained on the training data set, and prediction error is then assessed on the test data set. we search on a grid of 1, 2 values and choose the value of 1, 2 that minimizes the cross - validated errors. the prediction error is measured with the brier score, a measurement of the accuracy of probabilistic predictions defined as the euclidean distance between sample response and its estimated probabilities. the purpose of the simulation is to show that the structure - constrained model dominates the alternative models that do not use such prior information in terms of parameter estimation and prediction. for each scenario presented, we first select the optimal tuning parameters through a five - fold cross - validation on the training set. with the selected tuning parameters, a final model is built on the whole training set and then tested on the test set. for each setting, we run 50 simulations and calculate several criteria to evaluate the performance of the proposed model. the numbers of total predictors are 20 for small and 200 for large models, and the numbers of relevant ones are 4 and 10, respectively. the predictors are continuous and follow a multivariate normal distribution with mean 0 and the p p correlation matrix =(12 p11 p221 p3p1p2p3 1),where = 0.5. we divide the predictors into a few subsets (subnetworks). in the small model, 20 predictors are divided into 5 subnetworks evenly, and the 4 relevant predictors constitute the first subnetwork. in the large model, 200 predictors are divided into 20 subnetworks evenly, and the 10 relevant predictors form the first subnetwork. ideally, we assume full connection within each subnetwork and no connection between them ; that is, the corresponding adjacency matrix is a block diagonal matrix, with the main diagonal blocks being all - ones square matrices and the off - diagonal blocks zero matrices. we then construct the coefficient matrix, a 3 p matrix whose rows are indexed by all but the reference categories of the response and columns are indexed by predictors. the columns corresponding to the irrelevant predictors are filled with zeros. for the relevant columns, the first setting requires equal coefficients in each category for relevant predictors. in the case of similar coefficients, entries in each category share the same sign but have different values, indicating that all the relevant predictors impact the response in the same direction but with different magnitudes. their absolute values are independently drawn from the set { 0.05, 0.10,, 0.50 }, and the sign for each category is random. random coefficients are independently drawn from the set { 0.50, 0.45,, 0.05, 0.05,, 0.50}. in this case, the prior structure information is not useful, since we assume that the coefficients within each subnetwork should be at least similar. this scenario serves as an example of model misspecification. to further study the effects of structure misspecification, we also test our models in cases of incorrect networks and overlapping networks. in incorrect network setting, the large and small adjacency coefficients are randomly drawn from (0.4, 1) and from (0, 0.6) instead of being constant 1 and 0. in the overlapping scenario, based on the multinomial logistic model, the actual probabilities can be derived and the class label is then randomly drawn from a multinomial distribution for each observation. in addition, the intercept is set to zero for the sake of a relatively balanced design. to see the improved performance of using prior structure information in terms of parameter estimation and prediction accuracy, we compare the variants of the proposed model, network - constrained multinomial logit model with group lasso penalty (ngl - mlm) and the one with adaptive group lasso penalty (ngl - mlma), to two traditional multinomial logit models with lasso (l - mlm) and group lasso (gl - mlm), respectively, implemented in the package of glmnet in r.16 to measure the estimation accuracy, the mean - squared error (mse) between true parameter values and the estimated ones is used. in addition, the performance of prediction on test data is evaluated with brier score, the euclidean distance between sample response and the estimated probabilities, and prediction accuracy, the proportion of correctly predicted class labels. we first simulate ideal network structure ; that is, all the relevant variables come from a fully connected subnetwork. as expected, the structure information improves estimation significantly, especially for large models, which is particularly relevant for real applications. the estimation of the adaptive method (ngl - mlma) outperforms others substantially. in case of random coefficients, where prior network does not provide any useful information, the proposed model is comparable to models without using the network information (l - mlm, gl - mlm), and sometimes even better. figure 2 shows that the prediction accuracy is also higher for the proposed model in almost all scenarios. when brier score is used (fig. 3), a similar trend follows : the network - constrained model always performs better when we simulate ideal and similar coefficients, and is comparable to traditional models without using structure information in case of random coefficients. to investigate the impact of structure misspecification we simulate a medium - sized data set with 100 predictors, 10 being relevant. the performance of our models is still satisfactory because of the flexible tuning parameter on the structure - constraint term (fig., the prediction accuracy of ngl - mlm is comparable to that of gl - mlm in both situations, whereas, in terms of parameter estimation and brier score, the structure - constrained models ngl - mlm and ngl - mlma outperform the other two. in summary, our structure - constrained multinomial logit model has better performance in terms of parameter estimation and prediction when the prior network knowledge is at least partially correct, and the performance is comparable to traditional models when the network knowledge is incorrect. this is because the gl - mlm is a special case of ngl - mlm with 2 = 0. cross - validation tends to select 2 = 0 when the prior assumption is not correct. one important application of our method is cancer subtype prediction and relevant subnetwork identification on large - scale gene expression data. we apply all four candidate methods, l - mlm, gl - mlm, ngl - mlm, and ngl - mlma, to a large - scale tcga (the cancer genome atlas) gbm subtype prediction problem, which contains the expression data of 11,861 genes across 202 samples and four categories, with 40, 46, 58, and 58 samples in each category. the network was built from a variety of sources, including reactome, kegg, as well as the inferred gene - interactions from protein interactions, gene co - expressions, protein domain interactions, and text - mined interactions. the outcome is one of the four subtypes of gbm.31 the data set, the network information, as well as the subtype information were downloaded from http://bioen - compbio.bioen.illinois.edu / ncis/. since the number of genes in the gbm data set is much larger than the number of samples, which may lead to computation instability, we carry out gene screening before analysis. starting with 11,861 genes, we screen genes based on the prior weights resulting from the ncis algorithm,31 by including the 599 most highly weighted genes. to construct the network smoother for model building, we tailor the original network subject to the remaining 599 genes. then the laplacian matrix is constructed based on the tailored subnetwork. to compare the prediction performance of the four methods, 202 samples are randomly divided into two subsets, a training set of 150 samples and a test set of 52 samples. the random division is kept only when test samples have good representation of each category (1535% for each category). otherwise, we discard that division. feature selection and parameter estimation in model building are completed strictly on the training set, and then the fitted models are tested on the test set. in practice, 50 valid divisions are obtained, and model building and testing are carried out on all pairs of data sets to assess variability, the results being summarized in table 1. the tuning parameter of the network - constraint controls the impact of the prior structure knowledge on model building. the network information will have no effect if the tuning parameter is set to zero. among the 50 models built by ngl - mlm, the network tuning parameter is chosen as zero in 28 models, whereas ngl - mlm is reduced to gl - mlm in this specific case. in contrast, 48 ngl - mlma models choose non - zero tuning parameter for the network- constraint, which indicates that the structure knowledge is useful for prediction, explaining the higher prediction accuracy rate for ngl - mlma. next, we apply ngl - mlma, the best model in both simulation and the application to gbm subtype analysis, on the whole sample set of gbm gene expression data and investigate the selected subnetwork. it selects 35 predictors, among which 21 are non - singletons and form a subnetwork, shown in figure 5. for example, the most connected gene akt1 plays an important role in the pathogenicity of gbm. akt1 is a downstream serine / threonine kinase in the rtk / pten / pi3k pathway, and large - scale genomic analysis of gbm has demonstrated that this pathway is mutated in many but not all gbms.32 therefore, the akt1 can be potentially used to define gbm subtypes. cancer subtype prediction is of critical importance in the understanding, diagnosis, and treatment of cancer. we introduced a classification model on the basis of multinomial logit regression to identify cancer subtypes from high - throughput gene expression data. the group lasso penalizes the coefficients linked to a common predictor at a group level so that it facilitates variable selection by shrinking all elements within a group to zero simultaneously. in addition, the network - constraint improves the smoothness of coefficients with respect to the prior structure information and results in more interpretable identification of genes and subnetworks. the proposed model and its adaptive extension are compared to lasso and group lasso multinomial logit models with no network - constraint involved. from the results of simulation and the application to gbm gene expression data, we find that the proposed model is superior given correct prior network information and is comparable to traditional models given incorrect network information. a key challenge to the future study is to correctly specify the networks. in the application to real data, we may include too many misspecified edges on the network because of incomplete knowledge of pathways. one possible solution is to use problem - specific network for a particular type of cancer, rather than using a general molecular interaction network. the proposed method can be extended by using a non - convex sparsity penalty to reduce estimation bias. scad (smoothly clipped absolute deviation) and mcp (minimax concave penalty) are two potential candidates.3335 the applications of the method also go beyond the cancer subtype prediction. for example, it can also be used to predict the disease subtype based on the human microbiome data, where the phylogeny structure can be efficiently used.28,36,37 | classic multinomial logit model, commonly used in multiclass regression problem, is restricted to few predictors and does not take into account the relationship among variables. it has limited use for genomic data, where the number of genomic features far exceeds the sample size. genomic features such as gene expressions are usually related by an underlying biological network. efficient use of the network information is important to improve classification performance as well as the biological interpretability. we proposed a multinomial logit model that is capable of addressing both the high dimensionality of predictors and the underlying network information. group lasso was used to induce model sparsity, and a network - constraint was imposed to induce the smoothness of the coefficients with respect to the underlying network structure. to deal with the non - smoothness of the objective function in optimization, we developed a proximal gradient algorithm for efficient computation. the proposed model was compared to models with no prior structure information in both simulations and a problem of cancer subtype prediction with real tcga (the cancer genome atlas) gene expression data. the network - constrained mode outperformed the traditional ones in both cases. |
for increasing the success rate of implant surgery, various scaffolds and methods have been developed to augment atrophic alveolar ridge. generally, autologous bone augmentation has been penetrating as a golden standard bone augmentation ; however, most of the patients might not be feasible for extracting their own bone, just because it is a healthy part. to avoid aforementioned high - invasive treatments, cell therapy cultured mesenchymal cells introduced into ceramic scaffolds exhibits robust osteogenic potential, with bone forming into pore regions of scaffolds. after this report, numerous reports using various cells to regenerate bone and sophisticate reviews for bone regeneration of craniofacial site have been published [27 ]. usage of tooth, including periodontal ligament or pulp, has also been reported that multipotential stromal cells which are composed above mentioned were exploited in bone or periodontal regeneration [8, 9 ]. although bone augmentation is mostly fundamental to elderly, they unfortunately follow to edentulous patients in aging society. thus, this paper focuses on adult mesenchymal cells that could be able to expand from edentulous jaw. figure 1 shows the tissues we describe in this paper by sectional scheme of edentulous alveolar ridge. osteoblasts - like cells migrated from alveolar bone chips have generally high osteogenic activity. essentially, mammalian bones are in the form of two different ways : long bones via endochondral ossification and flat bones via intramembranous ossification. orofacial bone is mainly formed via intramembranous ossification, and a part of mandibular is formed via endochondral ossification. harvesting bony chips from various sites implicate that origins of the osteoblastic cells (from maxilla or mandibular, from cortical or trabecular bone) are distinct from each reports ; furthermore osteogenic activity, expression of surface antigens, or ability for ectopic bone formations might be different among each report, beside cell isolation protocols are different among each report. majority of culture protocol of osteogenic cells from alveolar bone are wash bone specimens in pbs, scrape to remove attached soft tissue and periosteum, brake into small pieces, and wash with collagenase (1 to 2 mg / ml) dissolved in culture medium [11, 12 ]. in some reports, osteogenic cells were collected without collagenase [1315 ]. however, despite harvesting bony chips from healthy site is essential when we use these in clinic, it is not feasible for all patients just because of the invasive operation. in addition, it is still not clear how amount of bony chips is enough to regenerate each part of alveolar ridge and which part of bone cells are suitable to keep augmented bone volume on long prognosis. the reason why iliac crest bone marrow is the most documented bone marrow transplantation is because they have been corrected for bone marrow transplantation in clinic as usual. bone marrow stromal cells (bmscs) have been reported their ability of multipotent differentiation to bone, cartilage, tendon, muscle, adipose tissue, and neuronal tissue [1618 ]. bone regenerative clinical studies using bmscs, collected from iliac crest to reconstruct jaw defects, have been reported [19, 20 ]. kawaguchi. reported that iliac crest bmscs enhance periodontal tissue regeneration as well [21, 22 ]. alveolar bmscs, however, is essentially different from axial bmscs from their differential potential or their gene expression pattern [23, 24 ]. embryologically, alveolar tissues including alveolar bone marrow are originated from neural crest cells, but other bone marrows are from mesoderm [25, 26 ]. cherubism, treacher collins syndrome, craniofacial fibrous dysplasia, and hyperparathyroid jaw tumor syndrome affect only jaw bones, indicating that orofacial bone development differs from that of axial and appendicular bone formation. whitaker 's group have reported that membranous bone underwent less resorption than endochondral bone in monkey model, and they found the rapid vascularization on membranous onlay bone grafts in rabbit model. in human alveolar cleft defects, chin bone was better incorporated, significantly less resorbed than iliac crest bone [33, 34 ]. in histomorphometry, autologous grafts obtained from calvarial sources for sinus lift procedure present a significantly higher degree of bone volume in contrast to bone harvested from the iliac crest. in in vitro and in vivo study, akintoye. have investigated skeletal site - specific phenotypic and functional differences between orofacial (maxilla and mandible) and axial (iliac crest) human bmscs. compared with iliac crest cells, orofacial bmscs proliferated more rapidly with delayed senescence, expressed higher levels of alkaline phosphatase, and demonstrated more calcium accumulation in vitro. orofacial bmscs formed more bone in vivo, while iliac crest bmscs formed more compacted bone that included hematopoietic tissue and were more responsive in vitro and in vivo to osteogenic and adipogenic inductions. comparing with the osteogenic properties of bmscs attached on titanium for evaluating the avidity bone to implant exhibited that there was no difference in the affinity of maxilla and iliac crest bmscs to titanium. titanium - attached maxilla bmscs, however, were apparently more osteogenically responsive than iliac crest cells based on calcium accumulation and gene expression of alkaline phosphatase and osteopontin. have also studied the skeletal site - specific osteogenic response of bmsc to bmp-2 stimulation. they reported orofacial bmsc displayed high expression of osteogenic markers in response to bmp-2 in contrast to the low response of adult iliac crest bmsc. they also reported that mandible bmscs were more susceptible to bisphosphonates than iliac crest bmsc ; orofacial bmsc survived higher radiation doses and recovered quicker than iliac crest bmsc. osteoclastogenic potential of jaw and long - bone - derived osteoclasts have different dynamics, and this might primarily due to differences in the cellular composition of the bone site - specific marrow. established a protocol for rat mandible and long - bone marrow stromal cell isolation and culture. upon implantation into nude mice, mandible bmscs formed 70% larger bone nodules containing three - fold more mineralized bone compared with long - bone bmscs. alveolar bmscs are obtained from older individuals, and the donor age has little effect on their gene expression pattern. according to aforementioned studies, usage of alveolar bmscs might have high advantages for alveolar bone regeneration compared with iliac bmscs ; however, establishing the protocol of harvesting bmscs in low invasive way is still unclear. usage of periosteal cells from periosteum was originally reported by breitbart. on rabbit experiments. outgrowthed periosteal cells were cultured with dexamethasone contained medium, and cell / polyglycolic acid non - woven fiver scaffolds complex showed significant bone formation on calvarial defect compared with scaffold only. adult human periosteal cells from tibia include multipotent clonogenic cells, while, in the case of oral tissue, periosteal cells isolated from the mandibular angle of human with -tricalcium phosphate granules have shown that combined treatment with bfgf and bmp-2 can make periosteal cells a highly useful source of bone regeneration. in nude mouse subcutaneously model, acid - treated ha block cultured with human periosteal cells complex had significant osteogenic potential at the site of implantation in vivo, while in canine model for peri - implant bone regeneration, periosteum - derived cells in conjunction with e - ptfe membranes did not provide additional benefit. comparing with proliferated periosteal cells, cicconetti. they concluded that both periosteal cells and marrow stromal cells showed comparable phenotypic profiles and both cell populations formed bone upon ectopic in vivo transplantation. harvesting periosteal cells is relatively invasive treatment ; for instance, it is still unclear the collection quantity for required bone regeneration and also periosteal tissue is hard to collect by general practitioners. during dental surgery, gingival tissue could be obtained frequently as a discarded biological sample. wound healing within the gingiva is characterized by markedly reduced inflammation, scarless healing, rapid reepithelialization, contrary to the common scar formation present in skin [50, 51 ]. recently, several reports have indicated the presence of progenitor cells in gingival connective tissue [52, 53 ]. tang. reported gingival tissue contains tissue - specific mesenchymal stem cell population and is an ideal resource for immunoregulatory therapy, using human normal and hyperplastic gingival tissues. the ratio of these progenitors in gingival fibroblasts, however, might be very low rather than bone marrow, periosteal tissue, or bone chips. nevertheless, enrichment of progenitor cells that show characteristics with of differentiation as osteoblastic cells entail for usage in bone tissue engineering. bone marrow - derived cells, called mononuclear cells or marrow mesenchymal cells, are essentially different from osteoblastic cells derived from bone chip. in a case of small animal model, it is hard to separate mandibular bone marrow cells to bone lining osteoblastic cells. in bulk, however, this bone / bmscs possess unique stem cell properties that the size of alveolar bone has restrained the precise analysis of bmscs phenotype. as we use outgrowth cells from tissues on the other side, the population cultured cells is influenced by their migration ability. treating tissue with enzyme to disperse cells from tissue, however thus, developing stable and universal methods to harvest and culture osteogenic cells for bone regeneration is prerequisite. | most of the cases of dental implant surgery, especially the bone defect extensively, are essential for alveolar ridge augmentation. as known as cell therapy exerts valuable effects on bone regeneration, numerous reports using various cells from body to regenerate bone have been published, including clinical reports. mesenchymal cells that have osteogenic activity and have potential to be harvested from intra oral site might be a candidate cells to regenerate alveolar bone, even dentists have not been harvested the cells outside of mouth. this paper presents a summary of somatic cells in edentulous tissues which could subserve alveolar bone regeneration. the candidate tissues that might have differentiation potential as mesenchymal cells for bone regeneration are alveolar bone chip, bone marrow from alveolar bone, periosteal tissue, and gingival tissue. understanding their phenotype consecutively will provide a rational approach for alveolar ridge augmentation. |
we report a case of kawasaki disease with para aortic lymphadenopathy, as an unusual feature in this disease. this 2.5 year old girl presented with persistent high grade fever, erythematous rash, bilateral non purulent conjunctivitis, red lips, and edema of extremities. laboratory results included an elevated erythrocyte sedimentation rate, leukocytosis, anemia, and positive c - reactive protein. on second day after admission she developed abdominal pain. ultrasonography of abdomen revealed multiple lymph nodes around para aortic area, the largest measuring 12mm6 mm. ultrasonography and ct scan of abdomen performed one week later showed disappearance of the lymph nodes. there are few previous reports of lymphadenopathy in unusual sites such as mediastinum in kawasaki disease. para aortic lymph nodes enlargement might be an associated finding with acute phase of kawasaki disease. in these patients a close observation and ultrasonographic follow up will prevent unnecessary further investigation. kawasaki disease (kd) is one of the most common causes of multisystem vasculitis in childhood. because of its predilection for the coronary arteries, kd is now recognized as the first cause of acquired heart disease in children in the developed world. the diagnosis of disease requires the presence of fever lasting 5 days or more as well as at least four of the five physical findings including bilateral conjunctival injection, polymorphous rash, cervical lymphadenopathy, mucosal changes, and changes in the extremities. the acute phase of the disease in 50% of cases is associated with anterior cervical lymphadenopathy, which occurs less commonly in the posterior cervical and axillary area. the present report describes a case of kd with para aortic lymphadenopathy which has never been reported. this was a 2.5 year - old girl admitted to the hospital with a 5 day history of high grade and persistent fever. the patient received amoxicillin (50mg / kg / day) for 3 days and acetaminophen (15mg / kg / dose every 4 - 6 h), but she no improvement was noted. she had red lips, strawberry tongue, bilateral non suppurative conjunctival injection, and edema of extremities. laboratory findings : white blood cell (wbc) 1410/l, hemoglobin 10.9 gr / dl, platelets 12610/l, c - reactive protein 192 mg / dl, erythrocyte sedimentation rate 80 mm / hr, albumin 2.8 gr / dl, serum glutamic pyruvic transaminase (sgpt) 24 u / l, serum glutamic oxaloacetic transaminase (sgot) 25 u / l, alkaline phosphatase 451 u / l, lactate dehydrogenase (ldh) 430 u / l, creatine phosphokinase (cpk) 124 and total billirubin 8 mg / dl. kd was diagnosed based on the presence of clinical criteria, and lesion of coronary artery. she was treated with aspirin (100mg / kg / day), and intravenous gamma globulin (2 gr / kg). on day 2 from admission she developed abdominal pain. ultrasound examination of abdomen revealed presence of multiple lymph nodes in paraaortic area distributed from below the pancreas to the bifurcation of the aorta ; the largest was 6 mm 12 mm (fig. the fever was not subsided, so another dose (2gr / kg) was given. four days thereafter we reduced aspirin dose (4mg / kg / day) and discharged the patient in good condition. one week later blood tests revealed platelet count 48010/l, wbc count 6.710/l and plasma albumin follow up 8 weeks later, erythrocyte sedimentation rate decreased to 13mm / hr and echocardiography showed disappearance of ectasia of the coronary artery, so aspirin was discontinued. the patient was followed by cardiologist for 1 year and the last echocardiography has been normal. kd is one of the most common vasculitides in children. owning to lack of diagnostic tests, the present case had high grade, persistent fever, and four characteristic signs and symptoms of kd : erythematous macular rash, bilateral non purulent conjunctival injection, oro pharyngeal changes, and edema of extremities. in addition to the established signs and symptoms which constituted the basis for diagnosis of the disease, the case had paraaortic lymphadenopathies. the patient had no other laboratory or clinical criteria of macrophage activation syndrome, such as elevated levels of aspartate aminotransferase, decreased wbc, central nervous system dysfunction, hemorrhage or hepatomegaly. the patient had thrombocytopenia though not common in kawasaki but if present, is associated with an increased risk of coronary artery aneurysm and myocardial infarction. because of rising platelet count and disappearance of symptoms and signs following treatment, bone marrow aspiration was not performed. as the second ultrasonography of abdomen was normal without a sign of lymphadenopathy, so ct scan of abdomen was done to confirm disappearance of lymphadenopathy that was normal too. it occurs most commonly in anterior cervical and less commonly in posterior cervical and axillary areas. the disappearance of lymphadenopathy upon conventional treatment indicates that lymphadenopathy was associated with the disease. in these patients a close observation and ultrasonographic follow up will prevent unnecessary further investigation. | backgroundkawasaki disease is an acute vasculitis that occurs mainly in children. cervical lymphadenopathy is one of the major presenting manifestations of kawasaki disease. we report a case of kawasaki disease with para aortic lymphadenopathy, as an unusual feature in this disease.case presentationthis 2.5 year old girl presented with persistent high grade fever, erythematous rash, bilateral non purulent conjunctivitis, red lips, and edema of extremities. laboratory results included an elevated erythrocyte sedimentation rate, leukocytosis, anemia, and positive c - reactive protein. on second day after admission she developed abdominal pain. ultrasonography of abdomen revealed multiple lymph nodes around para aortic area, the largest measuring 12mm6 mm. treatment consisted of aspirin and high dose intravenous -globulin. ultrasonography and ct scan of abdomen performed one week later showed disappearance of the lymph nodes.conclusionthere are few previous reports of lymphadenopathy in unusual sites such as mediastinum in kawasaki disease. para aortic lymph nodes enlargement might be an associated finding with acute phase of kawasaki disease. in these patients a close observation and ultrasonographic follow up will prevent unnecessary further investigation. |
morphogenesis during development is fundamental to the differentiation of several cell types. as neurite outgrowth marks neuritogenesis, formation of filopodia precede the formation of dendrites and axons. while the structure of filopodia is well - known, the initiation of filopodia during neurite outgrowth is not clear. sh2b1 is known to promote neurite outgrowth of pc12 cells, hippocampal and cortical neurons. as a signaling adaptor protein, sh2b1 interacts with several neurotrophin receptors, and regulates signaling as well as gene expression. our recent findings suggest that sh2b1 can be recruited to the plasma membrane and f - actin fractions by irsp53. irsp53 bends plasma membrane and facilitates actin bundling to set the stage for filopodium formation. we further demonstrate that sh2b1-irsp53 complexes enhance the formation of filopodia, dendrites and dendritic branches of hippocampal and cortical neurons. while the molecular mechanism underlying filopodium initiation is not clear, we propose that sh2b1-neurotrophin interacting sites may mark the putative sites of filopodium initiation. |
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for analysis of frontal sinus configuration, individuals who came for paranasal sinus radiograph investigation were selected. the study population comprised thirty individuals (15 males and 15 females) between 20 and 30 years of age without any history of sinusitis, surgery, or any trauma. paranasal sinus views were taken using planmeca oy 2002 cc machine (helsinki, finland). loaded cassette of size 8 inch 10 inch the height of the cephalostat was adjusted until the positioning cones at the ends of the ear posts were at the level of the patient 's ears. the patient is positioned between the two ear posts so that he / she will be facing the cassette. the tilt of the patient 's head was adjusted until the ala - tragal line was 3540 from the horizontal. the exposure parameters used were 78 kv and 12 ma with an exposure time of 1.2 s. exposure was made and the film was developed and dried which was then mounted on a view box for radiographic analysis of the frontal sinuses [figure 1 ]. the radiographs were analyzed for area size, bilateral asymmetry in area size, unilateral superiority of area size, outline of upper border of frontal sinus (left and right side), presence or absence of partial septa or supraorbital cells [figure 2 ]. tracing image of paranasal sinus view the area of the sinus, which falls above the line drawn tangentially to the superior border of two orbits, was taken into consideration, and area of the frontal sinus was estimated as the combined sum of both the right and left sinuses. according to area size, the frontal sinuses were classified as small, middle, large, and very large and were assigned the class number 1, 2, 3, and 4, respectively. the formula for asymmetric indices was given by : asymmetric index = a1/a2 100 where a1 is the area of the smaller sinus and a2 is the area of the larger sinus. according to the degree of bilateral symmetry, they were classified as symmetry and almost symmetry (10080), slight asymmetry (8060), moderate asymmetry (6040), strong asymmetry (4020), extreme asymmetry (> 20) and were assigned the class number 1, 2, 3, 4, and 5, respectively. unilateral superiority of the upper border of the frontal sinus was also used as a parameter. case in which the left side was superior to the right one was assigned as 1 and the reverse case as 2. the outline of the upper border of the frontal sinus in each side was divided into different categories as absent, smooth, scalloped with two arcades, scalloped with three arcades, scalloped with four arcades, and scalloped with five arcades and were assigned class number 0, 1, 2, 3, 4, and 5, respectively. the presence or absence of partial septa and supraorbital cells was taken into consideration and classified as absent, present on the left side, present on the right side, present on both sides and were assigned class number of 0, 1, 2, and 3, respectively. the frontal sinus pattern of a given person was formulated as a code number, which was determined by arranging the class numbers in order as the following : area size, bilateral asymmetry, superiority of side, outline of the upper border (left and right side), and presence or absence of partial septa and supraorbital cells. planmeca 2002 cc x - ray machinefilm cassette (8 10 inches)radiographic film (8 10 inches)processing solutionview boxmeasuring scalelead pencil. planmeca 2002 cc x - ray machine film cassette (8 10 inches) radiographic film (8 10 inches) planmeca 2002 cc x - ray machinefilm cassette (8 10 inches)radiographic film (8 10 inches)processing solutionview boxmeasuring scalelead pencil. planmeca 2002 cc x - ray machine film cassette (8 10 inches) radiographic film (8 10 inches) the frontal sinuses were examined in thirty individuals (15 males and 15 females) after obtaining a paranasal sinus skull radiograph. the radiograph was examined for area size, bilateral asymmetry, superiority of side, outline of upper border (left side and right side), and partial septa and supraorbital cells. based on the above findings, each individual was assigned a code in the same sequence of examination and was evident that the code for each person was different, indicating that the frontal sinus pattern was unique for each individual. the frontal sinus areas ranged from 0.6 to 26.27 cm for females and from 1.60 to 23.11 cm for males. the frontal sinuses of both sexes were classified into four categories based on the sinus area measurements. among the thirty individuals, most of them belonged to the categories of small and middle and only 20% and 13.33% of individuals belong to the large and very large category, respectively [tables 1 and 2 ]. the mean value of the frontal sinus area for males was larger than that for females. the difference in means between both the sexes was not significant at the 0.05 level of confidence (t = 0.632, p = 0.533). area of the frontal sinus in the sample group of both males and females classification of area size of frontal sinuses and relative frequency distribution of area size in sample the classification of the degree of bilateral asymmetry of frontal sinus based on asymmetry indices is presented in table 3 and their relative frequency distribution is presented in table 4. in the thirty individuals examined, 15 appeared bilaterally symmetrical or almost symmetrical, of which ten were females and five were males. strong asymmetry was present in 2 cases (1 male and 1 female) and extreme asymmetry in 2 cases (1 male and 1 female). degree of bilateral asymmetry of the frontal sinus in the sample group of both males and females classification of the degree of bilateral asymmetry in frontal sinuses and relative frequency distribution of degree of bilateral asymmetry in samples in addition to bilateral asymmetry of sinus size, the unilateral superiority of area size in frontal sinuses has used a parameter of classification of sinus patterns. the class number was given as follows : the case in which the left sinus was superior to the right one was assigned 1 and the reverse was 2. in the thirty individuals, twenty belong to the class number 1 (11 females and 9 males) and ten belonged to the class number 2 (4 females, 6 males) [table 5 ]. superiority of side of the frontal sinus in the sample group of both males and females the outline of the upper border of frontal sinus on each side was divided into the following category as 0, 1, 2, 3, 4, and 5. the relative frequency distribution and outline of upper border in both sex and on each side outline of upper border of the frontal sinus in the sample group of both males and females classification of the outline of the upper border of the frontal sinuses and relative frequency distribution of the outline of upper border in samples the presence or absence of partial septa and supraorbital cells was classified into four categories and is presented in tables 8 and 9, respectively, with their relative frequency distribution in table 10. partial septa of the frontal sinus in the sample group of both males and females supraorbital cells of the frontal sinus in the sample group of both males and females classification of the presence or absence of partial septa and supraorbital cells and relative frequency distribution of them in samples based on the above radiographic findings of frontal sinus, a code number [table 11 ] is assigned to each individual corresponding to the class number of each finding in the above order, which indicates that the frontal sinus pattern is unique for each individual. code number for identification of individuals in the sample group based on the radiographic interpretation of frontal sinus of both males and females the above study gives an idea about the uniqueness of the frontal sinus and its application in the identification of an individual. the frontal sinus is usually a paired, irregularly shaped, pneumatized cavity located in the frontal bone, which develops embryonically from an ethmoidal cell. it varies enormously in its size and shape and is considered as one of the unique anatomical structure. they rapidly develop in puberty and complete their definitive configuration at the 20 year of life. considering this factor, our study group included individuals above 20 years of age and they were assessed for various parameters. the mean value of the frontal sinus area for males (10.69 cm) was found to be higher than the mean value of the frontal sinus area of females (9.09 cm) and this correlates with the previous study. although the sinus area of males tended to be large than that of females, the statistical difference of means between them was not statistically significant. thus, the measurement of frontal sinus area might be of no validity for determination of sex since sinus area measurements were very variable among the individuals, which is correlating with the earlier findings. various studies have been undertaken to assess if the configuration of the frontal sinuses is used as a factor for determining the sex of an individual. the studies have concluded that it lacks in the level of accuracy and may have limited application as the soul predictor of sex. the degree of bilateral asymmetry in frontal sinus was classified into five categories based on the asymmetry index. about 66.66% of females and 33.33% of males belonged to 80100, and about 26.66% of females and 20% of males belonged to 6080. in the previous study, 40% of the samples in each sex belonged to asymmetry index 4060, and in a study done by szilvassy among austrians, about 40% of males belonged to 6080 and 40% of females belonged to 80100. from these findings, it was suggested that the degree of bilateral asymmetry in frontal sinus differs from race to race. this is in correlation with the study done by yoshino. and szilvassy. in the present study, morphological characteristics of the upper border of frontal sinus showed no significant difference between both sexes. males and females were not significantly different in the presence or absence rates of partial septa and supraorbital cells. based on the radiographic findings of frontal sinus pattern, a code number was assigned to each individual and was found that the numbers of no two individuals resembled each other, which is in concordance with the previous findings that the frontal sinus patterns are unique for every individual. thus, the usage of configuration of frontal sinuses for identification of an individual is gaining wide acceptance. a study with large sample size along with the various advanced imaging modalities will aid in substantiating the uniqueness of frontal sinus pattern in an individual. a frontal sinus comparison is particularly useful when no other means of an individual identification are available. caution must be taken regarding the physiological and pathological changes (trauma, infection, old age, surgery, etc.) and postmortem changes and about the technical issues while taking a radiograph (distance, angle, orientation of the skull). in spite of all these issues | background : identification of an individual whether living or deceased is of paramount importance in maintaining the integrity of the society. a simple, reliable, and efficacious method always finds a way for easier acceptance and inclusion in any discipline. likewise, identification of an individual using the radiographic frontal sinus patterns is a simple technique which emphasized to conduct the study with a proven result.objective:the objective of the study is to evaluate the radiographic configurations of frontal sinuses for their uniqueness based on different parameters.study group and methods : study group consisted of thirty individuals (15 males and 15 females) of age between 20 and 30. individuals with the history of sinusitis, surgery, or any trauma were not included in the study. paranasal sinus views were taken using standard exposure parameters, and the radiographs were assessed for their uniqueness.results:the radiographs were assessed for area size, area asymmetry, superiority of the upper border, outline of the upper border, presence or absence of partial septa and supraorbital cells, and based on these results, a unique code number was assigned to each individual to prove the uniqueness.conclusion:a frontal sinus comparison is particularly useful when no other means of an individual identification are available. caution must be taken regarding the physiological and pathological changes (trauma, infection, old age, surgery, etc.) and postmortem changes and about the technical issues while taking a radiograph (distance, angle, orientation of the skull). in spite of all these issues, the configuration of frontal sinus is an excellent individualizing feature. |
cutaneous leishmaniasis (cl) is an infectious disease caused by the leishmania parasite that is transmitted through the bites of phlebotominae sand flies. cl is characterized by broad clinical polymorphism varying from localized to diffuse and disseminated lesions. localized cl (lcl) is the most prevalent clinical manifestation worldwide, characterized by the appearance of a skin lesion at the vector sting site. the lesion occurs after reproduction of the parasites into the dermis, which generates a granuloma. the granuloma is initially manifested as small papule that progressively increases in size to form a rounded nodule. as a consequence of the parasite virulence and host immune response, the nodules show necrosis in the center and become ulcers characterized by a round form, with high and clear borders, an indurated base and granular background, painless and with a scab firmly attached to the base. overinfection with bacteria or fungi, often caused by empirical treatments, produces purulent and painful ulcers. characteristic scars with hyper- or hypochromic edges, commonly called onion bulb scars, appear after treatment. nodular lesions sometimes become scaly epidermal plaques or warty lesions. in some patients, the parasite may spread via lymph and blood to the skin of different anatomical regions, particularly the face, chest and upper limbs, where the parasite reproduces widely and causes disseminated leishmaniasis (dl) or diffuse cutaneous leishmaniasis (dcl). although these two clinical forms can be clearly differentiated (see table 1), some authors consider them as synonymous, which makes a meta - analysis of the literature review difficult. here, we report a series of dl patients attended at the pecet, university of antioquia (medelln, colombia). a literature review focusing the attention on the available information to differentiate dl and dcl twenty - two patients (85%) were men ; 20 patients (77%) were of mixed race, 4 patients (15%) were black, and 2 patients were indigenous ; their age ranged from 4 to 61 years (mean 32 years). twenty - five patients (96%) were from the northwest of colombia (antioquia, choco and cordoba states), but only 2 patients came from the southeast of the country. only 1 patient evidenced hiv coinfection. parasite cultures were positive in 16 patients, while the remaining 11 cultures were negative. leishmania (viannia) panamensis was identified as the causative agent in 15 isolates ; the last isolate was identified as viannia subgenus, but no species was determined. this leishmania species distribution is similar to that observed for patients with lcl from the northwestern colombian region, where 70% of the strains circulating are l. (v.) panamensis and 30% correspond to l. (v.) braziliensis, whereas in the southeastern region, 80% of the circulating species are l. (v.) braziliensis as identified by monoclonal antibody techniques and pcr - rflp (unpublished data). all patients met the diagnostic criteria of having 10 or more polymorphic lesions in at least 2 parts of the body surface. the number of lesions for each patient varied between 12 and 294 (21 patients with 100 lesions or less, 3 patients had more than 100 lesions and less than 200, and 4 patients had more than 200 lesions ; table 3). the evolution time since the appearance of the first lesion to the spreading was more than 2 weeks. in most patients, the primary lesion was plaque, and the secondary lesions (disseminated) were nodules, papules and plaques, with different sizes but smaller than 2.0 cm in diameter. only 1 patient had frank ulcers (fig. 1, fig. 2, fig. 3, fig. seven patients (25%) had nasal mucosal involvement consisting of ulcers, scabs or septal perforation indicating an important frequency of mucosal involvement. the montenegro skin test was positive in 18 of 21 patients (84%) with an induration size that varied between 5 and 12 mm (average 6.8 mm ; table 2), suggesting no complete anergy of cellular immune response. cell proliferation and t cell subpopulation in peripheral blood samples were evaluated only in 4 patients who had absolute values of t cell and lymphoproliferative response within the reference values. however, the percentages of t cell cd3 +, cd4 + and cd3+cd4 + subpopulations were below the reference values, whereas the percentage of naturally occurring regulatory t cells (cd4+cd25+foxp3 +) were increased. five of the 27 patients (19.3%) had diabetes mellitus, obesity, vitiligo, hiv / aids and leukemia as baseline comorbidities, which could favor the spread of the parasite. in 92%, their immunosuppressive condition was not identified, which could facilitate the spread of the parasite since most of patients were farmers from rural areas. only 2 patients had a clinical condition - based immunosuppression (hiv and leukopenia). twenty - two patients were treated with intramuscular meglumine antimoniate at a dose of 20 mg / kg / day for 20 days (table 3). nineteen of these 22 patients (86.3%) were cured with a single treatment cycle of meglumine antimoniate, while 3 patients did fail. one of these 3 patients was cured with a second cycle with topical amphotericin b (anfoleish) applied twice daily for 28 days, and the other 2 patients were cured with a second cycle with oral miltefosine at doses of 22.5 mg / kg / day for 28 days. one of these 2 patients had been treated before with amphotericin b deoxycholate 1 mg / kg daily only for 9 days because of renal toxicity. among the remaining 7 patients, 2 of them were cured with 4 applications of intramuscular pentamidine at a dose of 3 mg / kg / day (table 3) ; another patient, who had 103 lesions, was cured after treatment with pentamidine combined with topical amphotericin b applied as before. another patient was cured with miltefosine alone, and the last patient, who had hiv / aids coinfection, drug abuse, kaposi 's sarcoma, extrapulmonary tuberculosis, cryptococcal meningitis and poor adherence to antiretroviral therapy, was treated with liposomal amphotericin b combined with miltefosine ; this patient showed partial clinical improvement but subsequent appearance of new skin lesions and extensive mucosal involvement. dl is characterized by an initial injury into the dermis followed by a high number of skin lesions (> 10 polymorphic lesions) in at least two parts of the body surface, with or without mucosal involvement [2, 3 ]. dl has been described in brazil, venezuela, guyana and colombia, with a significant increase in the number of cases in highly endemic places as the brazilian north, colombo - brazilian amazon and colombian northwest. this clinical manifestation is caused by leishmania species of the subgenus viannia, mainly l. (v.) panamensis, l. (v.) braziliensis and l. (v.) guayanensis. patients suffering dl describe the initial appearance of a single lesion (nodule or ulcerative plaque), presumably located in the vector bite site. weeks or months later, as a consequence of parasite dissemination, polymorphic lesions (isolated or confluent), different in size and growth rate, appear in remote anatomic areas from the initial lesion that may cover the entire body surface, except armpits and inguinocrural region ; lesions in the face may become disfiguring. associated to the skin lesions, mucosal involvement or symptoms such as fever, chills and lymphadenopathy may occur [2, 4 ]. dissemination may occur after therapy with immunosuppressive drugs. the pathogenesis of dl is not well established. however, partial inhibition of specific cellular immunity against the parasite has been demonstrated. a decrease in cd4 + t cells in peripheral blood with partial absence of t cell response to leishmania antigen, evidenced by a mildly reactive (3 or 4 mm induration) or positive (> 5 mm) montenegro skin test, is observed. a low production of the type th1 cytokines ifn-, tnf-, il-10 and il-5, which favor the spread of the parasite, is also registered. histological examination of the lesions can reveal the presence of a granuloma composed of lymphocytic infiltrates with very few or no parasites [4, 7, 8 ]. a decrease in the percentage of cd3+cd4 + t cells and an increase in the percentage of regulatory t cells could affect the immune response mediated by t cells because it has been shown that the regulatory t cells cd4+cd25+foxp3 + (known as natural regulatory t cells) are potent suppressors of acquired immune response, as is the immune response mediated by t cells [9, 10 ]. a decrease in the percentage of activated cd4 + t cells in the blood of patients may affect the cellular immune response associated with a decrease of the specific local cellular response against some leishmania species. the diagnosis of dl as well as other clinical forms of cl is based on the observation of the parasite in smears or aspirates from the lesion. in most cases, the treatment of dl is based on systemic administration of pentavalent antimonials (20 mg / kg / day for 20 days), miltefosine (22.5 mg / kg / day for 28 days) and amphotericin b both deoxycholate (0.71 mg / kg / day for 30 days) or liposomal (3 mg / kg / day for 1014 days), although the liposomal form is preferred for the lower toxicity. therapeutic response may also vary from poor to good, depending on the leishmania species. thus, for example, the percentage of cured patients with dl by l. (v.) braziliensis treated with 1 course of antimony therapy for 30 days in its highest dosage (20 mg / kg / day) is 24%, while in patients with dl by l. (v.) panamensis, the cure rate is 86.3%, as shown here. with liposomal amphotericin use (dose ranging from 17 to 37 mg / kg, as shown in this work, pentamidine at a dose of 3 mg / kg / day (4 applications) is also a good option to treat dl even in cases having a high number of lesions. in endemic areas for leishmaniasis, dl is considered as an opportunistic infection mainly in hiv positive patients in whom therapeutic failures and relapses are frequent. differential diagnosis of dl include diseases such as psoriasis, histoplasmosis (and other relevant deep mycoses in immunosuppressed patients), pemphigus, pityriasis rosea and lupus but also other cutaneous forms of leishmaniasis such as dcl and post - kala - azar dermal leishmaniasis (pkdl). dcl was described for the first time in venezuela by convit and lapenta in 1948. this clinical manifestation of dcl is associated with leishmania species of the leishmania subgenus, mainly l. (l.) amazonensis (in the new world) and l. (l.) aethiopica (in the old world) [17, 18 ]. dcl appears during the first decades of age, with periods of remission and frequent relapses. the montenegro skin test is predominantly negative, and histologically, granulomas with few lymphocytes and abundant parasites are observed. dcl is more severe because a complete inhibition of specific cellular immunity occurs and the therapeutic response is poor and therefore, relapses occur more frequently [19, 20 ]. in dcl, temporary improvement has been observed in venezuela using immunotherapy with bcg and heat - dead l. (l.) amazonensis promastigotes, combined with other drugs such as pentavalent antimonial, amphotericin b and miltefosine. however, relapses of lesions make it necessary to use multiple schemes of treatment [22, 23, 24 ]. in turn, pkdl is caused by l. (l.) donovani in east africa and india. this disease is characterized by the appearance of rash during weeks or months, followed by macules and plaques especially in the face, upper chest and upper limbs., pkdl requires specific treatment, while in african countries, the disease usually heals spontaneously. although dl has been showing an increase in the number of cases in recent years in countries such as brazil and colombia, this is a rare entity and poorly described in the literature. in this clinical manifestation, anergy is not absolute, lesions heal easily and patients show good response to drugs recommended by the who to treat lcl such as pentavalent antimonials and miltefosine, and therefore, patients have good prognosis. it is very important to distinguish it from other cutaneous manifestations, mainly from dcl and pkdl, because there are significant differences in etiology, treatment response and prognosis. the authors state that the patients gave their informed consent complying with all ethical guidelines for human studies. | disseminated leishmaniasis (dl) is a poorly described disease that is frequently misdiagnosed as other clinical manifestations of cutaneous leishmaniasis (cl) such as diffuse cl or post - kala - azar dermal leishmaniasis. twenty - seven cases of dl diagnosed between 1997 and 2015 are described. a higher prevalence was observed in men (mean age 32 years). the number of lesions per patient ranged from 12 to 294, distributed mainly in the upper extremities, face and trunk. the lesions were mostly plaques or nodules. seven patients had nasal mucous damage, 74% of the patients were of mixed race, 92% lived in northwestern colombia, and leishmania (viannia) panamensis was identified as the causative agent in 58% of cases. eighteen patients recovered with pentavalent antimonial. the importance of distinguishing dl from those other clinical presentations is based on the fact that disseminated, diffuse and post - kala - azar cl are very different in etiology, clinical manifestations and response to treatment and prognosis. |
according to the american cancer society, colorectal cancer (crc) is the third most common cancer diagnosed and the third leading cause of cancer - related mortality in both men and women in the united states (us). while these statistics are alarming, the death rate from crc has decreased for more than 20 years due to early detection of removable polyps by colorectal screening tests. according to the us preventive services task force (uspstf), routine screening may reduce the number of people who die of colorectal cancer. additionally, treatment for crc has vastly improved over the last several years, and as a result, there are now more than one million survivors of crc in the us. the risk of developing colorectal cancer in a lifetime is about 1 in 19 and a number of risk factors associated with crc have been identified. established risk factors include increased age, personal history of inflammatory bowel disease, colorectal polyps, and family history of colorectal cancer. in addition, certain behavioral factors such as smoking, heavy alcohol use, and obesity, have shown to be the strongest links to an increased risk of colorectal cancer. previous studies have reported an association between obesity and a wide range of chronic diseases including cancer. during a 10-year follow - up study by field., the incidence of diseases such as diabetes, hypertension, and heart disease have increased with increasing body mass in both men and women. moreover, in women, the incidence of colorectal cancer was positively associated with bmi. however, only a few studies have evaluated the association between bmi and screening for colorectal, breast, and cervical cancer [711 ]. bmi status and its associations with the rates of crc screening were reported, but obesity has been reported as both negative [79 ] and positive predictor for crc screening. even fewer studies used national level databases focusing on medicare beneficiaries in the us. as crc screenings are covered by the us medicare program as one of the preventive services, an understanding on factors associated with crc screening can provide important information to the program for improving access to care and reducing health care costs. with the literature of conflicting results that obesity has been reported as both negative and positive predictor for crc screening, the objectives of this study were (1) to describe crc screening rates by bmi status with a null hypothesis of equal rates of crc screening by obesity status and (2) to evaluate if obesity is a predictive factor for crc screening among medicare beneficiaries in the us. data from the 2005 medicare current beneficiary survey (mcbs) were used in this study. the mcbs data collection began in 1991 conducted by the office of strategic planning of the centers for medicare and medicaid services. this mcbs data utilizes survey samples of the medicare population that utilizes stratified multistage probability sampling procedure with three stages of selection [13, 14 ]. the first stage involves the selection of primary sampling units (psus) consisting of metropolitan statistical areas and groups of rural counties. the psus are selected with probabilities proportionate to 1980 population strata defined by census region, metropolitan status, and selected psu - level socioeconomic characteristics. the second sampling stage consists of the selection of zip code areas within each sampled psu. at the third and final stage of selection, beneficiaries within the sampled zip clusters are stratified by age and subsampled at rates designed to yield self - weighting, or equal probability, samples of beneficiaries within each of seven age groups. this mcbs data including survey samples from the three stages of sampling lead to a sample of 15,769 beneficiaries, representative for 39,337,911 us medicare beneficiaries. the mcbs annually produces two files, the cost and use and the access to care files. our study used the access to care file which includes information on the beneficiaries ' access to health care, their satisfaction with this care, and their usual source of care while the cost and use file provides expenditure and source of payment data. the use of mcbs data for this study was requested to the center for medicare and medicaid services through the research data assistance center. this file also contains a supplement that includes a survey of medicare beneficiaries, as well as a survey that measures beneficiaries ' sources of information regarding medicare. these variables include, but are not limited to, history of colorectal cancer, history of other cancer diagnosis, history of screening for colorectal cancer or any other cancer, and the beneficiary 's usual source of care providers and care settings. the study population of this study includes all survey samples from 2005 mcbs data consisting of all community - dwelling mcbs respondents aged 50 years and older that can be extrapolated to nationally representative estimates of us medicare beneficiaries when sampling weights are aggregated. the dependent variable, colorectal cancer screening (crc), was created using two variables, colhtest (surveyed person was given a home test for fecal blood) and colscopy (if the surveyed person ever had a colonoscopy or sigmoidoscopy) from the mcbs survey data. if the surveyed person experienced either event, they were coded as having a crc screening. body mass index calculation (bmi) for each subject was calculated using self - reported height and weight information available in the dataset using the standard formula of (weight in lb/(height in inches)). based on the definition of bmi classification made by the world health organization (who), each beneficiary 's bmi status was determined (table 1). for analytical purposes, bmi status was divided into the two categories of obese and nonobese. this study was approved and conducted in compliance with the requirements of the howard university institutional review board. the primary goal for analysis was to investigate the association between crc screening and bmi, while adjusting for potential confounding variables such as age, gender, race, insurance coverage, and usual source of health care. logistic regression analysis was used to model multivariable associations between bmi and colorectal cancer screening, while considering other confounding variables. all analyses in the study used weighted data to yield national level estimates reflecting all us medicare beneficiaries. the weighted estimates were calculated as means or proportions, with the corresponding 95% confidence intervals (ci) and standard errors (se). all analyses were done using sas version 9.1 (sas institute inc., cary, north carolina). in the 2005 medicare current beneficiary survey, 15,769 beneficiaries were included as sample (weighted estimates are 39,337,911 beneficiaries) aged 50 years and older, that is, eligible for colorectal cancer screening. the majority of these beneficiaries was identified as non - hispanic white (78.8%) and were predominantly female (56.2%) (table 2). about 37% had at least a high school or vocational degree and more than one half of the beneficiaries were married. most had private insurance (55.9%) as supplemental insurance and about bout 48.9% of the beneficiaries reported annual income of less than $ 25,000 (table 2). about 25% of the beneficiaries were classified as obese (bmi 30) and almost 38% were reported as overweight (25 bmi < 30) (table 2). of the total number of participants, 65.3% reported having one of the two types of crc screening (table 2). nonobese beneficiaries after controlling for confounding variables (oradj = 1.25 ; 95% ci : 1.121.39). female beneficiaries had greater odds of receiving a crc screening compared to men (oradj = 1.24 ; 95% ci : 1.151.34). gender as an interaction term was investigated in the logistic regression model but was found to not be statistically significant (p = 0.860). age was associated with crc screening, with beneficiaries aged 65 and older having greater odds of having a crc screening than younger ones. after controlling for confounding variables, only the other racial group had lower odds of crc screening than non - hispanic white beneficiaries (oradj = 0.74 ; 95% ci : 0.620.88). relative to beneficiaries with private supplemental insurance, those with medicaid or no supplemental insurance had lower odds of screening as well. beneficiaries with a usual source of healthcare had odds that were about six times greater for reporting a crc screening (oradj = 5.64 ; 95% ci : 4.696.80) (table 3). findings from our study indicate that obese patients have greater odds of being screened for crc than those who were classified as nonobese. a report from the centers for disease control and prevention indicated that 33.8% of adults in the us are obese. while a number of previous studies reported the association between obesity and cancer screening rates in other cancers like breast, cervical, prostate cancer, and colorectal cancer, very few studies used national level survey data. contradicting findings indicating that obese adults were less likely to be screened for colorectal cancer were also reported [79 ], but previously published studies used various types of data source, methodology, age of the study population, and clinical setting. unique features of our study include (1) the use of national level data representing medicare beneficiaries and (2) the inclusion of all medicare beneficiaries aged 50 years and older instead of limiting the study population as beneficiaries who are 65 years and older. the beneficiaries who are between 50 and 65 years of age are eligible for social security benefits due to chronic and total disability, having end - stage renal disease or having been diagnosed with amyotrophic lateral sclerosis. our findings not only provide obesity status as a predictive factor for crc screening to the body of literature but also report a lower level of access to crc screening in this unique subpopulation of medicare beneficiaries with specific clinical conditions of chronic and total disability. obesity is one of the well - established risk factors for developing of and dying from crc. with the increased prevalence in obesity, we believe that potential reasons for the higher crc screening rates among obese beneficiaries from our study may be related to an increased likelihood for obese patients to receive more frequent surveillance while the obese patients visit their physicians for other chronic diseases. however, obesity can be equally recognized as challenges because outstanding health concerns other than cancer screening may be perceived as more urgent matters. it is also possible that the needs for better managing and accommodating obese patients are acknowledged by care providers. in fact, the importance of improving access to care and sensitivity for obese patients was highlighted in a publication made by the national task force on the prevention and treatment of obesity with a set of advice to health care professionals. however, it is beyond the scope of our study in identifying the reasons for higher crc screening rates for obese patients and we believe more research is needed to better understand the relationship between obesity and cancer screening. limitations of this study. there are several limitations to our study, primarily due to the data source being self - reports. given the way the question of whether or not a beneficiary had ever been screened for colorectal cancer was presented in the mcbs survey, there is no indication as to whether or not the beneficiary had been screened regularly as recommended by national clinical guidelines. an affirmation of having a history of colorectal cancer screening may not correlate to regular screening behavior of the beneficiary. furthermore, the history of crc screening outcome does not give any information as to when the beneficiary may have undergone a screening procedure for crc. as a result, there may be temporal issues between obesity and other crc screening related variables and therefore, adherence patterns of crc screening to national guidelines by medicare beneficiaries can not be evaluated in our study. our study described the crc screening rate by obesity status among medicare beneficiaries using recently available and large national - level survey data. unlike findings from previous studies reporting lower prevalence of crc screening among obese or morbidly obese patients, our study findings are encouraging in that obesity status was not found to be a hindering factor, but rather an assisting factor, for crc screening among medicare beneficiaries. in order to gain better insight on adherence of crc screening to national guidelines, future studies evaluating physicians ' ordering of screening tests compared to screening claims among medicare beneficiaries are needed. | background. findings from previous studies on an association between obesity and colorectal cancer (crc) screening are inconsistent and very few studies have utilized national level databases in the united states (us). methods. a cross - sectional study was conducted using data from the 2005 medicare current beneficiary survey to describe crc screening rate by obesity status. results. of a 15,769 medicare beneficiaries sample aged 50 years and older reflecting 39 million medicare beneficiaries in the united states, 25% were classified as obese, consisting of 22.4% obese (30 body mass index (bmi) < 35) and 3.1% morbidly obese (bmi 35) beneficiaries. almost 38% of the beneficiaries had a body mass index level equivalent to overweight (25 bmi < 30). of the study population, 65.3% reported having crc screening (fecal occult blood testing or colonoscopy). medicare beneficiaries classified as obese had greater odds of crc screening compared to nonobese beneficiaries after controlling for other covariates (oradj = 1.25 ; 95% ci : 1.121.39). conclusions. findings indicate that obesity was not a barrier but rather an assisting factor to crc screening among medicare beneficiaries. future studies are needed to evaluate physicians ' ordering of screening tests compared to screening claims among medicare beneficiaries to better understand patterns of patients ' and doctors ' adherence to national crc screening guidelines. |
the goal is to provide optimal healthcare to the global population and clinical laboratory practice is inexorably moving towards harmonization. as stated by greenberg, an increasingly important objective in laboratory medicine is ensuring the equivalency of test results among different measurement procedures, different laboratories and health care systems, over time (1). this requires harmonization and metrological traceability of assays to provide equivalence of results derived from different analytical systems (2). this has not been possible historically because assays provided by industry have not been sufficiently comparable due to a lack of established reference materials and methods to anchor tests. as noted by miller and myers, true and precise routine measurements of quantities of clinical interest are essential if results are to be optimally interpreted for patient care. additionally, results produced by different measurement procedures for the same measurand must be comparable if common diagnostic decision values and clinical research values are to be broadly applied (3). a patient s test history would be consistent if a single clinical lab performed all testing (i.e., same methodology, stable analytical performance, etc.) so a significant change in concentration (decrease or increase) would signal a meaningful clinical change. in reality, patients are increasingly mobile and two or more laboratories may test their samples. if the tests performed by different laboratories are sufficiently harmonized so as to produce essentially equivalent results (not necessarily quantitatively equal, but clinically equivalent), changes in concentration can be correctly interpreted by a healthcare provider. as explained by gantzer and miller clinical laboratory measurement results must be comparable among different measurement procedures, different locations and different times in order to be used appropriately for identifying and managing disease conditions (4). harmonization is needed to use of electronic medical records / electronic health records (emrs / ehrs) to capture all of a patient s lab results in an electronic file available to patients and healthcare providers. clinical laboratory results typically account for much of the information in emrs but the benefit is negated if the cumulative values in emr for the same analyte are not comparable. perhaps not a problem for traceable analytes, e.g., electrolytes and glucose, but very much an issue for immunoassays such as thyroid and fertility hormones and cancer markers. interpretation of sequential values using common reference intervals and medical decision levels (mdls) is difficult, if not impossible. hallworth has challenged that blanket statement but allows the value of laboratory medicine in patient care is unquestioned (5). that value is greatly diminished without comparability of test results. cholesterol is a prime example of successful harmonization. creating a reference measurement system (rms) for this key lipid over about 30 years (1970 2000) coincided with a major reduction in mortality rates for coronary heart disease (chd) in the us and also achieved a huge savings in healthcare dollars (1). the consequences of the lack of harmonization was demonstrated by an nist report on calcium (ca) that estimated the cost of a 0.1 mg / dl ca bias can cost $ 8 - $ 31 for additional, but unnecessary, patient follow up testing (6). a bias of 0.5 mg / dl could results in an additional $ 34 - $ 89/patient. on an annual basis, a 0.1 mg / dl bias could translate into $ 60 - $ 199 million / year for about 3.55 million patients screened for ca. in this paper harmonization is used interchangeably with standardization, though there is a distinction between the two (4). standardization means results are traceable to higher metrological order reference materials and/or methods and ideally can be reported using si units. harmonization means results are traceable to some declared reference but accepted higher order reference materials and/or methods are not available and si units are not applicable. harmonization ensures comparability of results, enables application of clinical best practice guidelines and reference intervals, increases patient safety, and decreases medical care costs. harmonization requires the cooperation of laboratories, academia, professional societies, metrological institutes, government agencies, eqa / pt providers, and industry. two recent harmonization (actually, standardization) success stories mediated by industry are creatinine and glycated hemoglobin (hb a1c). field assays for both of these analytes feature complete traceability chains and are firmly anchored by reference measurement systems. that said, ironically results for both assays are still typically reported in different units, creatinine in mg / dl (conventional units) and mmol / l (si units), and hb a1c in % hb a1c (ngsp units) and mmol / mol (si units). the in vitro diagnostics directive (ivdd) of 2003 applies to europe for the purposes of the ce mark, but has global implications. metrological traceability is defined in the vim, clause 2.41 as the property of a measurement result whereby the result can be related to a reference (a standard) through a documented unbroken chain of calibrations, each contributing to the measurement uncertainty. (1) the ivdd does nt provide specifics but iso 17511 (metrological traceability of values assigned to calibrators and control materials) applies (7 ; see fig 1.). the ivdd requirements are incorporated in iso 15189 (medical laboratories- particular requirements for quality and competence) (8). as white explains metrology, the science of measurement, provides laboratory medicine with a structured approach to the development and terminology of reference measurement systems which, when implemented, improve the accuracy and comparability of patients results (9). for example, the tietz textbook of clinical chemistry (third edition, 1999) made no mention of uncertainty or commutability (10). the fourth edition (2006) mentioned uncertainty and commutability but only a definition of commutability was given (11). the fifth edition (2011) includes a discussion of uncertainty along with commutability (12). as noted by de bievre, discussions with analytical chemists have revealed that basic concepts in metrology, including traceability, are generally not an integral part of university or college curricula and are not treated in most text books of analytical chemistry (13). metrology must be adapted to the clinical laboratory, but a practical approach is advisable due to differences between the disciplines. for example, metrology is a pure science contrasting with the mixed science of clinical chemistry (combines several diverse sciences / technologies). national metrology institutes are ivory towers in comparison to clinical laboratories (the trenches). metrology tests pure, well - defined analytes in simple matrices but clinical labs test complex, ill - defined analytes in challenging matrices (serum, plasma, urine, etc.). metrology estimates expanded uncertainty (bias eliminated) while clinical labs focus on total error allowable (tea = bias + imprecision). metrology seeks absolute scientific truth by reference method analysis but clinical labs deal in relative truth by field method analysis. good metrology does not necessarily equal good clinical laboratory science but the clinical laboratory field needs to adapt metrology concepts and translate them for practical application. in anticipation of the ivdd, the joint committee for traceability in laboratory medicine (jctlm) was formed in 2002(1). reference measurement procedures (rmp), 2. reference materials (rm), and 3. the jctlm maintains a searchable database for all three on the international bureau of weights and measures (bipm) web site (14). the laboratory community has identified three other pillars in response to harmonization : 1. universal reference intervals and medical decision levels (mdls), 2. accuracy based grading eqa / pt programs to ensure traceability of field assays is maintained and analytical bias is minimized or meets established criteria (e.g., cap pt requirement of + /-6% of the ngsp target value for hb a1c), and 3. harmonization of clinical laboratory practice and the total testing process (ttp), e.g., standardized nomenclature / terminology, reporting units, eblm, etc. the jctlm goal is comparability of patient test results from different methods to ensure appropriate medical decision - making and optimal healthcare (15, 16). type a (well defined ; concentration in si units ; results not method dependent ; full traceability chain), and 2. type b (not well defined, heterogeneous, present in both bound and free state, not traceable to si, rigorous traceability chain not available). the jctlm provides a list of higher order rms and rmps and reference laboratories (17). commutability is defined as a property of a reference material, demonstrated by the closeness of agreement between the relation among the measurement results for a stated quantity in this material, obtained according to two given measurement procedures, and the relation obtained among the measurement results for other specified materials (4). in other words, fresh patient samples and materials such as calibrators many secondary rms are not commutable with native clinical samples and have failed to accomplish the intended goal of achieving harmonized results (4). commutability is not a universal property of reference materials and must be proven with every field method. well recognized by metrology, commutability is not so widely appreciated in routine clinical laboratories. historically, the commutability reference materials and calibrators prepared from them or traceable to them has not routinely been established. noncommutability results in significant biases with field assays due to matrix effects, use of non - human forms of analyte, lack of antibody specificity, or other causes. the jctlm now requires a commutability assessment of reference materials to be listed in its database. clsi ep30 (characterization and qualification of commutable reference materials for laboratory medicine) is a recent guideline (18). metrology defines measurement uncertainty, or simply uncertainty, as a non - negative parameter characterizing the dispersion of the quantity values being attributed to a measurand, based on the information used (4). it is roughly equivalent to imprecision but ideally assay bias is eliminated prior to estimating uncertainty. clsi ep29 (expression of measurement uncertainty in laboratory medicine) is another recent guideline (19). the fourth pillar of traceability- universal reference intervals- can not be erected without the adoption of reference measurement systems and assay harmonization. reference intervals for some analytes can be affected by various partitioning factors, e.g., age, gender, ethnicity, bmi (body mass index), and thus universal ranges may not be feasible. but such decisions ca nt be made until harmonization has been achieved. to meet the ivdd traceability requirement for result trueness and comparability requires the fifth pillar : validation of manufacturers metrological traceability by eqa / pt. eqa / pt programs using commutable samples with reference method target values allow accuracy based grading (20). far too many laboratories consider proficiency testing just a necessary evil, little more than periodic pass fail exercises we perform solely to meet regulatory requirements. matrix effects, in that samples used for testing often react differently from native patient samples. therefore, comparisons must be made only to peer groups, rather than to the eqa / pt has typically been used to measure proficiency at performing a test and not the trueness of the test method or its performance relative to other method. for this reason, miller concludes traditional pt materials are not suitable for field - based postmarketing assessments of a method s trueness (21). in one study, commutable serum - based material was assigned target values by reference methods for six enzymes (alt, ast, ck, ggt, ld, and amylase) and was tested by 70 labs in germany, italy, and the netherlands using six field methods (22). performance for the other five enzymes was variable and all methods demonstrated significant bias for ck. overall, it appears clear that method bias should be reduced by better calibration to the internationally accepted reference systems (22). plebani observed although the focus is mainly on the standardization of measurement procedures, the scope of harmonization goes beyond method and analytical results : it includes all other aspects of laboratory testing, including terminology and units, report formats, reference intervals and decision limits, as well as test profiles and criteria for the interpretation of results (23). harmonization of reporting units would seem easy to achieve but that s not the case. even a change in the unit of hemoglobin (hb) expression could potentially affect patient safety. findings in a recent survey conducted in the uk revealed that 80% of laboratories were using g / dl, although g harmonization of basic terminology and units is necessary but the international clinical laboratory community has yet to reach agreement. for examples of disharmony, embracing metrological concepts and harmonization represents a paradigm shift for the in vitro diagnostics community. manufacturers traditionally sought to differentiate themselves from competitors (e.g., by claiming a greater dynamic range, lower lod, better precision, smaller sample size, etc.), and producing comparable patient results was not a priority. lack of harmonization among field assays is evident from review of eqa / pt data, often of necessity reported by peer group (as opposed to accuracy based grading). in an era of harmonization manufacturers are responding by : providing calibrator traceability / uncertainty information, restandardizing assays, testing commutability, etc., and they work with many professional organizations and each other to attain harmonization, but this is a new approach and challenge for the industry. manufacturers have an integral role in educating customers about harmonization of assays, harmonization and clinical laboratory practice in general. of course the age old question remains : where do manufacturers obligations end and the obligations of lab directors begin ? manufacturers must provide fit for purpose tests, but labs must use the assays properly and effectively. when an assay failure occurs (and failure can apply to myriad issues and causes) does the fault lie with the manufacturer or with the lab and its use of the test ? a major challenge for manufacturers is to choose a total allowable error (tea) goal from the many available options : clia requirements (u.s. specific) ; cap ; rcpa, rilibk, or other eqa / pt provider specifications. a popular approach is to define tea based on biological variability targets, but there are three targets from which to choose : minimum : tea < 1.65(0.75 cvi)+0.375(cvi + cvg) desirable : tea < 1.65(0.5 cvi)+0.25(cvi+ cvg) optimum : tea < 1.65(0.25 cvi)+0.125(cvi + cvg) cvi = individual biological variability ; cvg = group biological variability an ifcc initiative is the working group on allowable error for traceable results (wg - aetr). this group concluded although manufacturers are compelled by the european ivd directive, 98/79/ec, to have traceability of the values assigned to their calibrators if suitable higher order reference materials and/or procedures are available, there is still no equivalence of results for many measurands determined in clinical laboratories (24). for some common analytes, such as sodium, current assays are too imprecise to meet tea targets based on biological variation. the aim of harmonization is equivalent results but unfortunately, due to cost and limited resources, ivd manufacturers do nt always follow full traceability steps to value assign every new calibrator lot but rely on value transfer from an internally stored (master) calibrator material. in most cases, this procedure is probably valid, but a common complaint is calibrator lot to lot variability. the wg - aetr noted that when there are two traceability paths for a measurand, calibrators from different manufacturers may both be derived from valid traceability chains but produce non - equivalent results, as illustrated by fig. equivalent results from two systems may be possible by using a correction factor determined by a correlation study. a prime example is the aacc s ichclr (international consortium for harmonization of clinical laboratory results) (2). the ichclr prioritizes analytes globally for harmonization and development of rms and rmps for listing in the jctlm database, which will allow for comparable results irrespective of the laboratory, method, or the time when testing is performed. ichclr stakeholders include : clinical lab and medical professional societies, ivd manufacturers, metrology institutes, public health organizations, regulatory agencies, and standard - setting organizations. pathology harmony states : as we move towards full electronic reporting of pathology results, we appreciate more fully that variations in things such as test names, reference intervals and units of measurement associated with our results is something that hinders progress. in australia, there is the rcpa (royal college of pathologists of australasia) pitus (pathology information terminology and units standardisation project) program that is dedicated to harmonization (26). industry support can be optimized when the harmonization initiatives are coordinated and prioritized. from the industry perspective device manufacturers all have substantial product development priority lists and development schedules and personnel and financial resources are committed over long term periods to achieve strategic goals. the development process for a new product can be measured over years in our highly regulated environment. reprioritization is possible and welcomed by industry when the results will provide benefit to the clinician, patient and healthcare system. stellar examples such as creatinine, hemoglobin a1c and cholesterol have been pointed out in this manuscript. the global drive for harmonization creates competing project priorities for companies. as manufacturers sign on to support harmonization projects, timelines that reflect development cycles (years) allow companies to reprioritize resources while maintaining projects that drive innovation, product health and portfolio development. meeting the criteria of country specific regulatory agencies comes with additional considerations and complexities beyond the harmonization initiative. registration timing is not equivalent in all countries and multiple products for a given measurand may need to be supported for an extended period of time. it is imperative there be close coordination of industry, professional bodies and the global leaders of harmonization initiatives to ensure harmonization is successful. if companies could contribute to the prioritization of projects, design of experiment and contribute to the inputs we would be assured changes requiring product re - registration would be successful device manufacturer s typically register products using a predicate device to demonstrate product acceptance. in such cases proof of substantial equivalence. if a reference assay is a laboratory developed test the path to regulatory registration and the ability to commercialize the assay brings with it additional complications. lastly, a major consideration is whether the harmonization initiative provides benefit to the public. while accuracy is important, there are situations where existing assays may be relatively harmonized yet the reference method is very different from the commercialized assays. under these special circumstances the cost of harmonization which includes physician education, patient safety and investment in product redevelopment must be carefully weighed to understand the benefit of harmonization. | at the start of the 21st century, a dramatic change occurred in the clinical laboratory community. concepts from metrology, the science of measurement, began to be more carefully applied to the in vitro diagnostic (ivd) community, that is, manufacturers. a new appreciation of calibrator traceability evolved. although metrological traceability always existed, it was less detailed and formal. the in vitro diagnostics directive (ivdd) of 2003 required manufacturers to provide traceability information, proving assays were anchored to internationally accepted reference materials and/or reference methods. the intent is to ensure comparability of patient test results, regardless of the analytical system used to generate them. results of equivalent quality allows for the practical use of electronic health records (ehrs) capture a patient s complete laboratory test history and allow healthcare providers to diagnose and treat patients, confident the test results are suitable for correct interpretation, i.e., are fit for purpose and reflect a real change in a patient s condition and not just analytical noise. the healthcare benefits are obvious but harmonization of test systems poses significant challenges to the ivd industry. manufacturers must learn the theory of metrological traceability and apply it in a practical manner to assay calibration schemes. it s difficult to effect such a practical application because clinical laboratories do not test purified analytes using reference measurement procedures but instead deal with complex patient samples, e.g., whole blood, serum, plasma, urine, etc., using field methods. harmonization in the clinical laboratory is worth the effort to achieve optimal patient care. |
capsular block syndrome (cbs) has been known to occur as a rare complication of cataract surgery with continuous curvilinear capsulorhexis (ccc) and a posterior - chamber lens implant. typically, it presents with reduced vision in the early postoperative period and is characterised by a forward displacement of the posterior - chamber intra - ocular lens and an accumulation of intra - capsular opaque material. management of cbs is usually by nd : yag laser capsulotomy. we describe here a unique case of very - delayed - onset cbs with good visual acuity, occurring 8 years after surgery. a 75-year - old lady was referred to the eye department with a 6-month history of misty and fuzzy vision in her right eye. eight years earlier, she had undergone uncomplicated bilateral cataract surgery by phacoemulsification with lens implants. upon examination, her logmar scale best - corrected visual acuity (bcva) was 20/20 in both eyes. refractive error was 1.50 ds for the right eye and + 0.25 ds for the left eye, which suggested a myopic shift in the right eye. the left eye was normal, but there was a hazy appearance in the right eye in the retro - lenticular space between the posterior surface of the lens optic and the posterior capsule. the space was homogenously whitish and slightly opalescent, characteristic of a milky, turbid fluid (fig. 1). there were no signs of inflammatory activity. on clinical examination, the lens implant did not appear to have shifted forward and the fundus examination was normal. intra - operatively, a 30-gauge needle mounted on a 1-ml syringe was passed through the sclera and pars plana, 3.5 mm behind the limbus in the infero - temporal quadrant. the needle tip was gently passed through the posterior capsule and 0.2 ml of the turbid fluid was aspirated and then sent for microbiological culture and analysis. intra - vitreal vancomycin (1 mg/0.1 ml) was administered at the end of the procedure. she was reviewed after 5 days and there were no signs of inflammation or endophthalmitis. in addition, her myopic shift had disappeared and she now had a + 0.25 ds for both eyes, both still with a bcva of 20/20. two months afterwards, at follow - up, she was asymptomatic and reported complete resolution of her misty vision. examination showed a bcva of 20/20, with a clear papillary axis and an intact posterior capsule and lens implant. it is mainly known to occur with ccc and phacoemulsification, but has also been reported with can - opener - type capsulorhexis, extracapsular cataract extraction and intra - ocular lens implantation in the sulcus [4, 5 ]. our case did not have reduced visual acuity and only had symptoms of haziness and mistiness, which is quite unusual and, to the best of our knowledge, has not been reported in the literature before. miyake. divided the condition into 3 different types, depending upon the time of onset : intra - operative, early post - operative and late post - operative. they postulated that, in the late post - operative period, the cortical cells undergo metaplastic changes and proliferate in the bag, which leads to posterior capsular opacification and also causes occlusion of the capsular opening by sealing off the gap between the anterior capsule and the lens implant. these metaplastic cells also cause the release of a turbid fluid, which gets retained in the retro - lenticular space. upon electrolysis, this fluid has been found to be made of alpha - crystallin and collagen released from the necrotic and/or apoptotic autolysed cells [7, 8 ]. eifrig described 3 cases with capsulorhexis - related lacteocrumenasia (lacteo = milky and crumen = small bag or coin purse). the aspirated fluid was examined with electrophoresis and showed a large amount of alpha - crystallin and a small amount of albumin. the study concluded that it was unlikely to be a result of an antigen - antibody reaction in the capsular bag as there was no gamma globulin detected. there have been a few reports of late - onset cbs being linked to propionibacterium acnes [9, 10 ]. carlson and koch reported a case of p. acnes - associated endophthalmitis after nd : yag laser capsulotomy. these reports led us to err on the side of caution, and we therefore opted to perform the aspiration of the fluid via the pars plana, rather than using the conventional approach of nd : yag laser capsulotomy. our case had the unique feature of a very late presentation 8 years after an uncomplicated phacoemulsification and ccc. in addition, in comparison to most of the reported literature where the visual acuity was dramatically reduced, our patient had a bcva of 20/20 and only had symptoms of misty vision with a reduction in her contrast sensitivity, which improved after the aspiration of the fluid. in summary, late - onset cbs can present after a long time and may cause significant symptoms. the usual treatment for the condition is nd : yag laser capsulotomy, but an infective aetiology could be present and this may cause a dissemination of intra - ocular infection. we have described a pars plana removal technique with simultaneous administration of intra - vitreal antibiotics, which we believe is a safe approach for this condition. | capsular block syndrome (cbs) has been known to occur as a rare complication of cataract surgery with continuous curvilinear capsulorhexis and a posterior - chamber lens implant. typically, it presents with reduced vision in the early postoperative period and is characterised by a forward displacement of the posterior - chamber intra - ocular lens and an accumulation of intra - capsular opaque material. management of cbs is usually by nd : yag laser capsulotomy. in this report, we describe a unique case of very - delayed - onset cbs with good visual acuity, occurring 8 years after surgery. it was treated successfully with surgical removal of the opaque material. |
the heat exposure will initially create the heat strain but in long - term exposures, it will cause some problems such as muscle cramps, heat stroke, heat syncope, heat exhaustion, less productivity, more accident rate and less safety level in workplaces. there are various strategies in the field of engineering and administrative controls to decrease heat strain. the usage of personal cooling equipment is one of the strategies for decreasing heat strain in hot environments. in general, cooling equipment can be divided into five groups : evaporative cooling equipment, equipment based on phase change materials (pcms), compressed air equipment, liquid circulation equipment, and thermoelectric equipment. among cooling equipment, pcms cooling vest are of the cheapest, most mobile, and easiest equipment to wear. pcms as a medium for the storage of the latent heat energy are capable to change their phase in a certain temperature range during phase transitions between two states. they are chemically stable, without phase segregation, with minimum sub - cooling during repetitive phase change, nontoxic, not corrosive, with low vapor pressure, odorless, and easily available. furthermore, paraffin materials have disadvantages such as low thermal conductivity and low thermal storage density. pure paraffin materials are expensive but their commercial compound, which are consist of different hydrocarbons, are cheap in compared to pure paraffin materials. on the other hand, the mixture of the materials can provide operational pcms to possess a different thermal freezing - melting range and close to skin temperature. therefore, this study aimed to design the individual cooling vest using the cheap commercial paraffin compounds and then evaluation its effect on the heat strain parameters during physical effort under a hot condition in the climatic chamber. this study included three stages : preparing the paraffin compound with a proper melting point, designing the cooling vest, and evaluation the cooling effect of the cooling vest. for the appropriate performance of the pcms, they should have a melting point lower than skin temperature and close to the skin temperature (1535c) to absorb sufficiently the heat and do not cause the skin irritation due to high coldness. in this stage, it was prepared a composition of paraffin with a proper melting point in the range of 1535c from the cheap commercial paraffin compounds with a different melting point. it was used differential scanning calorimeter (dsc) technique to measure the accurate thermal properties of the paraffin such as melting point and latent heat. to analyze the analysis was performed with the constant increase in the temperature (10c / min) at the thermal range 2090c and under a nitrogen gas flow of 50 ml / min, according to the method of astm d314 - 12. for calibrating the set,, it was prepared packs of paraffin. because of the low thermal conductivity of paraffin, the packs were made of aluminum with a thickness of 0.125 mm and a thermal conductivity 237 w/(m k). each pack weighed 140 g and the total weight of cooling vest was 2.5 kg. since the fabric of the cooling vest was polyester in the number of studies to make the cooling vest, the cooling vest in our study was made of the polyester fabric and designed with an adjustable pattern based on the size of different individuals. it was considered 17 pockets (8 pockets on the front and 9 pockets on the back of the torso) in the inside layer of the cooling vest for fitting the cooling packs. the designed paraffin cooling vest and the pack of it for evaluating the effectiveness of cooling vest, an experimental study was carried out on ten healthy male students of the medical university of isfahan, in 2015, in the climatic chamber of health school. inclusion criteria included no history of cardiovascular, respiratory, neuromuscular, and musculoskeletal diseases, epilepsy, seizure, diabetes, nonconsumption of high blood pressure drugs and medications affecting the heart rate, nonconsumption of coffee, caffeine, and alcohol from 12 h before the test. exclusion criteria included the increased heart rate over 180 beats / min, oral temperature over 38.5c and fatigue. candidates were examined by a physician for inclusion criteria and then the participants were selected. also, they were asked to fill out the consent form developed by the medical ethics committee of the medical university of isfahan. also, they were talked about exclusion criteria and how to fill out the questionnaire and the test stages were clearly explained for them. it was gathered the participants demographic information such as age. to assimilate the conditions of the cooling vests usage, all of the participants were asked to wear similar types of work clothes (30% polyester, 70% cotton). to charge cooling packs, the study was conducted on the participants under hot and dry conditions (air temperature [ta ] of 40c, relative humidity [rh ] of 40%) in the climatic chamber. they were tested in the following stages : without cooling vest with light activities, with paraffin cooling vest with light activities, without cooling vest with moderate activities, with paraffin cooling vest with moderate activities. the light activity was done on a treadmill (kettler model) with the speed of 2.8 km / h and the slope of 0%, and the moderate activity was performed on the same treadmill with the speed of 4.8 km / h and the slope of 0%. in each stage of performing the test, at first, the participants rested and relaxed for 30 min, and their oral temperature and heart rate were measured. after that, the participants worked out on a treadmill for 30 min. to evaluate the heat strain, the heart rate, oral temperature, and skin temperature were measured. during the test, the heart rate was measured every 2 min (using a sports polar tester) and the oral temperature (using oral thermometer of beurer with accuracy of 0.1c) and the skin temperature (using a device for measuring the skin temperature model sina rt-923 with accuracy of 0.01c) were measured every 4 min. for measuring the amount of sweating, the participants weight was measured before and after each stage of the activity (using a digital scale of hamilton model with an accuracy of 0.1 kg). the participants did not drink anything during the activities. to calculate the percentage of the reduced sweating during activities with cooling vest, the following formula was used : % rsw = 100 [(swa - swb)/swa ] % rsw : percentage of the reduced sweating during activities with cooling vest. finally, data were analyzed using the statistical package for the social sciences (spss) 16. smirnov was performed to check the normality of data distribution, and repeated measurement anova test was used to determine the p value. for the appropriate performance of the pcms, they should have a melting point lower than skin temperature and close to the skin temperature (1535c) to absorb sufficiently the heat and do not cause the skin irritation due to high coldness. in this stage, it was prepared a composition of paraffin with a proper melting point in the range of 1535c from the cheap commercial paraffin compounds with a different melting point. it was used differential scanning calorimeter (dsc) technique to measure the accurate thermal properties of the paraffin such as melting point and latent heat. to analyze the analysis was performed with the constant increase in the temperature (10c / min) at the thermal range 2090c and under a nitrogen gas flow of 50 ml / min, according to the method of astm d314 - 12. for calibrating the set, at first, it was prepared packs of paraffin. because of the low thermal conductivity of paraffin, the packs were made of aluminum with a thickness of 0.125 mm and a thermal conductivity 237 w/(m k). each pack weighed 140 g and the total weight of cooling vest was 2.5 kg. since the fabric of the cooling vest was polyester in the number of studies to make the cooling vest, the cooling vest in our study was made of the polyester fabric and designed with an adjustable pattern based on the size of different individuals. it was considered 17 pockets (8 pockets on the front and 9 pockets on the back of the torso) in the inside layer of the cooling vest for fitting the cooling packs. for evaluating the effectiveness of cooling vest, an experimental study was carried out on ten healthy male students of the medical university of isfahan, in 2015, in the climatic chamber of health school. inclusion criteria included no history of cardiovascular, respiratory, neuromuscular, and musculoskeletal diseases, epilepsy, seizure, diabetes, nonconsumption of high blood pressure drugs and medications affecting the heart rate, nonconsumption of coffee, caffeine, and alcohol from 12 h before the test. exclusion criteria included the increased heart rate over 180 beats / min, oral temperature over 38.5c and fatigue. candidates were examined by a physician for inclusion criteria and then the participants were selected. also, they were asked to fill out the consent form developed by the medical ethics committee of the medical university of isfahan. also, they were talked about exclusion criteria and how to fill out the questionnaire and the test stages were clearly explained for them. it was gathered the participants demographic information such as age. to assimilate the conditions of the cooling vests usage, all of the participants were asked to wear similar types of work clothes (30% polyester, 70% cotton). to charge cooling packs, the study was conducted on the participants under hot and dry conditions (air temperature [ta ] of 40c, relative humidity [rh ] of 40%) in the climatic chamber. they were tested in the following stages : without cooling vest with light activities, with paraffin cooling vest with light activities, without cooling vest with moderate activities, with paraffin cooling vest with moderate activities. the light activity was done on a treadmill (kettler model) with the speed of 2.8 km / h and the slope of 0%, and the moderate activity was performed on the same treadmill with the speed of 4.8 km / h and the slope of 0%. in each stage of performing the test, at first, the participants rested and relaxed for 30 min, and their oral temperature and heart rate were measured. after that, the participants worked out on a treadmill for 30 min. to evaluate the heat strain, the heart rate, oral temperature, and skin temperature were measured. during the test, the heart rate was measured every 2 min (using a sports polar tester) and the oral temperature (using oral thermometer of beurer with accuracy of 0.1c) and the skin temperature (using a device for measuring the skin temperature model sina rt-923 with accuracy of 0.01c) were measured every 4 min. for measuring the amount of sweating, the participants weight was measured before and after each stage of the activity (using a digital scale of hamilton model with an accuracy of 0.1 kg). the reduced sweating during activities with cooling vest, the following formula was used : % rsw = 100 [(swa - swb)/swa ] % rsw : percentage of the reduced sweating during activities with cooling vest. finally, data were analyzed using the statistical package for the social sciences (spss) 16. smirnov was performed to check the normality of data distribution, and repeated measurement anova test was used to determine the p value. the results from paraffin thermal analysis by dsc technique showed that the latent heat of melting from the prepared composition of the paraffin was 108 kj / kg. the range of phase change temperature was 2434c and the peak of the melting point was 30c. the mean and standard deviation of the participant 's age was 25.1 and 3.66, respectively. the values of the range, means, and standard deviation of the measured physical parameters are illustrated in table 1. smirnov test showed that the distribution of the data was normal (p > 0.05). moreover, the result of the statistical analysis showed that there was not significantly difference between the dry temperature and the rh with cooling vest and without cooling vest in both light and moderate activities and the conditions were equal in both steps. the means and the standard deviation of the dry temperature for both activities were 40.04 and 0.38, respectively. the means and the standard deviation of the rh for both activities were 40.74 and 0.45, respectively. values of range, means and standard deviation of the measured physical parameters in light and moderate activities the mean values of the measured heart rate during 2 to 30 min every 2 min with cooling vest and without cooling vest in two physical activity levels are presented in figure 2. the results showed that the difference for the mean values of the heart rate with cooling vest and without cooling vest during light activity (p 6c. moreover, some of the studies show that there is a good correlation between body temperature and the heart rate. these assumptions are supported by our results to indicate the skin temperature of the torso, the oral temperature, and the heart rate have significantly reduced in light and moderate activities with cooling vest than activities without cooling vest. furthermore, the diagrams in the results section show that the skin temperature, oral temperature, and heart rate increase with lower speed during the light and moderate activities with cooling vest than activities without cooling vest, and it could be due to the above reasons. on the other hand, the results showed that the reduced sweating rate was 42% and 52% during light activity and moderate activity with the cooling vest, respectively which can prevent dehydration during activity. the dehydration can cause fluid and electrolyte imbalance in the body and lead to the heat - related illnesses. in general, the results showed that the cooling vest can reduce the heat strain by controlling the above parameters. since torso has the highest capacity for the dissipation of the heat in compared to the other areas of the body. there are several studies that show the effectiveness of the paraffin cooling vest on heat strain. in the study of jovanovic., in 2013, was used the pure paraffin (n - hexane) with the range of phase change temperature of 13.821.2c, the peak of the melting point of 18c and the latent heat of melting of 237 kj / kg. the said study conducted on 10 male trained soldiers under the temperature of 40c on treadmill with speed 5.5 km / h. the results showed that the cooling vest was able to reduce the physiological strain through reducing the skin and rectal temperatures. of course, the pure paraffin is expensive and has not commercial and industrial usage. while the designed paraffin cooling vest was manufactured from the cheap commercial material. the results of this study showed that the latent heat of the prepared paraffin compound was 108 kj / kg. this latent heat is lower than the latent heat pure paraffin (n - hexane) in the study of jovanovic., this low latent heat of designed cooling vest limit the duration time of usage. however, the higher range of phase change temperature and the higher peak of the melting point in designed cooling vest than n - hexane increase the duration time of cooling effect and reduce irritation by chill. also, chou. examined the performance of the pure paraffin cooling vest on eight students under the firefighting cloth in ta of 30c and rh of 50%. the result showed that the pure paraffin cooling vest with melting temperature of 28c was effective in reducing the rate of heat storage in subjects. in other study, bendkowska. examined the cooling effects of the pure paraffin cooling vests (hexadecane with melting temperature at 18c and octadecane with melting temperature at 28c) on a thermal manikin in ta of 20c and rh of 50%. the results of the present study were agreement with the results of the above studies, but the latent heat of the designed cooling vest was lower than pure paraffin cooling vest in other studies. moreover, the results showed that the skin temperature was close to the natural skin temperature when the participant used cooling vest. hence the cooling packs do not cause the skin irritation due to extreme coldness. because this prepared paraffin compound has a high melting point, the low energy is needful for recharging the prepared paraffin compound and the paraffin packs only require a refrigerator to recharge. in general, a disadvantage for the prepared commercial paraffin compound was the low latent heat in compared to the pure paraffin materials. of course, the price of the commercial paraffin is very lower than the pure paraffin. the low latent heat reduces the duration of the cooling effect but, on the other hand, the duration of the recharging decreases. the pure paraffin is expensive and has not commercial and industrial usage. because of this however, the results showed that the use of cooling vest containing the commercial paraffin compound can reduce the heat strain through reducing heart rate, oral temperature, skin temperature, and sweating rate. this cooling vest with the proper melting point close to the skin temperature hence, this type of cooling vest with low cost can be used to prevent heat strain and to increase the physiological stability against the heat. however, the disadvantage of the commercial paraffin compound was the low latent heat in compared to the pure paraffin materials. hence, a matter with high latent heat such as ice must be used on side the commercial paraffin compounds in the packs of cooling vest for increasing latent heat. | background : the phase change materials (pcms) have the appropriate properties for controlling heat strain. one of the well - known pcms is paraffin. this study aimed to design the cooling vest from the cheap commercial paraffin compound and evaluation of its effectiveness under laboratory hot conditions.methods:the cooling vest was made of the polyester fabric and it had 17 aluminum packs. the each of aluminum packs was filled by 135 g of prepared paraffin with a proper melting point in the range of 1535c. an experimental study was conducted on ten male students under warm conditions (air temperature = 40c, relative humidity = 40%) in a climatic chamber. each participant was tested without cooling vest and with cooling in two activities rate on treadmill to include : light (2.8 km / h) and moderate (4.8 km / h). the time of this test was 30 min in each stage. during the test, the heart rate, the oral temperature, the skin temperature were measured every 4 min. finally, data were analyzed using the kolmogrov smirnov and repeated measurement anova test in spss 16.results:the latent heat of the prepared paraffin compound and the peak of the melting point were 108 kj / kg and 30c, respectively. the mean and standard deviation of heart rate, oral temperature, and skin temperature with cooling vest in light activity were 103.9 (12.12) beat / min, 36.77 (0.32)c, and 31.01 (1.96)c and in moderate activity were 109.5 (12.57) beat / min, 36.79 (0.20)c, and 29.69 (2.23)c, respectively. there is a significant difference between parameters with a cooling vest and without cooling (p < 0.05).conclusions : the designed cooling vest with low cost can be used to prevent thermal strain and to increase the physiological stability against the heat. however, the latent heat of this cooling vest was low. |
stroke is one of the leading causes of death among middle - aged men, along with cancer and cardiovascular disease1. stroke is categorized as either cerebral hemorrhage, which arises from the rupture of blood vessels in the brain, or cerebral infarction, which is caused by blocked or narrowed blood vessels2. limitations in activities of daily living (adl) are found after the occurrence of stroke. an extended bedridden state can lead to sarcopenia and reduced muscle strength, muscle endurance, and cardiopulmonary function3, 4. motor disabilities due to stroke - induced hemiplegia and hemiparesis limit physical activity and motor function, and can lead to the accumulation of visceral fat, which is a metabolic risk factor7. the accumulation of visceral fat results in the excessive release of free fatty acids (ffa) and glycerol, which are degradation products of triglycerides (tg)7, when ffa and glycerol are excessively introduced into skeletal muscle cells, they interfere with insulin - dependent glucose absorption. this process leads to the development of hyperglycemia, hyperlipidemia, and insulin resistance. in cases of abdominal obesity resulting from visceral fat accumulation, blood and tissue concentrations of ffa increase simultaneously. ffa reduces insulin sensitivity in the liver by suppressing insulin - dependent glucose uptake. this disorder reduces the amount of glucose that available for glycogen synthesis and increases tg build up. an increase in glucose and ffa levels increases insulin secretion from the pancreas, causing hyperinsulinemia. hyperinsulinemia leads to increased sodium re - uptake, sympathetic nervous system (sns) activation, hypertension development, and circulating ffa levels8. in addition, low concentrations of high - density lipoprotein cholesterol (hdlc) are more closely related to the risk of stroke than high tg9. low hdlc increases the risk of all arteriosclerotic diseases such as myocardial infarction, stroke, and sudden death. particularly in stroke patients, improvements in hdlc concentration are considered an important goal in the treatment and reduction of these multiple risks10. since metabolic risk factors have various components and interactions, multifaceted prevention and treatment methods are necessary. in particular, stroke patients have a significantly higher chance of metabolic syndrome (mets) than to individuals without a stroke history or other specific disease states. studies have suggested that stroke and mets require multifaceted care, rather than separate treatment of each condition11. ffa serves as the link in a vicious cycle of metabolic risk factors ; therefore, it can not be ignored. it was recently reported that exercise is a non - pharmacologic treatment method that may be effective at preventing brain diseases, enhancing cerebral function, and improving diabetic neuropathy12,13,14. recent cohort studies have highlighted the importance of metabolic risk factors, as the improvement of these risk factors in stroke patients reduced total stroke recurrence by 1930%11. however, in previous studies, the primary goal of exercise programs for stroke patients was the enhancement of functional capacity, as measured by markers such as walking ability, falls, adl, and quality of life15. in addition, there were very limited applications of exercises that addressed cardiovascular risk factors or metabolic factor modulations intended for the improvement of metabolic abnormalities. therefore, the present study aimed to specifically investigate the effects of aerobic exercise as a means of reducing metabolic risk factors of stroke patients. this study was conducted as a pretest - and - posttest and control group study. the subjects of the present study were chronic stroke patients diagnosed as having stroke - induced hemiplegia with onset at least 12 months previously. twenty male patients aged 4759 years without orthopedic conditions that would have affected the study were selected. subjects were included in the study if they could walk 10 meters within 60 seconds without supplementary assistance16, were able to understand and follow verbal commands, and agreed to participate in the exercise program. those currently taking drugs were not excluded. selected subjects were placed into either the exercise group (n = 10), which performed an aerobic exercise program in addition to receiving rehabilitative therapy, or the control group (n = 10), which received utilized only the rehabilitation. all subjects completed the pretest before starting the exercise program, as well as the posttest at the conclusion of the 12-week exercise program. all participants provided their written informed consent to participation in this study prior to their enrollment. kyungwoon university approved this study, which complies with the ethical standards of the declaration of helsinki. the physical characteristics of the subjects in each group are shown in table 1table 1. physical characteristics of the participants of this experimentvariableage (years)height (cm)weight (kg)bmi (kg / m)time since stroke(months)stroke type(ischemia / hemorrhage)hemiplegic side(rt / lt)exercise (n=10)53.73.6167.85.886.25.225.71.547.16.64 / 63 / 7con (n=10)52.84.1166.45.082.36.224.72.444.87.37 / 32 / 8data are means sd.. body measurements were made as follows : height (cm) and weight (kg) were measured and then used to calculate the body mass index (bmi) with the equation bmi = weight (kg)/height squared (m) ; and waist and hip circumference were measured with a tape measure to calculate the waist - to - hip ratio (whr). blood pressure was measured after patient had rested in a sitting position for 5 minutes, using an automatic blood pressure monitor (ft-500r, jawon medical, seoul, korea) to obtain the systolic (sbp) and diastolic (dbp) blood pressure. blood pressures measurements were obtained twice, and the average values were used in the data analysis ; the time between measurements was 3 minutes. mean arterial pressure (map) was calculated as map = dbp + (sbp dbp)/3. for the intervention, all subjects received physical therapy consisting of posture, gait and strength training for 30 minutes / day which was conducted by a physical therapist with more than 5 years of bobath technique experience. the exercise was conducted using a stationary bicycle (full body exercise active, super dynamic 3, seoul, korea), a rehabilitative exercise machine designed to move the arms and legs together. the exercise program utilized the heart rate reserve (hrr) method to achieve an exercise intensity corresponding to 5070% of the target heart rate (thr). the program lasted for 30 minutes and was conducted 5 times per week for a total of 12 weeks. the exercise intensity was initially set at thr 50%, and was increased in 5% increments at 3-week intervals to the maximum of thr 70%. during exercise, the heart rate was continuously observed using an automatic pulse monitor by the patient, a professional physical therapist, and an exercise specialist to determine when the thr was achieved. in prior studies, although this equation is easy to use due to its simplicity, the rate of change is high. therefore, hrmax is underestimated in men and women less than 40 years of age, whereas it is overestimated in men and women aged 40 years. the recent american college of sports medicine (acsm) guidelines17 recommend the equation of gellish.18 as the most accurate equation. the present study used this recommended equation to calculate the subjects thrs as follows : thr = (206.9 [0.67 age ] hrrest) % intensity + hrrest. blood samples were drawn before and after completion of the 12-weekexercise program, under identical conditions and at the same time of day. serum was separated from the collected blood using a centrifuge. then, the concentrations of tg (vitros trig dtd, johnson & johnson, new brunswick, nj, usa), total cholesterol (tc ; vitros chol dtd, johnson & johnson), and glucose (vitros glu dtd, johnson & johnson) were analyzed with a vitros chemistry dt60 ii (johnson & johnson) using slides. hdlc was analyzed using magnesium chloride and dextran sulfate to precipitate apo b - containing cholesterol ; hdlc (vitros hdlc dtd, johnson & johnson) slides were used for vitros chemistry dt60 ii analysis (johnson & johnson). ffa (nefa hr kit, wako, osaka, japan) was analyzed using a hitachi 7180 (hitachi, tokyo, japan). two - way repeated measure analysis of variance (anova) was conducted for all data variables in order to express the average and standard deviation values, and to test the group trends before and after the exercise program. comparisons of changes in the average values of measurement variables before and after the 12-week exercise program were analyzed using the paired samples t - test. in addition, the variation () between baseline measurements and measurements after 12 weeks of exercise was calculated, and pearson s correlation coefficient was used to test for correlations between the measurement variables (score = change in score between pre - test and posttest). all statistical analyses were conducted with the statistical package for the social sciences (spss)-pc (version 18.0 ; ibm corporation, endicott, ny, usa), and statistical significance was accepted for values of p0.05. table 2table 2. changes in obesity indices and blood pressure with 12-week exercise trainingmeasured variablegrouppretestposttestscoreweight (kg)exercise86.25.283.33.02.93.2con82.36.283.06.60.71.8bmi (kg / m)exercise25.71.524.80.80.80.9con24.71.424.91.40.20.5wc (cm)exercise94.07.688.58.15.42.8con92.36.792.46.80.10.8whrexercise0.940.030.890.040.050.05con0.960.040.960.060.010.03sbp (mmhg)exercise133.712.8127.95.45.89.3con131.315.6132.117.50.87.2dbp (mmhg)exercise87.110.482.44.54.68.8con85.711.286.111.80.45.1map (mmhg)exercise102.610.697.64.25.08.4con100.912.4101.413.40.55.3data are meanssd, score = change in the score p value of paired t - test (: p<0.05, : p<0.01, : p<0.001), p value of two - way anova test (grouptime, : p<0.05, : p<0.01, : p<0.001), bmi : body mass index, wc : waist circumference, whr : waist to hip ratio, sbp : systolic blood pressure, dbp : diastolic blood pressure, map : mean arterial pressure shows the comparative results of the changes in obesity indices and blood pressure of stroke patients after the 12-week exercise program. the obesity markers of weight (p < 0.01), bmi (p < 0.01), wc (p < 0.01), and whr (p < 0.01) showed a significant interaction effect. however, no significant effect was observed for blood pressure. data are meanssd, score = change in the score p value of paired t - test (: p<0.05, : p<0.01, : p<0.001), p value of two - way anova test (grouptime, : p<0.05, : p<0.01, : p<0.001), bmi : body mass index, wc : waist circumference, whr : waist to hip ratio, sbp : systolic blood pressure, dbp : diastolic blood pressure, map : mean arterial pressure table 3table 3. changes in serum lipids and free fatty acid (ffa) levels elicited by exercise trainingmeasured variablegrouppretestposttestscoretg (mg / dl)exercise172.666.8134.548.938.153.7con173.458.9161.551.511.951.8tc (mg / dl)exercise208.255.7168.827.439.446.2con205.534.1207.636.92.021.4hdlc (mg / dl)exercise41.011.253.08.212.010.3con39.88.543.48.43.62.7glucose (mg / dl)exercise101.713.894.24.37.514.3con102.513.4103.810.61.39.8ffa (eq / l)exercise431.4139.2324.373.2107.1146.4con449.3109.0411.180.438.287.2data are meanssd, score = change in the score between pretest and posttest, p value of paired t - test (: p<0.05, : p<0.01, : p<0.001), p value of two - way anova test (grouptime, : p<0.05, : p<0.01, : p<0.001), tg : triglycerides, tc : total cholesterol, hdlc : high density lipoprotein cholesterol, ffa : free fatty acid. shows the results of the changes in blood lipids and ffa in stroke patients after 12 weeks of exercise training. there was a significant difference in tg according to measurement time (p = 0.04), and an interaction effect was observed with tc (p < 0.01). hdlc, an important marker of improvement in metabolic risk factors, showed a significant interaction effect (p = 0.02) with exercise training. additionally, there was a significant difference in ffa levels according to the measurement time (p < 0.01). data are meanssd, score = change in the score between pretest and posttest, p value of paired t - test (: p<0.05, : p<0.01, : p<0.001), p value of two - way anova test (grouptime, : p<0.05, : p<0.01, : p<0.001), tg : triglycerides, tc : total cholesterol, hdlc : high density lipoprotein cholesterol, ffa : free fatty acid. table 4table 4. correlation between the measured factors and the degree of changes elicited by 12-weeks of exercise trainingmeasured variableweightbmiwctgtchdlcglucoseffaweight10.9990.2820.5050.4470.762 0.2970.667bmi0.99910.2860.5110.4280.762 0.2890.657wc0.2820.28610.0660.2200.3110.6400.064tg0.5050.5110.06610.5010.4570.6010.726tc0.4470.4280.2200.50110.5750.2740.669hdlc0.7620.762 0.3110.4570.57510.1940.776glucose0.2970.2890.640 0.6010.2740.19410.550ffa0.667 0.6570.0640.726 0.6690.7760.5501 correlation is significant at the 0.05 level (2-tailed), correlation is significant at the 0.01 level (2-tailed) shows the correlation between changes (score) in the measurement variables of the 12-week exercise program. ffa levels, a direct cause of metabolic risk factors, showed significant positive correlations with weight (r = 0.66, p < 0.05), bmi (r = 0.65, p < 0.05), tg (r = 0.72, p < 0.05), and tc (r = 0.66, p < 0.05), and a significant negative correlation with hdlc (r = 0.77, p < 0.01). conversely, the metabolic improvement factor hdlc showed significant negative correlations with the obesity indices of weight (r = 0.76, p < 0.05) and bmi (r = 0.76, p < 0.05), as well as a significant negative correlation with ffa (r = 0.77, p < 0.01). correlation is significant at the 0.05 level (2-tailed), correlation is significant at the 0.01 level (2-tailed) weight, bmi, wc, and whr are reported to be predictive factors of stroke. weight and stroke risk are positively correlated19, and it has been proposed that wc and whr are risk factors of obesity - related stroke occurrence2, 20. the results of the present study show that the each of variables of weight, bmi, wc, and whr demonstrated an interaction effect with the measurement time and group, indicating that regular exercise was effective at improving obesity indices. this finding is likely a result of implementing appropriate amounts of exercise through a program that considered individual patients ` abilities. however, no significant changes were observed in blood pressure (sbp, dbp, and map) in the present study. although even small reductions in sbp and dbp have been shown to significantly decrease stroke risk21, the difficulty in identifying the effect of exercise in the present study may be attributable to the fact that most of the study participants were using blood pressure - related medications. stroke - induced reductions in physical activity and abdominal fat accumulation reduce the action of lipoprotein lipase (lpl), suppressing the breakdown of tg and increasing blood ffa concentrations22. during the process in which cholesterol ester transferase converts cholesterol and tg from hdlc to very - low - density lipoprotein cholesterol (vldlc), the high tg content results in a reduction in hdlc concentration23. the results of the present study show there was a decrease in tg and ffa levels at the end of the 12-week intervention, and tc and hdlc showed an interaction effect. these results are consistent with those of carl.24 we consider that regular exercise suppressed vldlc synthesis, reducing tg concentrations and relatively increasing hdlc levels. in addition, exercised - induced increases in lpl activity in the muscle and adipose tissues are considered show improvement in lipid metabolism, due to the suppression of tc and tg synthesis in the liver, as well as decreased liver uptake of ffa25, 26. in the initial phase of exercise, the muscles use glycogen stored within the muscle as an energy source. compared to lipid utilization, however, this glycogen supply only provides energy for a short period of exercise27. thus, the main energy source of the muscles in the context of continuous exercise is fatty acids, which are oxidized within the mitochondria to provide the energy needed for the muscles28. an increase in fatty acid oxidation may therefore effectively improve metabolic risk factors by reducing circulating levels of blood ffa. consequently, improvements in lipid metabolism observed after regular exercise in the present study are likely related to ffa oxidation and suppression of tg synthesis. in addition, low concentrations of hdlc have been reported to be closely related to stroke risk9, and the significant exercise - induced increase in hdlc observed in the present study suggests that exercise may be an effective means of reducing the risk of stroke recurrence. in particular, the observed significant negative correlation between ffa and hdlc (r = 0.77, p < 0.01) indicates the need for systematic and regular exercise programs for stroke patients. the various types of metabolic factors examined in the present study are intricately connected in a metabolic cascade. unlike specific pharmacologic agents, exercise individualizing the exercise intensity to patient fitness and characteristics is the most important factor in achieving this effect. since stroke patients have concomitant limitations in physical activity and motor disabilities, andmay also have several metabolic comorbidities such as hypertension, diabetes, cardiovascular disease, and obesity, the intensity of the prescribed aerobic exercise must be determined through accurate testing. furthermore, detraining can occur because of personal disposition or various other factors, resulting in rapid changes in adapted physical responses. therefore, further research is necessary to develop exercise programs that patients can enjoy over the long term. | [purpose ] the present study investigated the effects of regular exercise on the improvement of free fatty acid (ffa) levels and metabolic risk factors of stroke patients. [methods ] the subjects were 20 male patients aged 4759 years who were diagnosed as having hemiplegia resulting from stroke. exercise was conducted using a stationary bicycle, a rehabilitative exercise machine. the exercise program utilized the heart rate reserve (hrr) method to create an exercise intensity amounting to 5070% of the target heart rate (thr). the program lasted for 30 minutes and was conducted 5 times per week for a total of 12 weeks. [results ] in stroke patients, 12 weeks of exercise training yielded a significant interaction effect with weight, body mass index (bmi), waist circumference (wc), and waist - to - hip ratio (whr). there were also significant differences in triglycerides (tg) according to the time of measurement, and an interaction effect was observed for triglycerides (tc). high - density lipoprotein cholesterol (hdlc), an important marker of improvement in metabolic risk factors, showed a significant interaction effect with exercise training. in addition, free fatty acids (ffa) showed a significant difference based on the time of measurement and showed a significant negative correlation with hdlc (r = 0.77). [conclusion ] the results of the present study suggest that regular exercise by stroke patients reduces their risk of metabolic complications and stroke recurrence by reducing obesity indices, improving serum lipid and ffa levels, and increasing hdlc levels. |
the determination of the width of the implant is the first key step to select shape and volume of the implant in breast augmentation. the aim of this study was to introduce a new method to determine the width of the implant (w) and explain the reasons to do so in details. cd4 theory to determine the width of breast implant (w) in dual plane i or ii breast augmentation cases through transaxillary or periareolar incision for 560 patients. cd is defined as the curved distance on skin from the midline of the sternal bone to the anterior axillary line (aal) on the lateral chest wall through the horizontal level on inferior mammary fold. the 560 patients used both round and anatomic implants with w from 10.5 cm to 12.5 cm. about 78% of the patients have got followed up from 1 month to 5 years postoperatively. except for four patients who got unilateral capsular contractions, all the other patients are satisfied with their nature new breast shapes and volumes. their new intermammary cleavages without bras are between 1 cm and 2.5 cm, and lateral borders of the breast are on the area of the aal. w (width of the implant) = cd 4 (cm) when doing dual plan i or ii breast augmentation. for the very thin patient, for patients who need to do breast augmentation, there is an appropriate proportion between the breast implant width and the patients thoracic width. the best implant width can produce not only the best intermammary cleavage, but also a suitable fullness on the lateral side of the breast. on the middle part of the sternal bone, the 3 cm wide no touch zone(ntz) dr. john b. tebbetts emphasized that the medium dissecting edge of the implant pocket should stop at the point 1.5 cm away from the midline of the sternal bone to avoid damaging the vessel perforators. this means the most medium edge of the implant should not exceed the point that is 1.5 cm away from the middle line of the sternal bone. according to the normal breast anatomy, most plastic surgeons choose the anterior axillary line (aal) as the most lateral margin of the newbreast. the above medium and lateral margins are the fundamentals of the following cd 4 theory. from january 2006 to june 2014, we had used cd 4 theory to choose the width of implants for 560 patients who got the dual plane i or ii breast augmentation through transaxillary or periareolar incision. we should take notice that the theory is not suitable for subfascia or subglandular breast augmentation. cd is defined as the horizontal curved distance at the level of inferior mammary fold (imf) from the middle line of sternal bone to the laterally aal. it should be measured with a tape ruler adhering to the skin [figure 1 ]. the aal is defined as the perpendicular line on the lateral chest wall from the anterior point of the axillary fold. the doctor should stand on the patient 's lateral side to draw the line while considering the vertical direction of the thoracic wall [figure 2 ]. the reason for measuring the curved skin distance with a tape ruler rather than a straight line with caliper is that the thoracic wall is curved, and the implant lies just on the curved ribs. the reason for measuring cd on the level of imf is that on this level, there is no influence of the breast tissue volume. w (width of implant) = cd 4 (cm) is suitable for the regular patients. if the patient is so thin that we could see the contour of her ribs on the chest wall, w = cd 3.5 (cm) if the patient has a certain amount of breast tissue, for example, the pinch test 2.5 cm, we could subtract 0.5 cm or 1 cm from w according to the patient 's desire. postoperatively, we measured the distances between the two medium margins of the patients breasts (without bras) to judge whether or not they got best intermammary cleavages. we also observed if or not the most lateral margins of the breast were on the area of aal. among 560 patients, 62 patients chose round implant from 175 ml to 300 ml with w from 10.5 cm to 12 cm. the other 498 patients chose anatomic implants from 220 g to 320 g with w from 10.5 cm to 12.5 cm. 78% of the patients have got followed up from 1 month to 5 years postoperatively. their intermammary cleavages are between 1 cm and 2.5 cm without wearing bras. if wearing tight bras, 90% of the patients got their two breasts connecting with each other, which was the most satisfied part of their new breasts. all patients are satisfied with their new nature breast shape and volume except for four patients who got unilateral capsular contractions. (a) typical case 3a : frontal view preoperatively w = cd (16 left/15.5 right) 4 = 12 (cm). we add 0.5 cm to the right w ; (b) lateral view 1 week follow - up postoperatively, the black arrow indicating the anterior axillary line moving forward about 1 cm ; (c) frontal view 6 months follow - up postoperatively ; (d) lateral view 6 months follow - up postoperatively, the most lateral margin of the breast is on the postoperative anterior axillary line. figures 4 and 5 (a, b, c, d are defined) shows the reasons. a (1.5 cm) is the ntz. as we mentioned before, any sharp dissection with electric cautery within the ntz could have a high possibility of damaging the vessel perforators and uncontrolled bleeding, especially through the transaxillary incision with endoscope. because the broken artery will shrink into the deep muscles and electric coagulation could not work probably, ligation of the artery is sometimes needed. dissection of ntz could also induce the palpation of the implant edge, even the symmastia. although some experienced doctors could reach out of the margin of ntz a little bit without complications, as a rule, the ntz should be highly respected. the a is 1.5 cm, the half of no touch zone. the point w (implant width) = cd (a + b + c + d) = cd (1.5 + 0.5 + 1 + 1 or 0.5) = cd 4 or 3.5 (cm). the serratus anterior muscle (flat red c) becomes upright because of the implant projection. the b is death zone that implant edge could not reach to. is lateral thickness of soft tissue, which is about 1 cm for regular patients and about 0.5 cm for very thin patients. b (0.5 cm) is the death cavity, which means the most medium part of the dissected implant pocket is an acute angle area. in contrast, the serratus anterior muscle on the most lateral implant pocket 's baseline part (red line in c of figure 5) could be elevated. when the implant is put into the pocket, what is c ? as we mentioned above, unlike lying on the ribs medially, the implant is lying on the part of serratus anterior muscle laterally. when the implant is inserted into the dissected pocket, because of the projection of the implant, the part of lateral serratus anterior muscle (c) will stand up to embrace the implant. we call this phenomenon width become height [figures 4 and 5 ]. e shown in figure 4, dissecting should be done about 1 cm more laterally on the top of serratus anterior muscle (red line of c) to let [figure 3c ], we could see the aal move forward (black arrow), which is another proof of width become height. even though the preoperative aal is moved forward a little bit after the implant is put in, the most lateral border of the new breast is still on the postoperative aal if the patient has a certain amount of lateral soft tissue. for those patients with relatively thin lateral soft tissue, the most lateral border of the new breast is probably about 1 cm away from the post - operative aal. but that is fine because if we add 1 cm to w, the implant will be too big for the patient. for example, if cd = 16 cm, then the w = cd 4 = 16 4 = 12 (cm). in this situation, for the allergan 410 implants, we should choose implants no bigger than 300 g. but if we add 1 cm to 12 cm, then we have to choose implants over 300 g, which is too big for patients with cd of 16 cm. as we know, most chinese women do not like too big and unnatural breasts. d represents the thickness of the soft tissue on the lateral side of the pocket, which is about 1 cm for regular patients and 0.5 cm for very thin patients. so finally, we got the formula : w = cd (a + b + c + d) = cd (1.5 + 0.5 + 1 + 1 or 0.5) = cd 4 (or 3.5) (cm). most of women like to have their two breasts contacting with each other when they are wearing bra. if we suppose the soft tissue in ntz is very flat and thin like a piece of paper, the narrowest intermammary distance should be 3 cm. to produce the best intermammary cleavage, the only thing that the doctor could do is to let the medium border of the implant reach the point 1.5 cm away from the middle line, in spite of the existing b part. the more soft tissue around the ntz, the narrower intermammary distance will be, and the more likely the two new breasts would contact with each other postoperatively. the doctor could not totally control it and should let their patients know the fact. if you do this kind of communication with your patients preoperatively, it will greatly improve your patients postoperative satisfaction rates. sometimes in order to get narrower intermammary cleavage, the authors had to use blunt dissecting technique to surpass the limit of ntz about 0.5 cm and found that under endoscope some perforators remain intact [figure 6 ]. we also recommend using blunt dissecting technique on the lateral side of the pocket to avoid the damage of the intercostal nerves. the more you dissect the lateral side of the pocket, the more likely you will damage the intercostal nerves. this is another reason why we choose the aal as the lateral border of the new breast. endoscopic view : the vessels are not broken after blunt dissection on medium part of implant pocket. most of the plastic surgeons get used to take bw (base width of the existing breast parenchyma) as the main factor to determine w. dr. dennis c hammond thinks : w = bw 1/2 (medium tissue thickness + lateral tissue thickness). the author does not use the method because of the following reasons : the concept of bw is not very clear. sometimes it is defined as the base width of the existing breast tissue, sometimes it is defined as the distance between the medium and lateral margins when displacing the breast parenchyma medially and laterally. the above two ways will produce different bws, and only those experienced surgeons could be skilled at it.the bw does not concern about the ntz. some patients medium points of the breast widths are within the ntz.for some patients with very small amount of breast parenchyma or contracted breast, the bw is either very small or it is hard to recognize the border of the breast base.bw is always measured with calipers, so it is a straight - line distance. the chest wall is not straight but curved, and the implant is lying on the curved chest wall, so curved distance is more reasonable. sometimes it is defined as the base width of the existing breast tissue, sometimes it is defined as the distance between the medium and lateral margins when displacing the breast parenchyma medially and laterally. the above two ways will produce different bws, and only those experienced surgeons could be skilled at it. for some patients with very small amount of breast parenchyma or contracted breast, the bw is either very small or it is hard to recognize the border of the breast base. bw is always measured with calipers, so it is a straight - line distance. the chest wall is not straight but curved, and the implant is lying on the curved chest wall, so curved distance is more reasonable. the cd 4 theory can not be applied to the patients who need to do subfascia or subglandular breast augmentation. in these cases, the patients will always have enough breast tissue for doctors to measure clear bws. in such situations, the w bw. when a patient 's chest wall is much wider on the upper part and narrower on the lower part, we need to add 0.5 cm to the cd. because we should consider that the transverse diameter of the implant should be at the nipples level, but not the imf level. finally, we should know that the theory cd 4 should be regarded as a principle of choosing w. it is not a precise or accurate formula that you must always obey in clinics. sometimes you could add or subtract 0.5 cm or even more from the cd 4 according to the patients situations and still achieve good results. | background : the determination of the width of the implant is the first key step to select shape and volume of the implant in breast augmentation. the aim of this study was to introduce a new method to determine the width of the implant (w) and explain the reasons to do so in details.methods:from january 2006 to june 2014, the authors have found and applied cd4 theory to determine the width of breast implant (w) in dual plane i or ii breast augmentation cases through transaxillary or periareolar incision for 560 patients. cd is defined as the curved distance on skin from the midline of the sternal bone to the anterior axillary line (aal) on the lateral chest wall through the horizontal level on inferior mammary fold. w = cd 4 (or 3.5) cm.results:the 560 patients used both round and anatomic implants with w from 10.5 cm to 12.5 cm. their cds are from 14.5 cm to 17 cm. about 78% of the patients have got followed up from 1 month to 5 years postoperatively. except for four patients who got unilateral capsular contractions, all the other patients are satisfied with their nature new breast shapes and volumes. their new intermammary cleavages without bras are between 1 cm and 2.5 cm, and lateral borders of the breast are on the area of the aal.conclusions:w (width of the implant) = cd 4 (cm) when doing dual plan i or ii breast augmentation. for the very thin patient, 4 should be 3.5. |
we studied 53 mdr m. tuberculosis strains isolated from different patients between 1993 and 2001 at the bangui pasteur institute. fourteen of these patients were hiv positive, 30 were hiv negative, and 9 were of undetermined status. a non - mdr, nonbiased control group, which included 263 m. tuberculosis and 2 m. bovis strains, was also studied. all 318 isolates were typed by using the spoligotyping method previously described (13). spoligotypes were obtained for 283 (53 mdr and 230 non - mdr strains) of the 318 isolates and were analyzed with bionumerics software (applied maths, kortrijk, belgium). dendrograms were constructed according to degree of similarity (dice coefficient) and comparison with known spoligotypes. seventy - nine different spoligotypes were identified : 55 included only a single strain, and 24 included 256 strains. twenty - five (47.2%) of 53 clustered in type e, which has characteristics of the t family (ancient m. tuberculosis strains with numerous spacers). the 230 spoligotype patterns of non - mdr strains were grouped into 22 clusters, and spoligotype e was not a major cluster (figure, panel b). its spoligotype is identical to the db3 pattern st47 characteristic of the haarlem family (15). spoligotypes 97%99% identical with profiles characteristic of the haarlem family of strains represent 155 strains. strain distribution into various clusters observed among 53 spoligotyped multidrug - resistant (mdr) strains (a) and 230 spoligotyped non - mdr strains (b). the clustering of mdr strains suggested an mdr outbreak ; therefore, we looked for other characteristics in cluster e isolates. first, we tested for diversity in the rpob region, which was likely to be responsible for rifampin resistance. five variants were found among the 26 mdr strains that constituted clusters e and f (table) : 9 had a ser-531 (tcg) to leu (ttg) substitution ; 8 and 5 strains contained a substitution of his-526 (cac) with tyr (tac) and arg (cgc), respectively ; 3 had an asp-516 (gac) to val (gtc) variant ; and 1 a leu-533 (ctg) to pro (ccg) substitution. all these variations are in the rifampin resistance - determining region frequently encountered in strains with a rifampin - resistant phenotype (8). these variants probably determine rifampin resistance and may have occurred independently, not necessarily corresponding to mdrtb transmission, even for strains of the same cluster with the same change in rpob. indeed, sociodemographic and epidemiologic characterization of the patients did not show any links between these mdrtb cases. therefore, rifampin resistance seems to have been acquired independently and repeatedly by cluster e and f strains. to find a way to reduce the dissemination of such strains likely to generate mdr isolates, we characterized strains of cluster e and f. in particular, we looked for single nucleotide polymorphisms (snps) in the putative genes mutt1, mutt2, mutt3, rv3908, muty, mutm, ada / alka, and ogt. sequencing was performed as previously described (11). with reference to published m. tuberculosis sequences, we found 1 synonymous snp in mutt1 corresponding to val 265 (gtc) to val (gta). this snp is only present in strains of cluster e and strains 27 (cluster f), 28, and 29 and is absent from all strains in other clusters. therefore, these mdr strains are characterized by a spoligotype pattern (st 52 spacer 11 or 12 to 15 and st 107) and the presence of the mutt1 snp 265. atb, antibiogram results, indicates strains resistant to rifampin (r), isoniazid (i), ethambutol (e), and streptomycin (s) ; nd, not determined. mdr strains of clusters e and f and strains 28 and 29 (a single difference in spacers between e and f, strain 28 or 29) corresponded to 9 new cases and 19 patients who had received previous treatment. mdr strains in other clusters corresponded to 9 new cases, 15 previously treated patients, and 1 case for which no history was available (3). we used 2 types of markers to study the genetic diversity of mdr m. tuberculosis strains isolated in bangui : spoligotyping and snps in a series of putative dna repair genes. many mdr strains were clustered in 1 spoligotype and carried the same snp in the anti - mutator gene mutt1. indeed, 25 of the 53 mdr strains were in cluster e. thirty - two percent of these mdr strains were from new cases of infection, and 40% were from hiv - infected patients. this cluster was not a major cluster among the 265 non - mdr isolates collected during a 5-month period. the same snp was found in all strains of cluster e and f tested and in 2 strains that differed by 1 spacer. these strains carry variants of rpob that confer rifampin resistance, which implies that these strains do not correspond to an mdr - tb outbreak. however, this finding is consistent with the possibility that these strains represent an mdr - prone family, members of which are often associated with mdr phenotypes in bangui. detection of strains characterized by the t family spoligotype and mutt1 snp 265 may be useful to identify patients at risk of developing mdr - tb. | we investigated multidrug - resistant (mdr) mycobacterium tuberculosis strains in bangui, central african republic. we found 39.6% with the same spoligotype and synonymous single nucleotide polymorphism in the mutt1 gene. however, strains had different rpob mutations responsible for rifampin resistance. mdr strains in bangui may emerge preferentially from a single, mdr - prone family. |
unlike wound healing, which is common to all animals, the capacity for regeneration varies between and within species and phyla. species with the potential to regenerate the heart essentially use two strategies ; progenitor cell proliferation, or dedifferentiation and subsequent division of the cells surrounding the injury (here and elsewhere we use the term dedifferentiation to mean the condition whereby a cell regresses from a fully differentiated form into a simpler state, an event coupled with cell cycle reentry). heart regeneration is remarkably efficient in adult zebrafish, as demonstrated by experiments involving resection of the ventricular apex (poss., 2002). new cardiomyocytes originate from progenitor cells that express epicardial markers, such as raldh2 and tbx18 (poss, 2007). zebrafish cardiac progenitor cells are successfully driven to regenerate by interaction with the epicardium, the thin epithelial layer enveloping the chambers. the epicardium is not simply a bystander to myocardial regeneration after injury ; rather, it exhibits a rapid and robust proliferation. cells close to the resection site invade the regenerating tissue through a process strongly reminiscent of the epithelial - to - mesenchymal transition occurring in the developing heart, contributing endothelial and smooth muscle cells for the new vessels (lepilina., 2006). the epicardium also regulates the addition of new myocardial and epicardial cells during homeostatic cardiac growth (wills., 2008), but it remains unclear whether newly formed cardiomyocytes originate from this source or from progenitor cells after epicardial - mediated activation. epicardial and myocardial cross - talk is mediated by fgf signaling, and inhibiting the fgf receptor blocks cardiac regeneration (lepilina., 2006). the same signaling pathway guides cardiac progenitor cells in regulating heart size, atrial - to - ventricular proportions, and ventricular cardiomyocyte numbers at later stages of development (marques., 2008). other signals may cooperate with fgf to induce myocardial proliferation, as suggested by the up - regulation of growth factors (pdgf, insulin - like growth factor, and delta - notch) during regeneration (poss, 2007). in adult salamanders and newts (notophthalmus viridescens), larval axolotl (amblystoma mexicanum), and teleost fish (danio rerio, zebrafish), regeneration after heart apex amputation involves blastema formation ; i.e., the accumulation of dedifferentiated cells near the edge of the lesion. these blastema cells originate either from the dedifferentiation of resident cardiomyocytes (salamanders) or from resident progenitor cells (zebrafish). in the newt, dividing cardiomyocytes partially disassemble sarcomeric structures (tate and oberpriller, 1989) and revert to cells that can renew and also differentiate into other cell types on appropriate inductive signals (laube., 2006). after injury, 75% of cultured cardiomyocytes synthesize dna and 60% progress to karyokinesis ; about half of the latter divide, whereas the other half generate multinucleated cells (bettencourt - dias., 2003), which indicates a heterogeneity in relation to the regulation of cell division. dedifferentiation of preexisting cardiomyocytes has also been postulated for the zebrafish heart, but it has not been supported by experimental evidence (lepilina., 2006). at variance with zebrafish and salamanders, the uninjured mammalian myocardium is traditionally considered incapable of self - renewal. using a sophisticated and very elegant inducible transgenic mouse model, hsieh. they also showed, however, that the adult heart achieves a modest, but nonetheless convincing, self - renewal after injury that was attributed to a pool of resident stem cells. over the past few years, several reports have described putative cardiac stem and progenitor cells in the adult heart. we give a brief account here of the major contributions in this field, referring the reader to several recent papers for a thorough overview of the topic (passier., 2008 ; segers and lee, 2008 three distinct cardiac stem cell populations have been described in the adult myocardium, based on the expression of the surface markers c - kit (tyrosine kinase receptor-1), sca-1, and the atp - binding cassette transporter (also called the side population or sp because these cells pump out hoechst stain ; beltrami., 2003 ; oh. (2003) cloned and expanded these cells from different species, and obtained significant cardiac regeneration (beltrami., 2003), angiogenesis, and arteriogenesis (tillmanns., 2008) after their injection into the infarcted myocardium. despite these findings, the ability of c - kit cells to activate and support spontaneous regeneration in the adult heart remains highly controversial. all studies are based on the transplantation of very large numbers of c - kit cells into the injured heart, so a cell lineage approach is required to address this issue conclusively. two other major concerns about these cells are their precise localization and number. using genetically engineered mice expressing gfp under the c - kit promoter, fransioli. (2008) demonstrated that c - kit cardiac cells decline markedly in the first two postnatal weeks and almost disappear after ten weeks. numerous efforts have been put forward in the last few years to investigate the nature of progenitor cells responsible for heart development because it is well established that the multiple programs controlling the onset of cardiogenesis are recapitulated in heart disease. fate - mapping studies have demonstrated that the developing heart contains isl1/nkx2.5/flk-1 multipotent mesodermal progenitors that can give rise to cardiac muscle, smooth muscle, and endothelial lineages (fig. 1 2005), the embryological anlagen from which the atria, outflow tract, and most of the right ventricle develop (kelly., 2001). however, a recent study suggests that these cells contribute to cardiac, smooth muscle, and endothelial cells in all four cardiac chambers (ma., 2008 ; for review see laugwitz., 2008). additionally, a bipotent nkx2.5/c - kit progenitor isolated from the developing mouse embryo has been shown to generate cardiac and smooth muscle cells in vitro and in vivo (wu., 2006). schematic representation of stem and progenitor cells in the developing and juvenile / adult rodent heart. cm, cardiomyocytes ; ec, endothelial cells ; smc, smooth muscle cells. a second potential source of stem cells in the developing heart is the epicardium (fig. investigations on the fate and differentiation of epicardium - derived cells (epdcs) indicate that they generate smooth muscle cells and fibroblasts of the coronary vessels, and the interstitial fibrous skeleton of the heart (winter and gittenberger - de groot., 2007). in avians, the origin of endothelial cells in the coronary vessels is still being debated (prez - pomares., 2002), as is the myocardial potential of epdcs (mikawa and fischman, 1992 ; mikawa and gourdie, 1996 ; gittenberger - de groot., 1998). in the developing mouse heart, recent data have shown that epicardial progenitors expressing tbx18 (cai., 2008) and wt1 (zhou., 2008), possibly derived from a common isl1/nkx2.5 ancestor (ma., 2008), generate new cardiomyocytes. it would be tempting to hypothesize that the essential properties of the epicardium can be recapitulated in the adult heart and used for regeneration. epicardial c - kit multipotent cells expressing nkx2.5 and gata4 have been identified in the human and mouse adult heart as a minority of all the epicardial cells. keeping the pericardial sac intact during infarction by ligating the coronary artery prevents myocardial tissue from deteriorating and results in foci of cardiac regeneration, as demonstrated by the presence of a small population of lentivirus - labeled epicardial cells expressing the cardiac marker -sarcomeric actin (limana., 2007). other reports indicate that epdcs do not acquire a cardiomyocyte phenotype when transplanted into infarcted mouse heart (winter., 2007). to summarize, it seems reasonable to suggest that the mammalian epicardium is composed of heterogeneous populations of cardiogenic progenitors with distinct potentials for differentiation. as in the zebrafish, the epicardium has a crucial stimulatory role essential for proper development, and possibly also for regeneration. one of the factors that influences this instructive role is thymosine 4 (t4), a g - actin monomer - binding protein implicated in cytoskeleton reorganization. t4 secreted from the developing myocardium stimulates the proliferation, differentiation, and inward migration of epicardial cells (smart. similarly, t4 is responsible for enhanced cardiomyocyte survival, and consequently for cardioprotection (bock - marquette., 2004 ; smart., 2007), in the event of injury. a third, and potentially more useful, source of cardiac stem and progenitor cells may be the vasculature. vessel - associated progenitor cells, called mesoangioblasts, have recently been identified in the juvenile mouse heart (fig. 1). these cells express endothelial and pericyte markers, and are committed to cardiac differentiation in vitro and in vivo (galvez., 2008). because they retain a remarkable potential for cardiac differentiation when implanted in the secondary heart field of a chick embryo, it is reasonable to postulate that they are recruited to become cardiomyocytes during postnatal growth. it is worth noting that cardiac progenitor cells, which are identifiable from their ability to grow as cardiospheres, have been obtained using similar technical procedures from both adult mouse heart explants (messina., 2004) and human endomyocardial ventricular biopsies (smith., 2007). in the developing heart, there may be a cell network that, for regeneration purposes, connects the epicardium to the myocardium via the coronary vessels, as shown in fig. 2. further investigations are needed to establish whether such a network persists in postnatal life. schematic representation of the hypothetical link between epicardial progenitor cells and the mesoangioblast / pericyte cell population surrounding the coronary vessels. a subpopulation of epicardial cells delaminates from the subendocardial space and migrates to the underlying myocardium using the vascular network as a guide. these progenitors may also be able to generate the intramyocardial clusters of c - kit / sca-1 stem and progenitor cells that persist into adulthood, but alternative sources of these cells can not be excluded., the mammalian heart contains a plethora of potential stem and progenitor cells, but their persistence in adulthood is still unclear. isl1 cells progressively disappear in the postnatal heart except where the cardiac autonomic nervous system intersects with the cardiac conduction system (laugwitz., vessel - associated progenitor cells, in contrast, persist in the postnatal heart, but their number and potential to regenerate over time has yet to be determined. cardiac c - kit+ stem cells are found in adult myocardium, but it is unclear whether they are a remnant of development or are recruited postnatally. in addition, it is possible that some of the many progenitor cells found in the developing heart have simply not yet been identified in the postnatal heart. what are the restrictions to cardiac regeneration and are they species - specific ? can these evolutionarily imposed restrictions be removed ? in answer to these questions, two barriers to regeneration in the mammalian heart have been identified : one involving the capacity of cardiomyocytes to proliferate, the other lying in the interactions between cardiomyocytes and interstitial cells. a final round of incomplete division occurs after birth and results in binucleated or multinucleated cardiomyocytes. cardiomyocytes can reactivate the cell cycle after various pathological stimuli such as a hypertrophic response (pasumarthi and field, 2002 ; rubart and field, 2006 ; van amerongen and engel, 2008). therefore, the histological detection of cell cycle and dna synthesis markers, such as ki67, histone h3 phosphorylation, and brdu incorporation, does not necessarily mean cell division. only karyokinesis and cytokinesis can be accepted as definitive proof of cardiomyocyte proliferation. using a combination of in vitro and in vivo models, it has been demonstrated that the myocardial cell cycle can be reprogrammed, at least to some degree. the majority of proteins that affect the cell cycle activity of cardiomyocytes are involved in transit through the restriction point. cdk4 and cdk6, and their activating partners d - type cyclins (pasumarthi., 2005), the phosphorylation state of the retinoblastoma pocket protein (maclellan., 2005), and the subsequent activation of the transcription factor e2f, have been manipulated to induce cardiac cell division. striking evidence of the functional impact of reactivating cardiomyocyte proliferation comes from the demonstration that cardiac - specific overexpression of cyclin d2 improves cardiac function after infarction in mice (hassink., novel signaling pathways that control cardiac cell division have recently been revealed with a view to developing new pharmacological strategies to counteract myocardial dysfunction. the extracellular matrix protein periostin reactivates mitosis in mouse cardiomyocytes via -integrin and phosphoinositide 3-kinase (khn., 2007), but why high periostin levels in the infarcted myocardium are unable to promote cell cycle activation in the resident cardiomyocytes remains to be explained. cardiac cell division is accompanied by structural modifications, resulting mainly in sequential myofibrillar disassembly and reassembly (ahuja., 2004). this complex process might provide a mechanistic explanation as to why postnatal cardiomyocytes stop dividing as the contractile apparatus matures. it may be that dedifferentiation favors cell cycle reentry, as in naturally dividing newt cardiomyocytes (bettencourt - dias., 2003). indeed, dedifferentiated cardiomyocytes (characterized by disassembly of the contractile apparatus, the acquisition of fetal markers, and anomalous mitochondria) have been seen in human diseases such as chronic hibernating myocardium or chronic atrial fibrillation, and in the border zone of infarction (heusch and schulz, 2000). we have found that adult cardiomyocytes cocultured in the presence of cardiac fibroblasts undergo dedifferentiation and cell cycle reentry (zaglia., 2008). the fine - tuning that enables cell cycle progression and completion after dedifferentiation remains to be elucidated. the differing abilities of zebrafish, salamander, and mammals to generate new cardiomyocytes might also depend on the microenvironment. a comparative discussion on the influence of the microenvironment on cardiac regeneration is complicated because different types of injury have been studied in different species. in mammals, cardiac regeneration has been studied mainly in acute or chronic ischemia, or ischemia / reperfusion induced by coronary artery ligation. in newt and zebrafish, the model used is partial cardiac amputation because zebrafish have only subepicardial vessels that open into lacunes (hu., 2000), and the newt has no coronary vessels at all. fibroblast distribution also varies : fibroblasts account for the majority of nonmuscle cells in mammals, but they are rare in zebrafish and newts, and occur randomly interspersed within the subepicardial layer. on the whole, the myocardial histology of these lower vertebrates is quite simple and its 3d structure resembles that of the trabeculated fetal heart of mammals. tissue organization is more complex in the adult mammalian heart : single cardiomyocytes are in contact with capillaries and interconnected with fibroblasts (fig. fibroblasts have an important role in developing the local and global myocardial response to mechanical, electrical, and chemical signals generated by cardiac damage (baudino., 2006). hematoxylin and eosin staining shows tissue organization in zebrafish (a), axolotl (c), and rat (e) hearts. vascular - specific gfp expression in transgenic fli1 (friend leukemia integration 1)-gfp zebrafish (lawson and weinstein, 2002) or immunostaining with specific antibodies show the distribution of the vascular network in these species. zebrafish contain epicardial vessels (b, arrows) and endocardial cells that circumvent the cardiomyocytes (b). axolotl have no epicardial vessels, whereas endocardial cells expressing the von willebrand factor (vwf) surround the cardiomyocytes (d). in the rat, the coronary vessels deepen into the myocardium, as shown by staining with antibodies to cd31 (endothelial marker) and sma (smooth muscle cell marker). (right) two schematic representations show the positional relationships between cardiomyocytes (cm) and surrounding endothelial (ec) or endocardial (enc) cells, fibroblasts (f), and pericytes (p). b, b, d, and f are confocal images of tissue sections. bars : (a, c, e, and f) 100 m ; (b and d) 50 m. the modulation of fibroblast activity can impact the balance of regeneration versus healing after cardiac injury. in zebrafish and newt, regeneration is accompanied not only by progenitor cell activation or cell dedifferentiation, but also by the up - regulation of genes encoding matrix metalloproteinases (vinarsky., 2005 ; lien., 2006) and tissue inhibitors of matrix metalloproteinases (stevenson., 2006). in limb reconstruction, these genes after heart injury in zebrafish, fibroblasts and cardiomyocytes are activated in a reciprocal manner. when cardiogenesis is blocked by a dominant - negative fgf receptor or mutation of the msp1 mitotic dominant checkpoint kinase, fibrin is retained, and a collagen - rich scar forms (poss., 2002 ; poss, 2007). this indicates that regeneration and repair sense each other and that the balance can be tipped toward one or the other, even when evolution has preserved the genetic capacity for regeneration. it also suggests that the extensive, well - organized network of fibroblasts in the mammalian heart is, by itself, an unfavorable condition for regeneration purposes for both cardiac stem cell differentiation and cardiomyocyte division. although this response gives the injured heart the chance to surround the damaged area with granulation tissue and extracellular matrix, thus limiting expansion of the injured area, it can damage the surviving cardiomyocytes and cardiac stem cells. it has been postulated from experiments in limb reconstruction that this less sophisticated adaptive immunity may be involved in conferring a marked regenerative potential to these species (harty., 2003 ; godwin and brockes, 2006 ; mescher and neff, 2006). unfortunately, studies on cardiac regeneration in newt / axolotl and zebrafish have failed to provide a detailed analysis of the inflammatory response. blocking inflammation would be particularly interesting in zebrafish, for which mutants with an aberrant accumulation or deficiency of some inflammatory cells are available (mathias., 2007). finally, the immature immunoinflammatory response and limited number of fibroblasts might also explain the great potential for regeneration of the mammalian heart in its early stages of development (blewett. if a 14-d - old fetal mouse heart is explanted, surgically damaged, and kept in serum - free medium as an organ culture, its tissue architecture is rapidly reestablished with no inflammatory response or scarring. by 18 or 22 d of gestation, fibroblasts and myofibroblasts have accumulated in the heart, and the trabeculated structure of the ventricle has been replaced by compact myocardium. if the organ is explanted at this point in development, a wound will heal by scarring. we reasoned that the differing regenerative capabilities of hearts from teleost fish and urodele amphibians compared with mammals stems from the existence of species - specific barriers. in particular, adult mammalian cardiomyocytes have an inherent inability to regenerate, either via stem cell mediated mechanisms or by reentry of the cell cycle, and, additionally, they show a marked interstitial response. we suggest that the limited regeneration potential of the mammalian heart is caused by an evolutionary prioritization of hemostasis and fibrosis. bleeding from the heart in a high - pressure circulatory system, which is practically unique to higher vertebrates, can seriously jeopardize survival. selective pressure probably favored the more rapid fibrous healing response because survival would be compromised in the time it took to seal the defect by regeneration. the fetal cardiac microenvironment is characterized by a low oxygen tension in mammals as well, in contrast with the high oxygen - based metabolic demand of the postnatal heart. because progenitor cell activation requires low oxygen levels in the microenvironment (simon and keith, 2008), the mammalian adult heart may be inherently incapable of activating its own resident cardiogenic progenitors after injury. cardiomyocyte proliferation could provide a mechanism for cardiac regeneration ; however, it is unclear whether it is better for cardiomyocytes to revert to proliferating progenitors or to dedifferentiate back to immature cardiomyocytes that should be capable of one or more cell divisions. the former option would enable them to proliferate in abundance and become pluripotent ; the latter would have a limited effect, but could prevent tumorigenic outcomes and might also favor cell cell contact and integration with preexisting tissue. evidence from infarcted hearts of mhc - cycd2 transgenic mice demonstrated that cardiomyocyte cell cycle reactivation resulted in newly formed myocardium that participated in a functional syncytium with surviving myocardium from outside of the damaged zone (hassink., 2008). cardiac stem cells remain an attractive possibility for regeneration, but the major identifying criteria for these cells in adult heart need to be fixed. first, clonogenic, self - renewal, and differentiation properties should be established in vitro and in vivo. in the latter case, a single cell should be transplanted into a cardiac region depleted of its own putative stem cell reservoir, as this is the gold standard that has been achieved in other systems such as skeletal muscle (sacco., 2008) and second, if cardiac stem cells differentiate according to a hierarchical model, it is necessary to establish the transcriptional signature at different stages of quiescence, amplification, differentiation, and maturation. the combined expression of stem cell markers and cardiogenic transcription factors should be investigated using a cell lineage approach. slowing the process of fibrosis may tip the balance of cardiac repair from healing to regeneration. therapeutic options to counteract fibrosis by controlling the inflammatory response have been largely discouraging (for reviews see hinz., 2007 ; wynn, 2008) ; however, new approaches need to be attempted because the mechanisms involved in fibrogenesis are now known to be distinct from those pertaining to inflammation. cardiac fibroblasts might also be manipulated genetically to control their capacity for proliferation and transition toward myofibroblasts. depressing fibroblast proliferation via the p53 and/or p16 retinoblastoma pathways leads to a reduced fibrosis in the liver in mice (krizhanovsky., 2008), which suggests that a similar approach is worth testing in the heart. it has recently been shown that somatic cells like adult fibroblasts can be reprogrammed to obtain a stable pluripotency using a combination of four transcription factors : c - myc, oct-4, sox-2, and klf4 (takahashi and yamanaka, 2006). we recently found that cardiac fibroblasts differ from those of other organs in that they already express a repertoire of cardiac genes, including gata4 (zaglia., 2008). whether this particular gene expression program makes them more responsive to cardiac reprogramming remains to be seen. it is worth adding that forcing the expression of myocardin in postinfarcted cardiac fibroblasts induces the expression of cardiomyocyte - specific proteins (van tuyn., 2007). on the whole, it is probably just as important to block fibroblast proliferation as to provide specific cues to induce myocardial proliferation. we need to reconsider regeneration as a process dependent on a concerted interplay between cardiomyocytes and the surrounding nonmyocardial cells, possibly as a structural and functional unit. it is interesting that in a mouse model of heart failure, specific inhibition of the interstitial fibrogenic response has an impact on cardiac hypertrophy and dysfunction, which suggests that fibroblasts control the cardiomyocyte response to pathological stimuli (thum., 2008). the complexity of this partnership varies considerably from one species to another, ranging from single cardiomyocytes with associated supporting capillaries and fibroblasts, as in mammals, to cardiomyocytes surrounded by endocardium with almost no fibroblasts or capillaries, as in zebrafish and axolotl (fig. 3). to summarize, the lesson that we have learned from comparison with regeneration - prone species is that the mammalian heart is inherently resistant to regeneration, possibly as a result of an evolutionary imposition. therefore we propose that a combined approach encompassing all the three strategies described in this review, namely cardiomyocyte cell cycle reentry, stem cell mediated regeneration, and controlled fibrogenic response, has the greatest chance to succeed in restraining or even reversing the progression of cardiac deterioration in disease. cardiomyocyte proliferation could provide a mechanism for cardiac regeneration ; however, it is unclear whether it is better for cardiomyocytes to revert to proliferating progenitors or to dedifferentiate back to immature cardiomyocytes that should be capable of one or more cell divisions. the former option would enable them to proliferate in abundance and become pluripotent ; the latter would have a limited effect, but could prevent tumorigenic outcomes and might also favor cell cell contact and integration with preexisting tissue. evidence from infarcted hearts of mhc - cycd2 transgenic mice demonstrated that cardiomyocyte cell cycle reactivation resulted in newly formed myocardium that participated in a functional syncytium with surviving myocardium from outside of the damaged zone (hassink., 2008). cardiac stem cells remain an attractive possibility for regeneration, but the major identifying criteria for these cells in adult heart need to be fixed. first, clonogenic, self - renewal, and differentiation properties should be established in vitro and in vivo. in the latter case, a single cell should be transplanted into a cardiac region depleted of its own putative stem cell reservoir, as this is the gold standard that has been achieved in other systems such as skeletal muscle (sacco., 2008) and second, if cardiac stem cells differentiate according to a hierarchical model, it is necessary to establish the transcriptional signature at different stages of quiescence, amplification, differentiation, and maturation. the combined expression of stem cell markers and cardiogenic transcription factors should be investigated using a cell lineage approach. slowing the process of fibrosis may tip the balance of cardiac repair from healing to regeneration. therapeutic options to counteract fibrosis by controlling the inflammatory response have been largely discouraging (for reviews see hinz., 2007 ; wynn, 2008) ; however, new approaches need to be attempted because the mechanisms involved in fibrogenesis are now known to be distinct from those pertaining to inflammation. cardiac fibroblasts might also be manipulated genetically to control their capacity for proliferation and transition toward myofibroblasts. depressing fibroblast proliferation via the p53 and/or p16 retinoblastoma pathways leads to a reduced fibrosis in the liver in mice (krizhanovsky., 2008), which suggests that a similar approach is worth testing in the heart. it has recently been shown that somatic cells like adult fibroblasts can be reprogrammed to obtain a stable pluripotency using a combination of four transcription factors : c - myc, oct-4, sox-2, and klf4 (takahashi and yamanaka, 2006). we recently found that cardiac fibroblasts differ from those of other organs in that they already express a repertoire of cardiac genes, including gata4 (zaglia., 2008). whether this particular gene expression program makes them more responsive to cardiac reprogramming remains to be seen. it is worth adding that forcing the expression of myocardin in postinfarcted cardiac fibroblasts induces the expression of cardiomyocyte - specific proteins (van tuyn., 2007). on the whole, it is probably just as important to block fibroblast proliferation as to provide specific cues to induce myocardial proliferation. we need to reconsider regeneration as a process dependent on a concerted interplay between cardiomyocytes and the surrounding nonmyocardial cells, possibly as a structural and functional unit. it is interesting that in a mouse model of heart failure, specific inhibition of the interstitial fibrogenic response has an impact on cardiac hypertrophy and dysfunction, which suggests that fibroblasts control the cardiomyocyte response to pathological stimuli (thum., the complexity of this partnership varies considerably from one species to another, ranging from single cardiomyocytes with associated supporting capillaries and fibroblasts, as in mammals, to cardiomyocytes surrounded by endocardium with almost no fibroblasts or capillaries, as in zebrafish and axolotl (fig. the lesson that we have learned from comparison with regeneration - prone species is that the mammalian heart is inherently resistant to regeneration, possibly as a result of an evolutionary imposition. therefore we propose that a combined approach encompassing all the three strategies described in this review, namely cardiomyocyte cell cycle reentry, stem cell mediated regeneration, and controlled fibrogenic response, has the greatest chance to succeed in restraining or even reversing the progression of cardiac deterioration in disease. | different vertebrate species have different cardiac regeneration rates : high in teleost fish, moderate in urodele amphibians, and almost negligible in mammals. regeneration may occur through stem and progenitor cell differentiation or via dedifferentiation with residual cardiomyocytes reentering the cell cycle. in this review, we will examine the ability of zebrafish and newts to respond to cardiac damage with de novo cardiogenesis, whereas rodents and humans respond with a marked fibrogenic response and virtually no cardiomyocyte regeneration. concerted strategies are needed to overcome this evolutionarily imposed barrier and optimize cardiac regeneration in mammals. |
the lung is a primary target of cell injury in patients receiving cytotoxic drugs, and in many cases the reaction is severe enough to produce diffuse pulmonary fibrosis. although many different agents may damage the lung, the patterns of cellular injury and repair are relatively constant. using bleomycin toxicity as an example, it has been shown that, after iv injection, the selective site of lung injury is the vascular endothelium ; this is followed by the accumulation of interstitial edema and later by necrosis of type i epithelial cells. in normal repair, rapid proliferation of type ii cells, followed by differentiation to type i, restores the epithelial surface without fibrosis. however, after bleomycin, type ii cell proliferation is frequently followed by abnormal differentiation to a metaplastic form of epithelium. fibroblast proliferation accompanies this abnormal epithelial response which is related either to selective retention of bleomycin by epithelial cells or to the toxic effects of administering more drug at the time of type ii cell division. it is concluded that diffuse interstitial fibrosis results from prolonged disturbance of the normal epithelial - mesenchymal interrelationships at the alveolar wall. disruption of the fibroblastic control system by extensive epithelial necrosis or by delayed or inappropriate repair may be the key factor in instigating fibroblast proliferation and subsequent deposition of collagen. this mechanism may account for the development of diffuse fibrosis or fibrosing alveolitis in response to a variety of pulmonary toxins.imagesfigure 1.figure 2.figure 3.figure 4.figure 5.figure 6.figure 7.figure 8.figure 9.figure 10. |
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to report a first case of bullous pemphigoid (bp) following intravenous fluorescein for fundus angiography. clinical features : a 70-year - old male patient he reported a history of fundus fluorescein angiography with a pre - diagnosis of senile macular degeneration 2 months prior to presentation. at that time, fluorescein extravasated at the antecubital region. following the procedure, pruritus and erythema began at the wrists bilaterally, and quickly spread to the entire body. the patient also reported a history of allergy to human albumin solution (plamasteril ; abbott) 15 years before, during bypass surgery. on dermatologic examination, erythematous patches were present on the scalp, chest and anogenital region. vesicles and bullous lesions were present on upper and lower extremities. on day 2 of hospitalization, the patient was treated with oral methylprednisolone 48 mg (prednol ; mustafa nevzat), topical clobetasol dipropionate 0.05% cream (dermovate ; glaxo smithkline), and topical 4% urea lotion (excipial lipo ; orva) for presumptive bullous pemphigoid. skin punch biopsy provided tissue for histopathology, direct immunofluorescence examination, and salt extraction, which were all consistent with bp. after 1 month, the patient was transferred to the intensive care unit with sepsis secondary to urinary tract infection ; he died 2 weeks later from sepsis and cardiac failure. to our knowledge, this is the first reported case of bp following fundus fluorescein angiography in a patient with known human albumin solution allergy. consideration should be made to avoid fluorescein angiography, change administration route, or premedicate with antihistamines in patients with known human albumin solution allergy. a 70-year - old male patient was admitted to the intensive care unit (icu) with bp and sepsis. the patient 's history was significant for fundus fluorescein angiography (ffa) 2 months prior to diagnosis of senile macular degeneration. the procedure was complicated by fluorescein extravasation at the antecubital region. at that time, the patient experienced pruritus and erythema that began at the wrists and spread quickly to cover the entire body. his past medical history was significant for essential hypertension, glaucoma, and senile macular degeneration. his medications included oral metoprolol 50 mg (beloc), oral aspirin 100 mg (coraspin), and brimonidine tartrate 0.2% (alphagan) ophthalmic solution. the patient also reported a history of allergy to human albumin solution (plamasteril ; abbott), which he had received during bypass surgery 15 years before. on dermatological examination, erythematous patches were present on the scalp, chest and anogenital region. the patient was treated for presumed bp with daily oral methylprednisolone 48 mg, topical clobetasol dipropionate 0.05% cream (dermovate ; glaxo smithkline), and topical 4% urea lotion (excipial lipo ; orva). regression of the lesions began at day 6, at which time daily oral azathioprine 150 mg (imuran) was added. following resolution of all lesions, the patient was discharged with prescriptions for oral methylprednisolone 40 mg, topical 4% urea lotion (excipial lipo ; orva) and oral azathioprine (imuran) 150 mg daily. one month later, he was admitted to the icu with sepsis secondary to urinary tract infection due to underlying decreased immune response. there has been considerable progress in understanding the physiopathology of bp during the past two decades. several cases of bp in patients with diabetes mellitus, inflammatory bowel disease, rheumatoid arthritis, hashimoto 's thyroiditis, dermatomyositis, lupus erythematosus, myasthenia gravis and autoimmune thrombocytopenia have been reported [3, 4 ]. although medications have been implicated in the development of bp, the mechanisms by which drugs can cause bp are unclear. one hypothesis is that drugs may trigger bp in patients with underlying genetic susceptibility, either by modifying the immune response or by altering the antigenic properties of the epidermal basement membrane. a case control study assessing long - term drug use and development of bp showed that diuretics (particularly aldosterone antagonists) and neuroleptics were used more frequently by bp patients than control patients. because of these associations, potential drug causes should be considered in all bp patients. fluorescein angiography is a relatively safe procedure, with a risk profile comparable to that of other intravenous radiocontrast media angiography [6, 7 ]. sodium fluorescein is a low - molecular - weight, highly water - soluble compound with fluorescent properties. because of the physical properties of fluorescein, it is routinely used for medical and laser treatment of chorioretinal diseases. the pathogenic and immune mechanisms that cause adverse reactions in 5% of patients receiving intravenous fluorescein are unknown. adverse drug reactions to fluorescein are classified as mild, moderate, severe, and fatal. severe adverse reactions (0.05%) include cardiovascular shock, myocardial infarction, laryngeal edema, and bronchospasm, and require prolonged, intensive care. patients with a previous reaction to colloidal plasma substitute have a 48% incidence of adverse events with subsequent tests. although these rates are obviously high, we have no recent studies about the present rates [8, 9 ]. adverse reactions following intravenous fluorescein are very unusual, but a history of dye allergy, diabetes, or systemic arterial hypertension increases the risk of allergic reaction to fluorescein. prophylactic treatment, fluorescein desensitization or oral fluorescein angiography should be considered for high - risk patients. other imaging techniques, like optical coherence tomography, should be considered as an alternative in selected cases. to our knowledge, this is the first reported case of fluorescein - induced bp in a patient with colloidal plasma substitute allergy. the association between fundus fluorescein angiography and bp should be further investigated with a formal epidemiologic study. | purposeto report a first case of bullous pemphigoid (bp) following intravenous fluorescein for fundus angiography. clinical features : a 70-year - old male patient was admitted to the intensive care unit with bp and sepsis. he reported a history of fundus fluorescein angiography with a pre - diagnosis of senile macular degeneration 2 months prior to presentation. at that time, fluorescein extravasated at the antecubital region. following the procedure, pruritus and erythema began at the wrists bilaterally, and quickly spread to the entire body. the patient also reported a history of allergy to human albumin solution (plamasteril ; abbott) 15 years before, during bypass surgery. on dermatologic examination, erythematous patches were present on the scalp, chest and anogenital region. vesicles and bullous lesions were present on upper and lower extremities. on day 2 of hospitalization, tense bullae appeared on the upper and lower extremities. the patient was treated with oral methylprednisolone 48 mg (prednol ; mustafa nevzat), topical clobetasol dipropionate 0.05% cream (dermovate ; glaxo smithkline), and topical 4% urea lotion (excipial lipo ; orva) for presumptive bullous pemphigoid. skin punch biopsy provided tissue for histopathology, direct immunofluorescence examination, and salt extraction, which were all consistent with bp. after 1 month, the patient was transferred to the intensive care unit with sepsis secondary to urinary tract infection ; he died 2 weeks later from sepsis and cardiac failure.conclusionsto our knowledge, this is the first reported case of bp following fundus fluorescein angiography in a patient with known human albumin solution allergy. consideration should be made to avoid fluorescein angiography, change administration route, or premedicate with antihistamines in patients with known human albumin solution allergy. the association between fundus fluorescein angiography and bp should be further investigated. |
the agricultural health study, a large prospective cohort study has been initiated in north carolina and iowa. the objectives of this study are to : 1) identify and quantify cancer risks among men, women, whites, and minorities associated with direct exposure to pesticides and other agricultural agents ; 2) evaluate noncancer health risks including neurotoxicity reproductive effects, immunologic effects, nonmalignant respiratory disease, kidney disease, and growth and development among children ; 3) evaluate disease risks among spouses and children of farmers that may arise from direct contact with pesticides and agricultural chemicals used in the home lawns and gardens, and from indirect contact, such as spray drift, laundering work clothes, or contaminated food or water ; 4) assess current and past occupational and nonoccupational agricultural exposures using periodic interviews and environmental and biologic monitoring ; 5) study the relationship between agricultural exposures, biomarkers of exposure, biologic effect, and genetic susceptibility factors relevant to carcinogenesis ; and 6) identify and quantify cancer and other disease risks associated with lifestyle factors such as diet, cooking practices, physical activity, smoking and alcohol consumption, and hair dye use. in the first year of a 3-year enrollment period, 26,235 people have been enrolled in the study, including 19,776 registered pesticide applicators and 6,459 spouses of registered farmer applicators. it is estimated that when the total cohort is assembled in 1997 it will include approximately 75,000 adult study subjects. farmers, the largest group of registered pesticide applicators comprise 77% of the target population enrolled in the study. this experience compares favorably with enrollment rates of previous prospective studies.imagesfigure 1.figure 2.figure 3.figure 4. |
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numerous publications present anti - hcv prevalence in particular groups : blood or plasma donors, hemodialyzed and post - transfusion subjects, intravenous drug users, subjects with high - risk behaviour, health care workers, and patients with liver diseases. among these groups only data from studies of blood donor volunteers can more or less be treated as a reflection of anti - hcv prevalence in the general population. such studies were conducted in many european countries shortly after hcv discovery [117 ]. taking into account risk groups, blood donors studies gave a picture of anti - hcv prevalence and routes of hepatitis c virus (hcv) spread in various parts of europe. a systematic review by esteban summarized those data up to 2007. according to this comprehensive review, in general european population the lowest anti - hcv prevalence occurs in northern (0.11.0%) and central europe (0.21.2%). in southern europe and spain anti - hcv prevalence the authors stressed that up to now there were no sufficient data about anti - hcv prevalence in eastern europe. naoumov (1999) reviewed and summarized data from eastern european studies of blood donors, medical personnel and hemodialysed patients. it was ascertained with reference to blood donor studies that the eastern european population has a high prevalence of hcv infection from 0.68% in czech republic to 4.9% in romania. later publications from this european region have confirmed this conclusion, but there are still no population - based studies published from eastern european countries [2022 ]. prevalence of hcv infection in lithuania has been studied in small groups of first - time blood donors, acute viral hepatitis patients and in risk groups (2.2% in first - time blood donors, 7.9% in commercial donors, 13.9% in commercial plasma donors, 48.3% in hemodialysed patients, 29.4% in prisoners, 9.4% in elderly nursing home residents, and 7.9% in hemodialysis staff). these data did not provide a clear picture of anti - hcv prevalence in the general population, but it allowed estimation of prevalence at from 2% to 3%. we present a descriptive epidemiological study, prepared according to consort and strobe recommendations, aimed at assessing age - specific risk factors for hcv acquisition and prevalence of anti - hcv in the general population of lithuania. an observational analytical cross - sectional study design was conducted to clarify the prevalence of anti - hcv and hcv transmission routes in the general population. cross - sectional survey of random anonymous volunteers and testing them for anti - hcv was conducted may 1722, 2010 in 5 cities of lithuania (vilnius, kaunas, klaipda, iauliai, and panevys) and corresponding rural regions where 75% of the population live (2 513 313 out of 3 329 039, according to the lithuanian statistical department ; http://www.stat.gov.lt/lt/). according to data on anti - hcv prevalence in eastern europe and the study by ambrozaitis. on lithuanian blood donors and some risk groups, we expected the prevalence of anti - hcv in lithuania to be approximately p=3%1%. after calculation of an adequate sample size with precision 1% (d=0.01) and level of confidence of 95% (z=1.96), a cohort of n=1118 subjects was estimated by the formula n=(zp(1p))/d. people over age 18 who were willing to be tested for anti - hcv were voluntarily enrolled into the study by a convenience sampling of shoppers at one of the biggest supermarkets in vilnius, kaunas, klaipda, iauliai and panevys. the only eligibility criteria were age 18 and residence in the selected cities and/or corresponding rural regions. during this study, anti - hcv testing was wrongly performed or questionable for 14 (0.9%) participants or a tested person refused or could not repeat it. anti - hcv tests was suitable for evaluation in a total of 1514 (99.1%) subjects (572 men and 942 women) 651 (241 men and 410 women in vilnius), 266 (128 men and 138 women in kaunas), 313 (115 men and 198 women in klaipda), 148 (55 men and 93 women) in panevys, and 136 (33 men and 103 women) in iauliai (figure 1). the study was performed according to the world medical association declaration of helsinki, and all procedures were approved by the lithuanian bioethics committee. all volunteers who wanted to know their anti - hcv status signed informal consent and answered an anonymous questionnaire with demographic data (age, sex, education, occupation, residence) and exposure to hcv risk factors (intravenous drug use, blood transfusions, blood donations, hemodialysis, tattooing, piercing, open trauma, surgery, multiple and the long - term hospitalizations, hcv infection among family members, dentistry, tooth removal, childbirth, abortions, and risky sexual behaviour). screening for anti - hcv was performed on the peripheral capillary blood using rapid lateral flow immunochromatography test (core hcv - wb ; core diagnostics, birmingham b2 5hg, uk) with 99.99% diagnostic specificity and 100% sensitivity. all anti - hcv - positive subjects were admitted for further testing at the centre of hepatology, gastroenterology and dietetics of vilnius university hospital santariki klinikos, where anti - hcv were confirmed with 2-step chemiluminescent microparticle immunoassay (architect system anti - hcv ; abbott, 65205 wiesbaden, germany). the following limitations of such data collection are : free anonymous testing for anti - hcv might be more attractive for having exposure to hcv risk (for example : intravenous drug users, subjects having family member infected with hcv) rather than for persons without suspected exposure. that is why detected anti - hcv prevalence can occur been higher than it really is. such subjects could also be not fully overt in indication of possible risk factors, although the questionnaire used was fully anonymous. that is why in a study cohort we had 37.8% men and 62.2% women ; although in the general population we have 45% men and 55% women. in the study cohort the anti - hcv rate was calculated separately for men and women on age - dependent strata. crude rate in age - dependent strata and in the study population overall were calculated as a mean of rates among men and women. age- and sex - dependent standardized anti - hcv rate in the general population was calculated using the direct standardization method and separate calculation of standard vectors for men and women in age - dependent strata (wj = nj / n where nj number of males or females on age - depended stratum, n population overall). for every statistically significant (.05) hcv route, an odds ratio was estimated using univariate logistic regression analysis with 95% confidence interval. differences in anti - hcv positivity between men and women on age - dependent strata were evaluated with fisher s exact test. an observational analytical cross - sectional study design was conducted to clarify the prevalence of anti - hcv and hcv transmission routes in the general population. cross - sectional survey of random anonymous volunteers and testing them for anti - hcv was conducted may 1722, 2010 in 5 cities of lithuania (vilnius, kaunas, klaipda, iauliai, and panevys) and corresponding rural regions where 75% of the population live (2 513 313 out of 3 329 039, according to the lithuanian statistical department ; http://www.stat.gov.lt/lt/). according to data on anti - hcv prevalence in eastern europe and the study by ambrozaitis. on lithuanian blood donors and some risk groups, we expected the prevalence of anti - hcv in lithuania to be approximately p=3%1%. after calculation of an adequate sample size with precision 1% (d=0.01) and level of confidence of 95% (z=1.96), a cohort of n=1118 subjects was estimated by the formula n=(zp(1p))/d. people over age 18 who were willing to be tested for anti - hcv were voluntarily enrolled into the study by a convenience sampling of shoppers at one of the biggest supermarkets in vilnius, kaunas, klaipda, iauliai and panevys. the only eligibility criteria were age 18 and residence in the selected cities and/or corresponding rural regions. during this study, anti - hcv testing was wrongly performed or questionable for 14 (0.9%) participants or a tested person refused or could not repeat it. anti - hcv tests was suitable for evaluation in a total of 1514 (99.1%) subjects (572 men and 942 women) 651 (241 men and 410 women in vilnius), 266 (128 men and 138 women in kaunas), 313 (115 men and 198 women in klaipda), 148 (55 men and 93 women) in panevys, and 136 (33 men and 103 women) in iauliai (figure 1). the study was performed according to the world medical association declaration of helsinki, and all procedures were approved by the lithuanian bioethics committee. all volunteers who wanted to know their anti - hcv status signed informal consent and answered an anonymous questionnaire with demographic data (age, sex, education, occupation, residence) and exposure to hcv risk factors (intravenous drug use, blood transfusions, blood donations, hemodialysis, tattooing, piercing, open trauma, surgery, multiple and the long - term hospitalizations, hcv infection among family members, dentistry, tooth removal, childbirth, abortions, and risky sexual behaviour). screening for anti - hcv was performed on the peripheral capillary blood using rapid lateral flow immunochromatography test (core hcv - wb ; core diagnostics, birmingham b2 5hg, uk) with 99.99% diagnostic specificity and 100% sensitivity. all anti - hcv - positive subjects were admitted for further testing at the centre of hepatology, gastroenterology and dietetics of vilnius university hospital santariki klinikos, where anti - hcv were confirmed with 2-step chemiluminescent microparticle immunoassay (architect system anti - hcv ; abbott, 65205 wiesbaden, germany). the following limitations of such data collection are : free anonymous testing for anti - hcv might be more attractive for having exposure to hcv risk (for example : intravenous drug users, subjects having family member infected with hcv) rather than for persons without suspected exposure. that is why detected anti - hcv prevalence can occur been higher than it really is. such subjects could also be not fully overt in indication of possible risk factors, although the questionnaire used was fully anonymous. that is why in a study cohort we had 37.8% men and 62.2% women ; although in the general population we have 45% men and 55% women. in the study cohort the anti - hcv rate was calculated separately for men and women on age - dependent strata. crude rate in age - dependent strata and in the study population overall were calculated as a mean of rates among men and women. age- and sex - dependent standardized anti - hcv rate in the general population was calculated using the direct standardization method and separate calculation of standard vectors for men and women in age - dependent strata (wj = nj / n where nj number of males or females on age - depended stratum, n population overall). comparison of different routes of exposure was evaluated with fisher s exact test. for every statistically significant (.05) hcv route, an odds ratio was estimated using univariate logistic regression analysis with 95% confidence interval. differences in anti - hcv positivity between men and women on age - dependent strata were evaluated with fisher s exact test. among 1514 tests, 37 (2.44%) anti - hcv - positive subjects were detected (23 men, 14 women), of these there were 3 (8.1%) people (2 from vilnius region and 1 from klaipda) who knew their anti - hcv status because hcv infection had been diagnosed previously and/or they were treated for chronic hepatitis c, and 34 (91.9%) had never been tested for anti - hcv (table 1). in our cohort, anti - hcv positivity did not depend on age (p=.7216), but more men than women had been exposed to hcv (crude rates : 4.02% vs 1.49%, p=.0030). the mean of anti - hcv rates was 2.76% and was similar to the rate in the general population (2.78%). after direct standardization of sex - adjusting rates by age distribution of standard european population prevalence of anti - hcv, a rate of 2.85% was obtained. we did not standardize the anti - hcv rate of our cohort to the world population because the population of lithuania is older and anti - hcv prevalence dependence on age was not ascertained. in the study cohort rural lithuanian regions are less affected by hcv infection than the capital and bigger cities (table 2). after analyzing the questionnaire data, we found that intravenous drug use is the most important risk factor for hcv spread in our population (table 3). blood transfusions, tooth removal, childbirth, and long - term or multiple hospitalizations can be assigned to hcv acquisition risks (table 3). statistically, other healthcare procedures (blood donation, hemodialysis, surgery, dentistry, abortions) are not associated with hcv acquisition. people who had a single surgery had generally similar rates of infection to the general population, but people having more than 2 surgeries were more often anti - hcv positive (table 4). among the 5 (19.2%) anti - hcv - positive people, 2 had hcv - infected brothers, 1 had an hcv - infected sister, 1 had an hcv - infected wife, and 1 had an hcv - infected husband. among the 4 (1.4%) anti - hcv - negative people, 2 had hcv - infected fathers, 1 had an hcv - infected brother, and 1 had an hcv - infected sister. being rather often declared, piercing was ascertained as a safe procedure concerning hcv spread, but the tattoo is not safe. among 1514 tests, 37 (2.44%) anti - hcv - positive subjects were detected (23 men, 14 women), of these there were 3 (8.1%) people (2 from vilnius region and 1 from klaipda) who knew their anti - hcv status because hcv infection had been diagnosed previously and/or they were treated for chronic hepatitis c, and 34 (91.9%) had never been tested for anti - hcv (table 1). in our cohort, anti - hcv positivity did not depend on age (p=.7216), but more men than women had been exposed to hcv (crude rates : 4.02% vs 1.49%, p=.0030). the mean of anti - hcv rates was 2.76% and was similar to the rate in the general population (2.78%). after direct standardization of sex - adjusting rates by age distribution of standard european population prevalence of anti - hcv, a rate of 2.85% was obtained. we did not standardize the anti - hcv rate of our cohort to the world population because the population of lithuania is older and anti - hcv prevalence dependence on age was not ascertained. in the study cohort rural lithuanian regions are less affected by hcv infection than the capital and bigger cities (table 2). after analyzing the questionnaire data, we found that intravenous drug use is the most important risk factor for hcv spread in our population (table 3). blood transfusions, tooth removal, childbirth, and long - term or multiple hospitalizations can be assigned to hcv acquisition risks (table 3). statistically, other healthcare procedures (blood donation, hemodialysis, surgery, dentistry, abortions) are not associated with hcv acquisition. people who had a single surgery had generally similar rates of infection to the general population, but people having more than 2 surgeries were more often anti - hcv positive (table 4). nine people had hcv - infected relatives. among the 5 (19.2%) anti - hcv - positive people, 2 had hcv - infected brothers, 1 had an hcv - infected sister, 1 had an hcv - infected wife, and 1 had an hcv - infected husband. among the 4 (1.4%) anti - hcv - negative people, 2 had hcv - infected fathers, 1 had an hcv - infected brother, and 1 had an hcv - infected sister. being rather often declared, piercing was ascertained as a safe procedure concerning hcv spread, but the tattoo is not safe. based on the results of this study we estimate that the anti - hcv prevalence in lithuania is 2.78%, and adjusted to the standard european population the anti - hcv rate is 2.85%. perhaps the free anonymous testing provided by our study was more attractive for people possibly having exposure to hcv (eg, intravenous drug use, having a family member with hcv infection) than for those without such risks. our estimated anti - hcv prevalence is similar to that in russia, but it is higher than in poland, estonia and latvia. however, the conclusion related to anti - hcv prevalence in eastern european countries was based on studies of blood donors, as in case of poland, or on the basis of personally communicated data, as in case of the baltic states. we could not find a single observational study presenting anti - hcv prevalence in the general populations of estonia, latvia, poland or in other neighbouring countries (russia, byelorussia or ukraine). in lithuania blood donors, some small risk groups and chronic hepatitis c patients were investigated [23,3134 ]. similar data are obtainable in latvia and estonia [3539 ]. the data on anti - hcv prevalence from a large cohort of 4216 healthy people from various regions of russia and mongolia were presented in 1996. according to this study, anti - hcv prevalence in those regions varied from 0.7% in central russia to 10.7% in mongolia. there is no current data about hcv infection spread in russia including the kaliningrad region where an explosion of intravenous drug abuse was observed during the last decade. a study of northern ukrainian schoolchildren and blood donors found a 0.8% anti - hcv prevalence for schoolchildren and 2.3% for blood donors ; anti - hcv prevalence directly correlated with the age people ages 4049 years had 4.0% positivity, while those older than 60 years had 6.25%. those data were published in 1993 and do not reflect the present situation of hcv infection in ukraine. in addition, the correlation of anti - hcv prevalence with age was not reported anywhere in the literature, nor did we find such correlation in our cohort. in byelorussia, 1.26% anti - hcv prevalence was reported without reference to the source. on the basis of the above - mentioned publications, esteban and co - authors presented anti - hcv prevalence in eastern europe (figure 1 in review). however, these data are not sufficient to provide a true picture of anti - hcv prevalence or the number of hcv carriers in this region. those authors presumed that anti - hcv prevalence could be higher not only in eastern europe, but also in the general european population because there are not sufficient current data. epidemiological studies of the general population would be useful in order to clarify the situation of hcv infection in europe in light of increasing intravenous drug abuse. according to the obtained data, the main hcv infection route in lithuania is currently intravenous drug use (or=42.5). in our country about 80% of intravenous drug users are infected with hcv and men more often than women engage in this behavior. this could explain the higher rate of anti - hcv positivity found in men of our cohort. intravenous drugs are more accessible in cities (especially in the capital) ; accordingly, we found lower anti - hcv prevalence in rural regions. observations and data of other investigators taken together reinforce the fact that intravenous drug use is now the principal route of hcv spread in the european population. former nosocomial routes such as blood donations, surgeries, and hemodialysis seem to be well controlled in our country. nevertheless, blood transfusion can still be a source of hcv (or=6.4). in lithuania every donor s blood has been tested for anti - hcv since 1994 and since 2004 for hcv rna. hcv rna is tested separately in every donation, not in pools, and nat sensitivity is 3.01 iu / ml. safety of donor s blood is still a worldwide health care problem which could be partially solved by introducing more sensitive nat methods. however, it will not be solved completely, especially in the case of paid donations. the possibility of hcv transmission through saliva is questionable, but it can not be ruled out. taking into consideration the hcv rna presence in saliva of 3555% of chronic hepatitis c patients, there is a possibility to transmit hcv to dentists and others [4449 ]. childbirth, being a risk factor for hcv acquisition, was an unexpected finding for us. anti - hcv prevalence among pregnant women and mother - to - child hcv transmission have been discussed in the literature [5054 ]. the number of anti - hcv - positive women in our cohort was only 14 and only 10 answered the question about childbirth 7 (70%) had children and 171 anti - hcv - negative women, answered a question about childbirth 54 (32%) had children. so, the small group of anti - hcv - positive women in our cohort may indicate why childbirth became statistically significant as an hcv risk factor. we failed to find a single study of anti - hcv prevalence in women before and after childbirth. such a study would be useful to confirm or deny our controversial finding and clarify a possible source of hcv infection if this exists in the maternity clinic. confirming that multiple and long - term hospitalization is an hcv risk factor raises the question of the specific route of transmission in hospitals. the possible infection source can be asymptomatic hcv carriers who are admitted for surgery or conservative treatment, for example, heart disease, and they are not tested for hcv by diagnostic algorithm. a health care worker infected with hcv but not diagnosed there were only 2 patients in the study cohort who underwent hemodialysis : 1 was anti - hcv positive and the other one was not. such limited data does not allow us to draw conclusions, but studies before 1995 indicated to a high prevalence of anti - hcv in hemodialyzed patients. it is necessary to pay attention to a new risk factor for hcv acquisition in our population nine people (5 anti - hcv - positive and 4 anti - hcv - negative) indicated presence of hcv infection in family members (usually it is sister or brother, but also it can be a spouse or parent) ; it confirmed a possibility of household and/or sexual hcv acquisition. however, risky sexual behaviour was not proved to be an hcv acquisition route. still, intrafamilial hcv spread is not well elucidated, it was only recognized that family members of hcv - infected patients had a 3- to 5-fold higher prevalence of anti - hcv than the general population and are often infected with the same hcv strain [6164 ]. open traumas with a big blinding area can be considered as a risk for hcv acquisition but its reasons are unclear (or=3.7). receiving a tattoo is a risk factor for hcv infection (or=4.4), possibly because of violation of sanitary norms during the procedure. our previous study of chronic hepatitis c cohort found a changing trend in hcv transmission, from nosocomial to less controlled intravenous drug use or hcv infection in family member. we found that older patients often get hcv during surgeries or long - term and multiple hospitalizations, while younger ones get it through intravenous drug use and tattooing. considering the asymptomatic course of infection, a high hcv rate in intravenous drug users and existence of their trusted networks, a new peak of hcv infection can be expected in the near future. this is important not only for lithuania, but also for other european countries where intravenous drug use is becoming a core of the hcv epidemic. hcv spread through intravenous drug use is not only a healthcare problem ; it is a social problem as well. it can not be resolved only with prevention or other healthcare tools, especially in the absence of effective immunization. only a complex of coordinated means directed to intravenous drug use prevention, public education and preventive efforts can be effective in prevention of hcv spread. in our population, anti - hcv prevalence is 2.78% ; adjusted to the standard european population, prevalence is 2.85%. this rate is higher in vilnius and kaunas in comparison with other regions of lithuania. the main hcv transmission route is intravenous drug use ; intrafamilial transmission is significant as well. blood transfusions, tattooing, open trauma, tooth removal, uncertain nosocomial transmission during multiple and long - term hospitalizations and childbirth are also hcv infection routes. despite active prevention of nosocomial hcv transmission, the incidence of hcv infection does not decrease, due to virus spread in trusted networks of intravenous drug users. nowadays, hcv transmission control can not be carried out without controlling intravenous drug use and active prevention educational tools. | summarybackgroundthe aim of this study was to assess risk factors for hcv acquisition and prevalence of anti - hcv in the general population of lithuania.material/methodsthe study enrolled 1528 randomly selected adults from the 5 biggest cities of lithuania and its rural regions. screening for anti - hcv was performed by analysis of peripheral capillary blood with lateral flow immunochromatography and confirmation of positive cases by peripheral venous blood testing with 2-step chemiluminescent microparticle immunoassay.resultsanti-hcv prevalence in lithuania is 2.78% and according to the standard european population the adjusted anti - hcv rate is 2.85%. it is more prevalent among men (crude rates : 4.02% males vs. 1.49% females, p=.0030) and this does not depend on age. vilnius and kaunas regions have higher infection rates than smaller rural regions (2.92% and 3.01% vs. 2.24%, 0.74% and 1.35%). nowadays among our population hcv infection spreads mainly via intravenous drug use (or=42.5, p<.0001). hcv transmission occurs through blood transfusions (or=6.4, p=.0002), tooth removal (or=4.1, p=.0048), childbirth (or=5.0, p=.0224), multiple and a long - term hospitalization (or=3.0, p=.0064), tattooing (or=4.4, p=.0013), open traumas (or=3.7, p=.0009) and intrafamilially (or=11.3, p=.0002).conclusions2.78% of the population is anti - hcv - positive. the anti - hcv rate is higher in vilnius and kaunas in comparison with other regions. hcv spreads mainly through intravenous drug use, but intrafamilial and some nosocomial routes are also important. the anti - hcv prevalence did not depend on age. despite active prevention of nosocomial hcv transmission, the incidence of hcv infection does not decrease due to virus spread mostly in trusted networks of intravenous drug users. |
neuroendocrine lung tumors arise from a population of neuroendocrine cells normally present in the bronchoalveolar structures and are characterized by secretory activity and ability to take up and decarboxylate the amine precursors (apud system cells). these neuroendocrine phenotypical and morphological characteristics are present within a broad spectrum of histologies of lung neuroendocrine tumors (nets), from relatively indolent typical carcinoids (tcs) to histologically high - grade, biologically aggressive tumors, as large - cell neuroendocrine carcinomas (lcnec) and small - cell lung cancers. the spectrum of pulmonary nets, according to the 2004 world health organization (who) categorization, includes 4 subtypes characterized by increasing biologic aggressiveness : tcs and atypical carcinoids (acs) are categorized together as carcinoids, lcnec is considered a subgroup of large - cell carcinomas and small - cell lung carcinoma (sclc) as an independent category. bronchopulmonary (bp) tcs and acs are uncommon, representing 1 - 2% of all pulmonary neoplasms ; these variants are associated with relatively slow growth, and generally show a favorable outcome. the tc subtype is predominant (80 - 90%), and occurs most frequently in the 5th and 6th decades of life. the most important difference between typical and atypical form is represented by the mitotic index. tcs have 10 mitoses/2 mm. nevertheless, diagnosis of bp carcinoids can be difficult and they may be mistaken for sclc. a review of pathologic material by an experienced pathologist is important prior to diagnosis and specific treatments in this type of tumors. in combination with the histologic appearance, the cell proliferation characterized by low labeling index ki67 (mib1) 50%). several peptide and amine markers, including chromogranin a (cga), neuron - specific enolase (nse), serotonin, synaptophysin, and adrenocorticotropic hormone (acth), can provide us with further tools in order to better establish a differential diagnosis. molecular genetic changes may be useful as an adjunctive element to differentiate tcs from acs. retinoblastoma gene (13q13) and p53 (17p13) mutations, multiple endocrine neoplasia (men) 1 gene activation (11q13), and telomerase activity are particularly frequent in acs if compared with tcs. we report a case of a young woman with misdiagnosis of bp net which has heavily influenced her clinical story. a 41-year - old symptomatic female patient, nonsmoker, following the onset of cough and low - grade fever despite empirical antibiotic therapy, underwent chest radiogram, which revealed an uneven area in the right lung, near the hilum. because symptoms persisted after empirical antibiotic therapy thorax ct scan documented a nodule in the right lung, near the hilum, 2 millimetric nodules in the left lung, in the lower lobe, and subcarinal lymphadenopathies. cytological examination of fine - needle aspiration specimens showed atypical cells immunohistochemistry - positive for cytokeratin mnf116, indicative of infiltration of small - cell lung cancer. after 5 cycles of chemotherapy with cisplatin and etoposide and radiotherapy on mediastinum, the patient was referred to surgery (sleeve bilobectomy, pericardial resection and mediastinal lymphadenectomy). histological examination showed an ac tumor (ki67 > 2%) with pathological staging pt2 n2. during the follow - up, the patient was subjected to ct scan every 6 months and then every year, to inspect the 2 millimetric nodules in the controlateral lung. after 8 years, ct scan documented an increase in the size of 1 of the 2 nodules in the lower lobe of the left lung, confirmed by pet (suv5). histological examination documented tc (ki67 < 2%), pt1 n1 as pathologic staging. ct and pet were negative for cancer, but blood test showed hypercalcemia (14 mg / dl). therefore, neck ultrasound and thyroid scintigraphy were performed, and both revealed an increase in size of the right parathyroid gland. the presence of 3 distinct histological types of bp nets in the same subject is unusual. sporadic lung carcinoids may occur as a component (~5%) of the familial endocrine cancer syndrome multiple neuroendocrine neoplasia 1 (men1) ; in contrast, in poorly differentiated lung nets (sclc and lcnec), this condition was rarely identified. analyzing the patient 's family history, we learned that her mother had zollinger - ellison syndrome, a cousin had pancreatic neuroendocrine carcinoma and parathyroid adenoma and an uncle underwent surgery for a parathyroid adenoma. thus, the patient and her children will be subjected to genetic counseling and evaluation for men1 gene mutation. consistent with our hypothesis, our histological review documented ac on the first cytological diagnosis, with immunohistochemical positivity for cga, synaptophysin and ki67 : 5.3%, while it confirmed the diagnosis of acs and tcs, after lung surgery, according to who classification (fig. 1, fig this case report demonstrates that diagnosis by cytologic sampling can be confounding and it alone does not allow a correct and complete characterization of pulmonary tumors. it is also important that the diagnostic iter must be followed, as far as possible, in a specialized center, especially when we are faced with rare neoplastic diseases, as in this case. | bronchopulmonary neuroendocrine tumors are an uncommon group of neoplasms, accounting for about 20% of all lung carcinomas, arising from stem cells of the bronchial epithelium known as kulchitsky cells. in the past, these tumors were grouped among benign or less aggressive malignant pulmonary tumors. currently, according to the 2004 world health organization categorization, these tumors are separated into 4 subtypes characterized by increasing biologic aggressiveness : low - grade (typical carcinoid ; tc), intermediate - grade (atypical carcinoid ; ac) and high - grade (large - cell neuroendocrine carcinoma, lcnec, and small - cell lung carcinoma, sclc). they differ by morphologic, immunohistochemical and structural features. at histopathologic analysis, these tumors share progressive increase in a number of mitotic figures per 10 high - power fields and in the extent of necrosis, with tc having the lowest values and sclc having the highest. tcs and acs make up approximately 12% of all primary lung tumors. differentiating acs from tcs or lcnec and sclc is clinically important because the treatment modalities and prognoses for these types of tumors are different. we report a case of misdiagnosis of bronchopulmonary neuroendocrine tumor in a young woman which has heavily influenced her clinical history. |
numerous studies have been conducted about stress and burnout in the healthcare professionals including doctors, nurses and dentists. however, there are few formal studies about stress in veterinary surgeons. is it linked to a lack of interest in the subject or to the fact that veterinary surgeons are not concerned with problems associated with stress ? according to the belgian veterinary surgeon association, and taking experiences gathered from veterinary surgeons into account, the first proposition seems to best fit the situation. before looking at the literature about occupational stress and work - home interference in veterinary surgeons, when measuring occupational stress, one must be careful not to confound the effects of stress or outcome variables and the sources of stress or the antecedent variables. to date, the literature suggests that there is no clear evidence of a common pattern of physiological effects of stress for all the sources of stress. for that reason, the authors ' approach to stress refers to a more transactional model based on cognitive theories and coping that emphasise the interaction between environmental demands and individual responses. we will define stress as " a process by which job demands are appraised by the worker as exceeding their own resources which results in undesirable health consequences ". according to the literature, the effects of stress can be mitigated by having high control, high satisfaction and high work commitment in a job. the study of stress at work should thus examine the effects of stress (outcome variables, e.g., job strain, burnout, job engagement) together with the sources of stress and situational factors which may decrease stress levels by improving an individual 's ability to cope with a stressful situation. ' work - home interference ' (whi) is defined in " a process in which a worker 's functioning (behaviour) in one domain (e.g., home) is influenced by (negative or positive) load reactions that have built up in the other domain (e.g., work) ". results from previous empirical research give support to some sources of stress inherent to the situation of veterinary surgeons. excessive workload can represent a real threat to practitioners ' health and safety, even more so than a feeling of too much job involvement or a lack of job satisfaction. veterinary surgeons facing the act of euthanasia involving pets they have grown fond of may suffer from emotional conflict and ambivalent feelings. a small percentage of clients complain, and new practitioners must be highly skilled in communicating with people to maintain proper relationships with clients. moreover, occupational risks are high in this profession, especially in relation to injuries inflicted by animals. however, it is hard to get reliable data about this, since veterinary surgeons can sometimes deal with injuries by themselves. another professional risk concerns diseases related to animal care (allergies, zoonoses, illnesses due to radiation, drugs and pesticides, etc.) or musculoskeletal disorders due to uncomfortable working positions, above all in rural practice. a survey published in the australian veterinary journal shows that the main stress factors are : relationships with angry or sad clients, the recovery of amounts of money not paid by clients, euthanasia and working conditions, especially working time problems. women are more likely to report job stressors in relation with job demands, interpersonal interactions with employers, peers and clients or the need to keep up technical skills and knowledge. job stressors for men are more particularly related to income, finances and career prospects. there have been, however, few formal studies on job strain and burnout in veterinary surgeons and the rare studies available present contradictory results. most of the literature reports moderate stress levels among respondents. in one study, rejula. reported that 73% of the veterinary surgeons perceived feeling ' rather ' or ' very stressed '. suicide among veterinary surgeons, for example, has been used as an indicator of the high stress level in this occupation. the studies reported in the literature present the limitation of using non standardised measures of wellbeing outcomes. concerning gender differences, gardner and hini reported that stress affects mostly young women in small animal practice (mainly because of the relationships with clients in clinical practice).. demonstrated that 71% of woman and 77% of men reported being ' rather stressed ' or ' very stressed ', mainly (65%) because of : being on call, administrative duties, insecurity of work, haste in work, heavy workload and unclear job description. in belgium, more and more veterinary surgeons are talking about high levels of stress and suicide in their profession. however, the authors have found no scientific research about wellbeing in belgian veterinary surgeons. over the last two decades, the profession is an aging one and belgium will soon face a lack of young bovine veterinary surgeons. furthermore, a decreasing number of farms has left a large part of the rural economic system under threat. aside from the administrative workload associated with bovine practice, the clinical work is also very physical, mainly due to the high number of caesarian sections performed in the belgian blue (bb) breed. each year, approximately 400,000 bb cattle calve in belgium, of which almost 99% are by caesarian section (f. rollin, personal communication). in addition, these caesarian sections are not equally distributed over the course of the year but are concentrated mostly between january and may. in belgium, around 1,400 veterinary surgeons it is thought that some complete more than 1,000 caesarian sections a year, on average ; the highest number reported is 27 caesarian sections in 24 hours (f. rollin, personal communication). the authors believe that this situation is specific to belgium, as they have not found a similar situation in other european countries. in the current study, the authors intend firstly to measure levels of job strain, job engagement, burnout and work - home interference in veterinary surgeons using self - reports and questionnaires, and secondly to further identify stressors and work characteristics to propose strategies for alleviating stress. according to the belgian context described above, bovine practice is compared to small animal and mixed activity as far as outcome variables are concerned. a comparative perspective will be used in this study through normative scores for job strain, job engagement and burnout and comparison with other samples of workers for work - home interference. numerous studies have been conducted about stress and burnout in the healthcare professionals including doctors, nurses and dentists. however, there are few formal studies about stress in veterinary surgeons. is it linked to a lack of interest in the subject or to the fact that veterinary surgeons are not concerned with problems associated with stress ? according to the belgian veterinary surgeon association, and taking experiences gathered from veterinary surgeons into account, the first proposition seems to best fit the situation. before looking at the literature about occupational stress and work - home interference in veterinary surgeons, when measuring occupational stress, one must be careful not to confound the effects of stress or outcome variables and the sources of stress or the antecedent variables. to date, the literature suggests that there is no clear evidence of a common pattern of physiological effects of stress for all the sources of stress. for that reason, the authors ' approach to stress refers to a more transactional model based on cognitive theories and coping that emphasise the interaction between environmental demands and individual responses. we will define stress as " a process by which job demands are appraised by the worker as exceeding their own resources which results in undesirable health consequences ". according to the literature, the effects of stress can be mitigated by having high control, high satisfaction and high work commitment in a job. the study of stress at work should thus examine the effects of stress (outcome variables, e.g., job strain, burnout, job engagement) together with the sources of stress and situational factors which may decrease stress levels by improving an individual 's ability to cope with a stressful situation. ' work - home interference ' (whi) is defined in " a process in which a worker 's functioning (behaviour) in one domain (e.g., home) is influenced by (negative or positive) load reactions that have built up in the other domain (e.g., work) ". results from previous empirical research give support to some sources of stress inherent to the situation of veterinary surgeons. excessive workload can represent a real threat to practitioners ' health and safety, even more so than a feeling of too much job involvement or a lack of job satisfaction. veterinary surgeons facing the act of euthanasia involving pets they have grown fond of may suffer from emotional conflict and ambivalent feelings. a small percentage of clients complain, and new practitioners must be highly skilled in communicating with people to maintain proper relationships with clients. moreover, occupational risks are high in this profession, especially in relation to injuries inflicted by animals. however, it is hard to get reliable data about this, since veterinary surgeons can sometimes deal with injuries by themselves. another professional risk concerns diseases related to animal care (allergies, zoonoses, illnesses due to radiation, drugs and pesticides, etc.) or musculoskeletal disorders due to uncomfortable working positions, above all in rural practice. a survey published in the australian veterinary journal shows that the main stress factors are : relationships with angry or sad clients, the recovery of amounts of money not paid by clients, euthanasia and working conditions, especially working time problems. women are more likely to report job stressors in relation with job demands, interpersonal interactions with employers, peers and clients or the need to keep up technical skills and knowledge. job stressors for men are more particularly related to income, finances and career prospects. there have been, however, few formal studies on job strain and burnout in veterinary surgeons and the rare studies available present contradictory results. most of the literature reports moderate stress levels among respondents. in one study, rejula. reported that 73% of the veterinary surgeons perceived feeling ' rather ' or ' very stressed '. suicide among veterinary surgeons, for example, has been used as an indicator of the high stress level in this occupation. the studies reported in the literature present the limitation of using non standardised measures of wellbeing outcomes. concerning gender differences, gardner and hini reported that stress affects mostly young women in small animal practice (mainly because of the relationships with clients in clinical practice).. demonstrated that 71% of woman and 77% of men reported being ' rather stressed ' or ' very stressed ', mainly (65%) because of : being on call, administrative duties, insecurity of work, haste in work, heavy workload and unclear job description. in belgium, more and more veterinary surgeons are talking about high levels of stress and suicide in their profession. however, the authors have found no scientific research about wellbeing in belgian veterinary surgeons. over the last two decades, the profession is an aging one and belgium will soon face a lack of young bovine veterinary surgeons. furthermore, a decreasing number of farms has left a large part of the rural economic system under threat. aside from the administrative workload associated with bovine practice, the clinical work is also very physical, mainly due to the high number of caesarian sections performed in the belgian blue (bb) breed. each year, approximately 400,000 bb cattle calve in belgium, of which almost 99% are by caesarian section (f. rollin, personal communication) in addition, these caesarian sections are not equally distributed over the course of the year but are concentrated mostly between january and may. in belgium, around 1,400 veterinary surgeons it is thought that some complete more than 1,000 caesarian sections a year, on average ; the highest number reported is 27 caesarian sections in 24 hours (f. rollin, personal communication). the authors believe that this situation is specific to belgium, as they have not found a similar situation in other european countries. in the current study, the authors intend firstly to measure levels of job strain, job engagement, burnout and work - home interference in veterinary surgeons using self - reports and questionnaires, and secondly to further identify stressors and work characteristics to propose strategies for alleviating stress. according to the belgian context described above, bovine practice is compared to small animal and mixed activity as far as outcome variables are concerned. a comparative perspective will be used in this study through normative scores for job strain, job engagement and burnout and comparison with other samples of workers for work - home interference. job strain, job engagement, burnout and work - home interference measures are considered in this study. firstly, job engagement and job strain are measured using the ' positive and negative occupational stress inventory ' (pnosi, 19 items ;). eight items assessed job engagement (e.g., " i feel stimulated by my work ", " my work gives me a lot of satisfaction ") and 11 items assessed job strain (e.g., " i feel overload by what i have to do ", " i feel nervous when at work ") on a four - level scale from one (" never " or " rarely ") to four (" almost always " or " always "). the reliability coefficients for our sample are 0.83 and 0.84, respectively. a moderate level of job strain / job engagement can vary from 40 to 60, with a mean equal to 50. burnout is a concept that consists of three dimensions : emotional exhaustion, depersonalisation and lack of personal accomplishment. practically, the use of the subscale of emotional exhaustion alone (nine items) appears to be a valid measure of professional burnout. the subject is asked to answer each item on a scale from one (" never ") to seven (" every day "). the level of burnout can vary between nine and 63 with a low level when the score varies from nine to 17, a moderate level from 18 to 29 and values higher than 29 characterising severe burnout. finally, the ' survey work - home interaction nijmegen ' (swing, 27 items) is used to measure four types of work - home interaction : (1) negative whi, referring to a situation in which negative load effects built up at work hampering functioning at home (nine items) ; (2) negative hwi, referring to negative load effects developed in the home domain that impede functioning at work (six items) ; (3) positive whi, defined as positive load effects built up at work that facilitate functioning at home (six items) ; and, (4) positive hwi, occurring when positive load effects developed at home facilitate functioning at work (six items). the reliability coefficients in our sample are 0.88, 0.79, 0.80 and 0.84, respectively. there are four response categories : " never " (0), " sometimes " (1), " often " (2) and " always " (3). a scale mean for each of the four whi - scales is calculated. as far as job stress factors are concerned, the veterinary stress inventory (vsi, 45 items) (biron, o.. unpublished dissertation, university of lige, belgium) has been developed for the purpose of this study. items were created on the basis of results from preliminary interviews with veterinary surgeons and a review of the literature, especially the questionnaire developed by cooper., to measure stressful job situations among general practitioners. examples include : " phone calls during the night and early in the morning ", " administrative formalities because of the new policy about medicines " or " euthanasia requests for non - suffering patients ". the response format is a four - point likert scale from " never a cause of stress " to " always a cause of stress ". this quantitative methodology was completed by an open question in which participants were asked to freely describe a maximum of three problematic events that they encounter in their daily work activities. in collaboration with the belgian professional union of veterinary surgeons, these questionnaires were sent to the 2,700 french - speaking veterinary surgeons through the belgian journal ' veterinaria '. in the first instance, analyses of variance were performed to test for differences in weekly working hours according to the type of activity and age categories. a t - test was used to compare the difference in weekly working hours for men and women. descriptive statistics including means and standard deviations are presented for job strain, job engagement, burnout and work - home interference. according to standardised scores, the three first outcome variables are categorised in small, medium or high level of score. comparisons of these three variables, according to the type of activity, gender and age categories, were performed using multivariate analyses of variance (manova). all paired comparisons were evaluated using the tukey post - hoc test. as regards work - home interference, paired sample t - tests were used to compare the differences between subscales of work - home interference (whi). in order to account for multiple comparisons, the authors used the bonferroni correction, which sets the alpha value for the entire set of n comparisons equal to by taking the alpha value for each comparison equal to /n i.e., in this case p 0.05). the problematic situations encountered by small animal practitioners were somewhat different when subcategories were considered. in the " client relations " category, the main problem was concerned with non payment (43%, n = 43). in the " time management " category, phone harassment during practice (22%, n = 18) was the most important source of stress. problematic situations (total n = 175) encountered by bovine practitioners nb : 10 non relevant situations were not included in the analysis. results to the vsi confirmed this panel of results as emergency / working time management (six items out of 10 related to this topic) and client relations (three items out of 10 related to this topic) appear as the two most important sources of stress for rural veterinary surgeons but also for mixed practitioners. table 5 also shows that small animal practitioners have to deal with different problems like ethical problems and difficulties in practice. : the multivariate analysis of the 15 items performed to compare the type of activity was significant, wilks ' lambda =.61, f(30, 348) = 3.27, p =.000. the aim of this study was to : (1) measure levels of job strain, job engagement, burnout and work - home interference in belgian veterinary surgeons using self - reports and questionnaires ; and, (2) to further identify stressors and work characteristics. the current study revealed a moderate level of job strain in belgian veterinary surgeons, no higher than in other professional groups. this is surprising when the inherent stressful working conditions described in the introduction, i.e., increasing responsibility of veterinary surgeons and the increasing workload, are taken into consideration. different authors have found that stress levels can be mitigated by having high authority and high satisfaction in the job. most of the veterinary surgeons sample showed high job engagement which, in turn, may have moderated the job strain levels. another important result concerns burnout : more than 15% of the veterinary surgeons sample reported a high level of emotional exhaustion. these professionals, like veterinary surgeons, seem to enjoy and feel stimulated by their job, but find certain working conditions difficult to cope with. for veterinary surgeons, in the context described above, administrative requirements, difficult relationships with clients and working time constraints can explain this feeling of emotional exhaustion. this result concerning burnout can be further explained by a much higher negative interference from work to home than what the authors found in other working populations. the unpredictability of the veterinary working schedule leads all practitioners, but especially bovine and mixed practitioners, to be on stand - by regularly. in addition, veterinary surgeons have to be easily reachable by phone and most of the time they do not feel free, especially if they are self - employed and isolated. their working time does not allow them to fulfil elementary family roles, which is perceived as difficult by practitioners, and probably even more so by their spouse and children. that, among veterinary surgeons, small animal and equine practitioners are the most affected by emotional exhaustion. job engagement, job strain and burnout were not different according to the age of the veterinary surgeon. the authors had thought that even if a practitioner was engaged and committed in the early years of his or her career, their work might have become monotonous and not status - enhancing. the response rate in our study was very small, mainly because it was not possible for the authors to send a reminder to potential respondents. the authors could also argue that only veterinary surgeons having more time to answer (and thus a lower workload) have participated in the survey. this could also explain why, despite an alarming context, the results do not confirm a high risk situation in veterinary surgeons as far as wellbeing is concerned. however, the size of the sample was acceptable for the statistical analyses performed on the data. moreover, the study is focused on french speaking veterinary surgeons working in belgium and there are few studies in the literature with which to compare the results. it would be interesting to replicate this study in other countries in a cross - cultural perspective. in this present study, belgian veterinary surgeons were involved in a survey about stressors and work characteristics, job strain, job engagement, burnout and work - home interference. the main results were a moderate level of job strain together with a high job engagement, a percentage of high burnout in the sample higher than 15% and a much higher negative interference from work to home than what was found by the authors in other working populations, especially for bovine and mixed practitioners. first, job engagement is a key variable in the way veterinary surgeons perceive their work situation. this is in line with the stress literature which has recently proposed a new theoretical model : the ' job demands - resources model '. this model refers to two processes, one linked with the positive (motivational process, i.e., job engagement), and the other with the negative (health impairment process, i.e., burnout) aspects of wellbeing, making this phenomenon easier to understand. second, results about work stressors in the veterinary profession are consistent all through research, including this study : long working hours, excessive workload, working time problems, ethical problems and euthanasia, relationships with clients, occupational risks, diseases related to animal care, uncomfortable working positions and recovery of amounts of money not paid by clients. third, negative work - home interference was used for the first time in veterinary research. in this study, bovine and mixed practitioners are particularly at risk, mainly because of the unpredictability of the veterinary working schedule and the flexible planning according to phone calls and stand - by duty. for these three new aspects, a sensitisation about job resources, such as external support for the planning, some extra help for administrative purposes and good communications skills, may be effective for improving wellbeing at work in the veterinary profession. bartram and baldwin also reported that several countries such as the uk, us, canada and norway have already developed stress management interventions, including a 24-hour peer - support telephone helpline service, programmes of mental health seminars in veterinary schools or training modules on professional key skills. in belgium, such a helpline service for farmers is organised by the agricall walloon association and the idea was recently extended to veterinary practitioners. bartram and baldwin also suggest incorporating training in the use of effective coping strategies for alleviating stress, burnout and negative work - home interference. in conclusion, the cross - cultural perspective could be interesting in terms of sensitisation and publication of best practices within the world concerning for example administrative requirements, training programmes in terms of communication skills or counselling for a better work - life balance among veterinary surgeons. | there have been few formal studies on stress in veterinary surgeons and, in the rare studies available, stress is not examined jointly through the levels of job strain and job engagement, the sources of stress in the issue of work environment and the work - home interference. the authors ' goal in this study was to analyse job engagement, job strain, burnout, work - home interference and job stress factors among 216 belgian veterinary surgeons. rural practice was compared to small animal and mixed activity. the mean job strain and job engagement level in veterinary surgeons was not higher than what we found in other working populations. however, 15.6% of the group were found to be suffering from high burnout. rural practitioners had a lower level of job engagement than small animal veterinary surgeons. these small animal practitioners had a lower level of job strain than the mixed practitioners. the level of burnout did not differ significantly across the three types of activity. in comparison to other belgian and dutch workers, veterinary surgeons perceived more negative work - home interference. bovine and mixed practitioners were the most concerned with this problem. the two most important sources of stress reported by bovine practitioners were relations to farmers and working time management (including emergencies and availability). |
multifocal motor neuropathy (mmn) is an immune - mediated disorder that is characterized by slowly progressive and asymmetrical weakness, but its pathophysiological mechanism is uncertain. the hypothesis that mmn is an immunological disease has been supported by the proven therapeutic effects of intravenous immunoglobulin and the detection of antiganglioside antibodies in mmn patients. the clinical manifestations coincided well with mmn : predominantly distal upper - limb weakness, asymmetric involvement, a progressive course, absence of sensory symptoms, absence of pyramidal signs, and sparing of the cranial muscles. the electrophysiological findings also supported a diagnosis of mmn, with motor nerve conduction block in the median, ulnar, and radial nerves, without sensory nerve involvement. the patient was simultaneously diagnosed as having hashimoto 's thyroiditis, which is a well - known immune - mediated disease. the concurrence of mmn and hashimoto 's thyroiditis in our patient is significant for understanding the immunological characteristics of the two diseases. multifocal motor neuropathy (mmn) is an immune - mediated disorder with predominantly distal, mainly upper limb, asymmetrical manifestations, and pure motor neuropathy.1 - 3 the hallmark of mmn is the presence of conduction block in motor nerve conduction studies (ncss). mmn is a treatable disorder, with about 80% of patients responding to intravenous immunoglobulin.2,4 for this reason, it needs to be distinguished from motor neuron disease, which is much more common but remains untreatable. the pathogenesis of the disease is still unknown, but there is some evidence, based mainly on the clinical improvement of patients after immunological treatments, that mmn is related to an immune disorders.3 mmn is also frequently associated with antiganglioside antibodies to monosialoganglioside (gm1), and disialoganglioside (gd1b) of the igm type.5 gangliosides are sialic acids containing glycosphingolipids that are particularly abundant in the neural membranes of the nervous system ; they are also detected in thyroid cells.3,5,6 antibodies against the main gangliosides, such as gm1 and gd1b, are found in patients with mmn, which suggests an autoimmune pathogenic mechanism. hashimoto 's thyroiditis is one of the most frequent causes of primary hypothyroidism and a well - known immune - associated disease ; circulating thyroid autoantibodies are usually detected.7 the histopathologic findings of hashimoto 's disease show widespread lymphocyte infiltration in the thyroid tissue.8 other autoimmune diseases may also occur in patients with hashimoto 's disease.9 however, the coincidence of mmn and hashimoto 's thyroiditis has rarely been reported, and the pathophysiology has been limitedly clarified. five years prior to admission, she had noticed mild weakness of insidious onset in her left hand. she began to experience difficulty in writing, handling chopsticks, and making a fist. the patient had severe weakness and muscle atrophy in both hands, without sensory change. she had attended school and had led an apparently normal life until she noticed neurological manifestations. relevant diseases, a familial history of neurological disease, smoking, and alcohol were not noted. she had taken an antihypertension drug and oral thyroid hormone at a local clinic. on examination, her vitals sign were stable, with a blood pressure of 140/90 mmhg and a heart rate of 72 beats / min. motor system examinations using the medical research council (mrc) grade revealed severe motor weakness in both hands (flexor pollicis longus, mrc grades 3/5 and 2/5 on the right and left, respectively ; flexor digitorum profundus and sublimis, mrc grades 3/5 and 3/5 ; flexor carpi ulnaris and radialis, mrc grades 3/5 and 3/5 ; extensor pollicis brevis and longus, mrc grades 3/5 and 2/5 ; extensor carpi radialis, mrc grade 3/5 and 2/5 ; extensor carpi ulnaris, mrc grades 3/5 and 3/5 ; extensor digitorum communis, mrc grades 2/5 and 2/5 ; abductor pollicis brevis, mrc grades 3/5 and 3/5 ; and abductor digiti minimi, mrc grades 2/5 and 2/5). the patient exhibited symmetric and intact responses to all sensory stimuli, and her cortical senses were intact. her neck was supple, no carotid bruit was audible, and the results of a cerebellar function test were normal. evaluations for vasculitis involving rheumatoid factor, anti - dsdna antibody, lupus anticoagulant, anticardiolipin antibody, antithrombin iii, antineutrophil cytoplasmic antibody, anti - ssa and ssb (sjogren syndrome a and b) antibody, complement 3/4, anti - jo-1 antibody, cryoglobulin, immunofixation electrophoresis / protein electrophoresis, anti - gm1 antibody, and anti - gd1b antibody were within the normal range or negative, with the exception of a mildly elevated fluorescent antinuclear antibody titer (1 : 40). the results of tumor screening tests involving alpha - fetoprotein, carcinoembryonic antigen, cancer antigen (ca)-19 - 9, and ca-125 were normal. hexosaminidases a and b, and creatine kinase were also within the normal ranges. however, a thyroid function test revealed an abnormally elevated antithyroglobulin antibody titer (794 u / ml, normal range : 0 - 100 u / ml) and antimicrosomal antibody (26,030 u / ml, normal range : 0 - 100 u / ml), but the thyroid hormone levels, including t3, free t4, and thyroid stimulating hormone, were normal. cervical magnetic resonance imaging revealed no prominent abnormalities except mild disc protrusion at the c5 - 6 and c6 - 7 levels. these examinations revealed motor neuropathies in the median motor nerve, ulnar motor nerve, and radial motor nerve, with definite motor conduction block manifesting as the compound muscle action potential amplitude being more than 50% lower for proximal than for distal stimulation across a standard peripheral nerve segment (fig. 2). however, the findings of sensory ncss in the upper extremities, and motor and sensory ncss in the lower extremities were normal. thus, even though the patient was negative for antiganglioside antibodies, the electrophysiological study and clinical manifestations resulted in a diagnosis of mmn. a biopsy of the patient 's thyroid tissue by ultrasound - guided aspiration revealed diffuse lymphocyte infiltration, a finding that is consistent with hashimoto 's thyroiditis (fig. when she visited our outpatient department 1 month later, the weakness and deformity in her right hand was considerably improved (fig. 1b) and the conduction block of the motor nerves were mildly improved, as evaluated by follow - up ncss (fig. the case presented herein is characterized by an association between mmn and hashimoto 's thyroiditis. the clinical presentation was typical for mmn, since such patients complain predominantly of progressively worsening upper - limb weakness (mainly distal), asymmetric involvement, absence of sensory symptoms, absence of pyramidal signs, and sparing of the cranial muscles.1,2 the electrophysiological findings also supported a diagnosis of mmn, with motor nerve conduction block in the median, ulnar, and radial nerves, without sensory nerve involvement. the most important investigations for determining mmn are electrophysiological studies, and in particular motor ncss, for which the main finding in mmn is a persistent multifocal conduction block.1,2,5 conduction block manifests as a lower compound muscle action potential amplitude or a smaller area on electrical stimulation of a nerve at a proximal site, compared to a distal site. although conduction block is characteristic of mmn, it may be found in several other disorders such as guillain - barr syndrome, chronic inflammatory demyelinating polyneuropathy, and nerve ischemia.10,15 therefore, the appropriate interpretation of a conduction block also requires consideration of the clinical presentation.10 our patient could be clinically differentiated from guillain - barr syndrome, chronic inflammatory demyelinating polyneuropathy, and nerve ischemia since she complained of slowly progressing, asymmetric motor weakness in the distal upper extremity, without sensory symptoms. according to the clinical manifestations, electrophysiological study findings, and response to intravenous immunoglobulin, a diagnosis of mmn in our patient was reasonable, although she was negative for antiganglioside antibodies related to mmn. elevated antiganglioside antibody titers are a typical finding ; however, the sensitivity of this measure is uncertain, reportedly varying between 30% and 80% (although usually lying between 40% and 50%).1,2,5 in addition, these antibodies are not specific for mmn, having been found in about 10% of cases of the pure lower motor neuron variant of motor neuron disease.5 therefore, the negative antiganglioside antibody finding did not rule out a diagnosis of mmn in the case reported here. the pathophysiology of mmn is believed to be focal motor nerve demyelination, resulting in conduction block. however, this remains uncertain, and an immune - mediated origin of mmn is suspected on the basis of the presence of antiganglioside antibodies and response to immune intervention. the ganglioside gm1, which is abundant in motor nerves at the nodes of ranvier and motor end plates, may be the target of an immune response, resulting in conduction block.3,5 in vivo and in vitro experiments have demonstrated that the serum from patients with mmn and anti - gm1 antibodies induces focal conduction block after intraneural injection.11 disruption of the blood - nerve barrier by these antibodies has also been hypothesized as a possible mechanism.12 however, about 50% of reported cases of mmn are negative for anti - gm1 antibodies.5 despite a probable autoimmune basis, the precise mechanism underlying the development of mmn is still poorly understood. on admission to our hospital, our patient was additionally diagnosed as having hashimoto 's thyroiditis, which is a well - known autoimmune disease and one of the most frequent causes of primary hypothyroidism. the prevalence of various diseases with autoimmune components in patients with hashimoto 's thyroiditis and in their relatives is higher than can be accounted for by chance.7 - 9 the plasma membranes of thyrocytes are also very rich in ganglioside, and these are particularly abundant in neural membranes, inducing specific antibodies for mmn.6,13 the high concentrations of gangliosides in both neural and thyroid tissues had led to some authors suggesting a pathogenic relationship between mmn and hashimoto 's thyroiditis and a potential immunological interaction between antiganglioside antibodies and both neural and thyroid tissues.14 unfortunately, antiganglioside antibodies were not detected in our case. the reported variations in the prevalence of these antibodies in mmn have been variably attributed to technical differences in the procedures adopted by different laboratories, differences in the controls used to establish normal reference values, and differences in the disease period tested for mmn. the reasons for such a wide variation in the percentage of abnormal findings remain to be elucidated. mmn is a rarely described, treatable, immune - mediated motor nerve disease ; however, there are many remaining uncertainties about the precise underlying pathophysiological mechanisms. therefore, the case reported here is significant in that it demonstrates a possible link and pathophysiological mechanism between mmn and a more common autoimmune disease, hashimoto 's thyroiditis. | backgroundmultifocal motor neuropathy (mmn) is an immune - mediated disorder that is characterized by slowly progressive and asymmetrical weakness, but its pathophysiological mechanism is uncertain. the hypothesis that mmn is an immunological disease has been supported by the proven therapeutic effects of intravenous immunoglobulin and the detection of antiganglioside antibodies in mmn patients. the coexistence of mmn with other immune diseases has been rarely reported.case reporta 37-year - old woman visited our hospital complaining of weakness in both hands. the clinical manifestations coincided well with mmn : predominantly distal upper - limb weakness, asymmetric involvement, a progressive course, absence of sensory symptoms, absence of pyramidal signs, and sparing of the cranial muscles. the electrophysiological findings also supported a diagnosis of mmn, with motor nerve conduction block in the median, ulnar, and radial nerves, without sensory nerve involvement. the patient was simultaneously diagnosed as having hashimoto 's thyroiditis, which is a well - known immune - mediated disease.conclusionsthe concurrence of mmn and hashimoto 's thyroiditis in our patient is significant for understanding the immunological characteristics of the two diseases. |
we conducted a systematic search of cinahl, pubmed, and psycinfo to identify all empirical studies published between january 1995 and april 2013 about african american women and genetic testing and/or counseling. the following search terms were used to identify all relevant publications : african american, black, breast cancer, ovarian cancer, genetic risk assessment, genetic testing, genetic counseling, and brca. eligible studies included either an african american sample or a mixed sample with sub - analyses conducted among african american women. studies addressing participation in both genetic counseling and testing were included in this review, as both are central to the genetic risk assessment process. empirical research findings from observational or correlational / descriptive studies, clinical trials, and longitudinal cohorts were included in this review ; reviews, editorials, and commentaries were excluded. also excluded were papers that only measured knowledge of genetic counseling and testing among african american woman, as this was extensively reviewed by halbert.) conducted the search, developed the coding form, and coded the studies ; the two other authors (s.m. and s.s.g.) independently reviewed the coded studies. disagreements among the coders and the reviewers were discussed until agreement was reached among all authors. we conducted a systematic search of cinahl, pubmed, and psycinfo to identify all empirical studies published between january 1995 and april 2013 about african american women and genetic testing and/or counseling. the following search terms were used to identify all relevant publications : african american, black, breast cancer, ovarian cancer, genetic risk assessment, genetic testing, genetic counseling, and brca. eligible studies included either an african american sample or a mixed sample with sub - analyses conducted among african american women. studies addressing participation in both genetic counseling and testing were included in this review, as both are central to the genetic risk assessment process. empirical research findings from observational or correlational / descriptive studies, clinical trials, and longitudinal cohorts also excluded were papers that only measured knowledge of genetic counseling and testing among african american woman, as this was extensively reviewed by halbert.) conducted the search, developed the coding form, and coded the studies ; the two other authors (s.m. and s.s.g.) disagreements among the coders and the reviewers were discussed until agreement was reached among all authors. twenty - four studies were retrieved for a more thorough evaluation, and a further six were excluded for not meeting review eligibility criteria. 1selection of included articles selection of included articles table 1 provides an overview of studies included in this review. across all studies, there was an average of 98 african american women participants (range, 13 to 266 women ; matthews. 1999). among the prospective studies, three recorded measurements at one time point and assessed subsequent risk assessment participation (halbert. 2005b ; hughes. 2003 ; thompson. 2002), four reported the findings from randomized control trials (halbert. 2006, 2010 ; lerman. 1999 ; charles. 2006) and six reported only baseline data as part of a larger intervention study (halbert. 1999 ; kessler. 2005 ; hughes. 1997 ; edwards. 2008 ; durfy. two studies used a qualitative approach (matthews. 2000 ; ford. 2007) involving focus groups with african american women.table 1characteristics of studies incorporating psychosocial predictors of participation in genetic susceptibility counseling and testing for breast cancer in african american womenauthorsnumber (% afam women ; number afam women)breast cancer risk criteriadesign / methodsmeasuresfindingsarmstrong. control study of 408 women, of whom 217 underwent genetic counseling and 191 did not.probability of brca mutation, socioeconomic characteristics, perception of breast / ovarian cancer risk, cancer worry, attitudes about genetic testing, and discussion of testing with primary care physician.afam women were significantly less likely to receive genetic counseling. result trends show afam women had greater perception of having a brca mutation and of breast / ovarian cancer risk. they also show a pattern for afam women to worry more about developing breast / ovarian cancer. (2006)54 (100 %) 510 % probability of having a brca1/2 mutationparticipants were offered genetic testing as part of a rct which compared the effects of culturally tailored genetic counseling (ctgc) and standard genetic counseling (sgc). satisfaction was evaluated via a survey following allocation to ctgc or sgc.clinical factors, perceived risk of having a brca1/2 mutation, satisfaction with the genetic counseling.96 % of women were very satisfied with genetic counseling ; however, only 26 % reported that their worries were lessened and 22 % reported that they were able to cope better. women who received ctgc were significantly more likely than women who received sgc to report that their worries were lessened (p < 0.05).donovan, tucker (2000)220 (49 % ; 108)no criteria specifiedcross sectional study. afam and caucasian women completed a survey regarding their knowledge and genetic risk for breast cancer, and their interest in genetic testing.perceived risk, knowledge about breast cancer, knowledge about genetic risk for breast cancer, perceived benefits, limitations and risks of genetic testing, and interest in genetic testing.caucasian women had significantly more knowledge about breast cancer and genetic testing compared with afam women, even when controlling for level of education and income.durfy. (1999)543 (7 % ; 36)family history of breast cancerexamined knowledge and opinions about genetic testing for breast cancer risk in women recruited for a rct of breast cancer risk counseling methodsfamiliarity with genetic testing for breast cancer risk, interest in such testing and opinions of it, and anticipated actions based on test results.mean perceived risk of study participants was higher than the mean actual risk for all groups. (2008)140 (56 % ; 74)personal and/or family history of breast / ovarian cancertelephone interviews were conducted to explore the relationship between temporal orientation and the pros and cons of genetic testing.temporal orientation, and pros and cons of genetic testing.results indicated an association between future orientation and perceived benefits of undergoing testing for both groups.ford. (2007)20 (65 % ; 13)above average riskfocus groups were conducted to determine factors influencing perceptions of breast cancer genetic counseling.factors (background, cognitive / psychosocial, social, and systematic) influencing perceptions of breast cancer genetic counseling.afam women who received counseling believed they had a small chance of developing breast cancer, and believed that changes in lifestyle activities could reduce likelihood of developing the disease.halbert, brewster. (2005)164 (100 %) 510 % probability of having a brca1/2 mutationevaluated the process of recruiting afam women into genetic counseling. women completed baseline interviews followed by genetic counseling prior to genetic testing.perceived risk of brca1/2 mutation, genetic counseling uptake.referral from oncology clinics was the only factor significantly associated with participation in genetic counseling ; no association between perceived risk and genetic counseling uptake.halbert, kessler. (2005)141 (100 %) 510 % probability of having a brca1/2 mutationexamined cancer - specific distress in afam women at an increased risk of hereditary breast and ovarian cancerdistress, history of cancer and avoidance.afam women aged 50 and younger, those who are unemployed and women with a personal history of breast or ovarian cancer may be the most vulnerable to experiencing elevated levels of distress during genetic counseling and testing.halbert, kessler, stopfer. (2006)157 (100 %) 510 % probability of having a brca1/2 mutationinvestigated acceptance rates of genetic testing results among afam women at increased risk for breast cancer.perceived risk of brca1/2 mutation, perceived certainty of risk, worry, genetic testing result acceptance.women with higher pre - testing beliefs about the probability of being a mutation carrier and those who had less certain beliefs about the certainty of developing cancer were more likely to accept genetic test results.halbert. (2010)198 (100 %) minimum 5 % probability of having a brca1/2 mutationrct of genetic counseling and testing (20032006) to evaluate effects of genetic counseling and testing in afam based on different levels of exposure : (a) women who were randomized to culturally tailored (ctgc) and standard genetic counseling (sgc) to women who declined randomization (non - randomized group) ; (b) participants and non - participants in genetic counseling ; and (c) brca1/2 test result acceptors and decliners.perceived risk of developing breast cancer and cancer worry.women randomized to ctgc and sgc did not differ in terms of changes in risk perception and cancer worry compared to decliners.hughes, gomez - caminero. (1997)407 (24 % ; 97)at least one fdr with breast and/or ovarian cancer ; no personal cancer historytelephone and structured interviews examined women s knowledge of breast cancer inheritance and attitudes about genetic testing for breast / ovarian cancer susceptibility in women at increased risk.ethnicity, genetic testing exposure, knowledge about breast cancer genetics genetic testing, attitudes about the benefits, limitations, and risks of genetic testing.compared to caucasian women, afam women had lower levels of knowledge about genetic testing. 23 % of afam women rated concern about the effect on their family as very important, compared with 13 % of caucasian women.hughes, fasaye. (2003)28 (100 %) minimum 10 - 20 % prior probability of having a brca1/2 mutationsociocultural influences on participation in genetic testing among afam women. a two week follow - up interview assessed associations between cultural beliefs and values and participation in genetic testing.attitudes towards benefits and limitations of genetic testing, fatalistic beliefs about cancer.women participating in genetic testing were more likely to have a high level of fatalistic beliefs about cancer, report a future temporal orientation, and view themselves as independent from family members, compared with non-participants.kessler. (2005)74 (100 %) 510 % probability of having a brca1/2 mutationevaluated attitudes about the benefits, limitations, and risks of genetic testing.clinical factors, beliefs about cancer, perceptions of risk and control, attitudes and intentions regarding genetic testing.higher levels of fatalistic beliefs about cancer were associated with greater consideration and uptake of genetic testing.lerman, hughes. (1999)228 (23 % ; 70)at least one fdr with breast and/or ovarian cancer ; no personal cancer historytelephone interviews in a rct were used to assess racial differences in responses to pre - test education strategies for brca1 genetic testing.risk comprehension, genetic testing intention, breast cancer anxiety.afam women benefited from the combined provision of genetic risk information and counseling more than caucasian women. afams who received the education and counseling intervention reported greater intentions to be tested in the future and were more likely to donate a blood sample for storage.lipkus. (1999)266 (100 %) at least one fdr with breast cancerexamined relationships among perceptions of, and concern about, getting breast cancer and interest in genetic testing.perceptions and attributions of risk, knowledge of risk factors, breast cancer concerns, interest in genetic testing.increasing perceptions of breast cancer risks and concerns were related to a greater interest in genetic testing.matthews, cummings. focus groups were conducted to learn more about factors influencing participation of afams in genetic testing.level of perceived cancer risk, worries about health, participation in annual cancer screening, discussion of cancer within families, impact of genetic information on medical decision making, knowledge about cancer, and factors affecting decisions to participate in genetic testing.several factors influence participation, including perceptions about cancer risk and survivability, lack of awareness about the role of genetic testing, and concern about how to emotionally deal with genetic risk feedback. concerns about being unable to handle testing and results, and feeling overwhelmed by anxiety, cited by women in particular.thompson, valdimarsdottir, duteau - buck. (2002)76 (100 %) at least one fdr with breast and/or ovarian cancer ; no personal cancer historyinvestigated predictors for genetic counseling and testing for breast cancer susceptibility. participants completed a questionnaire, and underwent genetic counseling and genetic testing.knowledge of breast cancer, breast cancer - specific emotional distress, perceived benefits and barriers of genetic counseling and testing.women declining genetic counseling or testing were less knowledgeable about breast cancer genetics than women receiving genetic counseling and testing.thompson, valdimarsdottir, jandorf. (2003)273 (42 % ; 115)no criteria specifiedinterviews explored genetic testing attitudes, and determined the extent to which ethnicity, awareness of genetic testing, and medical mistrust is associated with genetic testing attitudes.ethnicity, knowledge of genetic testing, medical mistrust, risks and benefits of genetic testingafam women strongly concurred more with concerns about perceived disadvantages (confidentiality and effects on family) and testing abuses (religion), compared with caucasian women. rct randomized controlled trial, afam african american, fdr first - degree relative characteristics of studies incorporating psychosocial predictors of participation in genetic susceptibility counseling and testing for breast cancer in african american women rct randomized controlled trial, afam african american, fdr first - degree relative overall, 10 studies included only african americans in the sample (matthews. ; lipkus. 1999 ; kessler. 2005 ; charles. 2006). of these, nine included only african american women ; one included both men and women in the study sample (matthews. fifteen studies included african american women who were at risk for developing breast and/or ovarian cancer ; the remaining three included a combined sample of at - risk and not at - risk participants. most studies (n = 14) evaluated predictors, or the process, of participation in genetic susceptibility counseling or testing ; far fewer studies (n = 4) examined the outcome of testing, counseling, or program participation (halbert. 2010 ; lerman. 1999 ; charles. 2006 ; ford. 2007). uptake of genetic testing and/or counseling was reported by eight studies (charles. the proportion of women who elected to receive their results varied considerably, with rates ranging from 25 % (halbert. 2006) to 61 % (hughes. 2003) of women who had undergone genetic testing and/or counseling. most studies (n = 11) recruited from clinical settings or oncology / medical facilities (halbert. 2005a, b, 2006, 2010 ; donovan and tucker 2000 ; hughes. 1999 ; thompson. 2002 ; lerman. 1999 ; armstrong. 2005 others recruited via a combination of clinics, self - referrals, and community settings (matthews. 1997 ; kessler. 2005) or via mass media advertisements (durfy. 1999). african american women s levels of breast cancer - related knowledge or awareness are generally low (donovan and tucker 2000 ; hughes. 1997 ; matthews. 1999), with many women holding inaccurate perceptions of breast cancer risk (matthews. this is particularly important as greater knowledge about cancer genetics is associated with higher participation in genetic risk assessment programs among african american women (thompson. for example, thompson. found that participants who declined counseling reported significantly lower levels of knowledge of breast cancer genetics compared with women who accepted both genetic counseling and testing. 1999), the association between perceived risk and participation in genetic risk assessment programs is somewhat inconsistent in an african american population. regarding the decision to undertake initial genetic counseling, one study found no association with perceived risk of having a mutation (halbert. 2005b). findings from four other studies, however, suggest a relationship between perceived risk of developing breast cancer and genetic risk assessment program interest and uptake (ford. 2007 ; armstrong. 2005 ; halbert. 2010 ; lipkus. found that african american women who perceived greater risk and were more concerned about breast cancer reported greater interest in genetic testing (lipkus. additionally, findings from a randomized controlled trial showed that women who received genetic counseling were significantly more likely to report reductions in perceived risk of developing breast cancer, compared with non - participants (halbert. 2010). collectively, these findings suggest that at - risk women have high levels of perceived risk prior to undergoing genetic counseling, although counseling reduces this concern. while two other studies of at - risk african american women showed a pattern that those who received genetic counseling had greater perceived risk, these findings were not subjected to statistical analyses and it is unclear when in the genetic testing process these findings were observed (armstrong. only one study examined the association between perceived risk and subsequent test result acceptance (halbert. evaluated acceptance of brca1/2 test results in 157 african american women at high and moderate risk for having a deleterious mutation who were offered genetic testing through a genetic counseling research program. they found that women who were less certain about their risk of developing breast cancer were approximately three times more likely to receive brca1/2 test results compared to women who reported greater certainty, suggesting that ambiguity reduction is a strong motivator of decision making (han. overall, african american women hold positive beliefs about genetic testing, compared with caucasians (hughes. 1997 ; donovan and tucker 2000). african american women believe that undergoing testing raises awareness of the need for additional cancer prevention measures (hughes. 1997), leads to greater motivation to carry out regular surveillance (e.g., breast self - examination), and enables them to help their daughters or sisters decide about future testing options (thompson. 2002). we found only one study which specifically examined the association between holding positive beliefs about genetic counseling and testing and actual participation (thompson. 2002). in this study, 76 african american women were offered free brca1/2 counseling and testing, thus removing any financial burden to participate. there were no differences among women who declined versus those who accepted counseling and/or testing in terms of the perceived benefits of undergoing this process, indicating that positive beliefs do not necessarily translate to increased rates of participation (thompson. only one study has examined the association between belief in one s ability to control breast cancer risk (rather than belief in the testing process itself) and counseling / testing participation. ford. found that women who received genetic counseling endorsed the belief that they were able to reduce breast cancer risk through lifestyle factors, including changes to diet, exercise, smoking, drinking, stress, and social involvement (ford. african american women are more likely than caucasian women to report family and confidentiality concerns as salient barriers to participation in this process (donovan and tucker 2000 ; thompson. 2003). perceived familial barriers to participation include worry about the mutation status of other family members, and possible guilt if other family members are identified as gene carriers (thompson. expectancies about confidentiality breaches, stigmatization, and abuse at the hands of the medical profession also preclude testing participation (thompson. for example, in a study conducted by thompson., 30 % of african american women were concerned about testing abuses, compared with less than 10 % of caucasians. findings from two studies indicated that expected stigmatization (thompson. 2002) and the belief that an individual should not view herself as independent from family members (hughes. 2003) are associated with lower genetic testing participation. this finding is inconsistent with the family related advantages of undergoing testing reported in thompson.s study (thompson. 2002), further supporting the notion that perceived benefits do not necessarily translate to testing participation rates. in addition to the specific beliefs and expectancies about genetic counseling, the role of cultural values and the context of african american women should be considered. 2003) highlighted three worldview values important to this population : fatalism, that is the belief that one is powerless to control the onset and progression of cancer ; temporal orientation, that is how events and their consequences are perceived in terms of past, present, and future implications ; and religiosity (hughes. 2003). both a future temporal orientation and high levels of fatalism are positively associated with testing and counseling uptake in african american women (edwards. for example, in one study, a future orientation was positively related to greater perceived benefits of genetic testing (edwards. african american women, higher levels of future temporal orientation and fatalism were found in women who accepted genetic testing, compared with those who declined (hughes. 2003). similarly, kessler. found that high levels of fatalistic beliefs were associated with greater consideration of genetic testing participation (kessler. 2005). regarding religiosity, hughes. reported no significant association between religious coping style and participation in the genetic testing process however, they did acknowledge a trend for women who reported coping with difficult situations by working together with god to be more likely to participate in genetic risk assessment and counseling (hughes. an important aspect of an individual s reaction to health risk information, such as genetic risk, involves the regulation of their emotional responses (miller. similar to caucasian women, african american women with an increased risk for developing breast cancer report a moderate cancer - related distress prior to undergoing genetic counseling and testing (durfy. indeed, two studies report that concerns of being unable to handle the testing and results, and feeling overwhelmed by anxiety, are reasons cited by african american women for not undergoing testing (matthews. anxiety about their health also prevents some women from seeking genetic testing following a family member s death from cancer (matthews. these findings suggest that a lack of self - regulatory skills to manage this anxiety may underlie non - participation. consistent with the c - ship model, which highlights the importance of managing emotional responses (i.e., self - regulatory capacity), lerman. reported that discussion of the emotional impact of being at risk for breast cancer leads to increases in testing intentions in african american women (lerman. importantly, while many at - risk african american women report high levels of cancer - related distress prior to participating in genetic risk assessment programs, actual participation may result in few, if any, deleterious outcomes. pre - test genetic counseling is associated with reductions in cancer - specific distress and greater decision satisfaction (halbert. 2012 ; lerman. 1999) furthermore, charles. found that high - risk african american women who participate in genetic counseling that incorporates their beliefs and values were more likely to report that their worries were lessened ; women who underwent genetic testing in this sample showed no evidence of negative psychological consequences following disclosure of results and reported high levels of satisfaction with the genetic testing process (charles. african american women s levels of breast cancer - related knowledge or awareness are generally low (donovan and tucker 2000 ; hughes. 1997 ; matthews. 2000 ; lipkus. 1999 ; durfy. 1999), with many women holding inaccurate perceptions of breast cancer risk (matthews. this is particularly important as greater knowledge about cancer genetics is associated with higher participation in genetic risk assessment programs among african american women (thompson. for example, thompson. found that participants who declined counseling reported significantly lower levels of knowledge of breast cancer genetics compared with women who accepted both genetic counseling and testing. 1999), the association between perceived risk and participation in genetic risk assessment programs is somewhat inconsistent in an african american population. regarding the decision to undertake initial genetic counseling, one study found no association with perceived risk of having a mutation (halbert. 2005b). findings from four other studies, however, suggest a relationship between perceived risk of developing breast cancer and genetic risk assessment program interest and uptake (ford. 2007 ; armstrong. 2005 ; halbert. 2010 ; lipkus. found that african american women who perceived greater risk and were more concerned about breast cancer reported greater interest in genetic testing (lipkus. additionally, findings from a randomized controlled trial showed that women who received genetic counseling were significantly more likely to report reductions in perceived risk of developing breast cancer, compared with non - participants (halbert. collectively, these findings suggest that at - risk women have high levels of perceived risk prior to undergoing genetic counseling, although counseling reduces this concern. while two other studies of at - risk african american women showed a pattern that those who received genetic counseling had greater perceived risk, these findings were not subjected to statistical analyses and it is unclear when in the genetic testing process these findings were observed (armstrong. only one study examined the association between perceived risk and subsequent test result acceptance (halbert. halbert. evaluated acceptance of brca1/2 test results in 157 african american women at high and moderate risk for having a deleterious mutation who were offered genetic testing through a genetic counseling research program. they found that women who were less certain about their risk of developing breast cancer were approximately three times more likely to receive brca1/2 test results compared to women who reported greater certainty, suggesting that ambiguity reduction is a strong motivator of decision making (han. overall, african american women hold positive beliefs about genetic testing, compared with caucasians (hughes. 1997 ; donovan and tucker 2000). african american women believe that undergoing testing raises awareness of the need for additional cancer prevention measures (hughes. 1997), leads to greater motivation to carry out regular surveillance (e.g., breast self - examination), and enables them to help their daughters or sisters decide about future testing options (thompson. we found only one study which specifically examined the association between holding positive beliefs about genetic counseling and testing and actual participation (thompson. 2002). in this study, 76 african american women were offered free brca1/2 counseling and testing, thus removing any financial burden to participate. there were no differences among women who declined versus those who accepted counseling and/or testing in terms of the perceived benefits of undergoing this process, indicating that positive beliefs do not necessarily translate to increased rates of participation (thompson. only one study has examined the association between belief in one s ability to control breast cancer risk (rather than belief in the testing process itself) and counseling / testing participation. ford. found that women who received genetic counseling endorsed the belief that they were able to reduce breast cancer risk through lifestyle factors, including changes to diet, exercise, smoking, drinking, stress, and social involvement (ford. african american women are more likely than caucasian women to report family and confidentiality concerns as salient barriers to participation in this process (donovan and tucker 2000 ; thompson. perceived familial barriers to participation include worry about the mutation status of other family members, and possible guilt if other family members are identified as gene carriers (thompson. 2002). expectancies about confidentiality breaches, stigmatization, and abuse at the hands of the medical profession also preclude testing participation (thompson., 30 % of african american women were concerned about testing abuses, compared with less than 10 % of caucasians. 2002) and the belief that an individual should not view herself as independent from family members (hughes. this finding is inconsistent with the family related advantages of undergoing testing reported in thompson.s study (thompson. 2002), further supporting the notion that perceived benefits do not necessarily translate to testing participation rates. in addition to the specific beliefs and expectancies about genetic counseling, the role of cultural values and the context of african american women should be considered. (hughes. 2003) highlighted three worldview values important to this population : fatalism, that is the belief that one is powerless to control the onset and progression of cancer ; temporal orientation, that is how events and their consequences are perceived in terms of past, present, and future implications ; and religiosity (hughes. both a future temporal orientation and high levels of fatalism are positively associated with testing and counseling uptake in african american women (edwards. for example, in one study, a future orientation was positively related to greater perceived benefits of genetic testing (edwards. african american women, higher levels of future temporal orientation and fatalism were found in women who accepted genetic testing, compared with those who declined (hughes. similarly, kessler. found that high levels of fatalistic beliefs were associated with greater consideration of genetic testing participation (kessler. 2005). regarding religiosity, hughes. reported no significant association between religious coping style and participation in the genetic testing process however, they did acknowledge a trend for women who reported coping with difficult situations by working together with god to be more likely to participate in genetic risk assessment and counseling (hughes. 2003). an important aspect of an individual s reaction to health risk information, such as genetic risk, involves the regulation of their emotional responses (miller. similar to caucasian women, african american women with an increased risk for developing breast cancer report a moderate cancer - related distress prior to undergoing genetic counseling and testing (durfy. indeed, two studies report that concerns of being unable to handle the testing and results, and feeling overwhelmed by anxiety, are reasons cited by african american women for not undergoing testing (matthews. 2000 ; donovan and tucker 2000). anxiety about their health also prevents some women from seeking genetic testing following a family member s death from cancer (matthews. these findings suggest that a lack of self - regulatory skills to manage this anxiety may underlie non - participation. consistent with the c - ship model, which highlights the importance of managing emotional responses (i.e., self - regulatory capacity), lerman. reported that discussion of the emotional impact of being at risk for breast cancer leads to increases in testing intentions in african american women (lerman. importantly, while many at - risk african american women report high levels of cancer - related distress prior to participating in genetic risk assessment programs, actual participation may result in few, if any, deleterious outcomes. pre - test genetic counseling is associated with reductions in cancer - specific distress and greater decision satisfaction (halbert. 2012 ; lerman. 1999) furthermore, charles. found that high - risk african american women who participate in genetic counseling that incorporates their beliefs and values were more likely to report that their worries were lessened ; women who underwent genetic testing in this sample showed no evidence of negative psychological consequences following disclosure of results and reported high levels of satisfaction with the genetic testing process (charles. this systematic review describes the psychosocial factors influencing the participation of african american women in genetic risk assessment programs. taken together, findings indicate that specific cognitive and affective factors influence an african american woman s interest in, and decision to undergo, genetic risk assessment. these factors include her perception of risk of developing breast cancer, the extent to which she endorses specific limitations of undergoing genetic testing, her fatalistic beliefs and temporal orientation, and her levels of cancer - related distress. overall, studies that have drawn direct comparisons between african american and caucasian women have noted significant differences regarding their knowledge about the genetics of breast cancer (donovan and tucker 2000 ; hughes. 1997), perceptions of risk (donovan and tucker 2000), endorsement of the benefits and limitations of undergoing counseling and testing (donovan and tucker 2000 ; thompson. 1997), and ability to manage emotional distress associated with the genetic testing process (donovan and tucker 2000). this suggests that targeted interventions to facilitate decisions regarding genetic counseling and testing participation should be tailored to the specific cognitive affective profile of an african american woman. in one study, african american women who received education about genetic risk and counseling reported a higher intention to be tested and were more likely to provide a blood sample for storage, compared with women who received education only (lerman. counseling involved discussion of the emotional impact of having a family history of cancer, psychosocial implications of a positive test result for participants and their family members, intentions to communicate results to friends and family, and anticipated reactions to possible test results., who found that african american women who received culturally tailored genetic counseling (discussing strategies for coping with cancer and family reactions to a cancer diagnosis) were more likely to report that their cancer - related worries were lessened, compared with those who received standard counseling (charles. (halbert. 2010) found that african american women who received tailored counseling centering on beliefs and values such as spirituality, temporal orientation, and communalism did not report changes in perceived risk or psychological functioning, perhaps suggesting that culturally tailored counseling may be effective only for women who hold specific beliefs and values regarding risk assessment. to date, no interventions have attempted to enhance the strategies required for african american women to manage their emotional responses throughout the genetic testing process. this is surprising, given that improved self - regulation has been shown to predict intention to undergo genetic testing across a range of illnesses (frost. 2001), and an inability to emotionally manage test results precludes testing participation in african american women (matthews. further research is required to evaluate the impact of emotional self - regulation on decision making for genetic testing in this population, and to implement these findings into future interventions. first, many studies recruited their samples through cancer clinics and hospitals, which may not be representative of all african american women. for example, in the studies which provided participant mean income figures, an average of 52 % of women earned above $ 35,000 per year, compared to an average annual income of $ 17,880 across us blacks in 2011 (us census bureau 2011). second, it is possible that, despite a systematic and thorough search, we may not have identified all studies that examined factors relating to participation in genetic risk assessment programs among african american women. our review provides an in - depth analysis of the cognitive and affective factors that influence an african american woman s interest in, and decision to undergo, genetic risk assessment. while the decision to participate in this process is complex and depends upon each individual s situation, understanding the psychosocial factors that underlie this choice is important in developing interventions to facilitate informed decision making among african american women. | breast cancer is a significant health concern for african american women. nonetheless, uptake of genetic risk assessment (including both genetic counseling and testing) for breast cancer gene mutations among these populations remains low. this paper systematically reviews cognitive (i.e., beliefs) and affective (i.e., emotions) factors influencing brca1/2 genetic risk assessment among african american women as well as psychosocial interventions to facilitate informed decision making in this population. a systematic search of cinahl, pubmed, and psycinfo was undertaken, yielding 112 published studies. of these, 18 met the eligibility criteria. african american woman are likely to participate in genetic risk assessment if they are knowledgeable about cancer genetics, perceive a high risk of developing breast cancer, have low expectancies of stigmatization from medical professionals, view themselves as independent from family, and have fatalistic beliefs and a future temporal orientation. anticipated negative affective responses, such as an inability to handle the results of testing, are barriers to uptake. specific perceptions, beliefs, and emotional factors are associated with genetic risk assessment among african american women. understanding these factors is key in the development of interventions to facilitate informed decision making in this population. |
its incidence worldwide is 10.1 in every 100,000 men and 2.5 in every 100,000 women. the highest incidence rate of bladder cancer in europe is seen in the western and southern regions, whereas eastern europe has the lowest incidence rate. the worldwide mortality rate is 4 in every 100,000 men and 1.1 in every 100,000 women. the mortality rate of bladder cancer in europe in the past decade was 16% in men and 12% in women. seventy percent of cases of newly diagnosed disease are superficial disease ; roughly 30% of newly diagnosed cases are muscle - invasive metastatic diseases. although local recurrence is significant despite treatment, the natural progression of non - muscle - invasive bladder cancer tends to remain superficial. about 5% to 20% of superficial tumors will develop into muscle - invasive disease despite treatment. the most common areas of distant metastases of urothelial carcinomas are typically the liver, lung, mediastinum, bone, and adrenal gland, respectively. we present a case of non - muscle - invasive bladder cancer that metastasized into the bone, mediastinum, iliac lymph node, and adrenal and thyroid glands. an 83-year - old male patient visited the urology department with complaints of macroscopic hematuria at intervals over 9 months. transurethral resection of the bladder (tur - b) was performed on the patient when a 17-mm14-mm mass was found in the ultrasound examination. repeat tur was applied to the patient 4 weeks later and high - grade urothelial carcinoma with lamina propria invasion was found, whereas muscularis propria invasion did not exist. positron emission tomography (pet) was performed during the second month because the patient had chest pain in the postoperative period, and multiple metastatic bone lesions, metastatic lymphadenopathies on the mediastinum and right external iliac area, a hypermetabolic lesion on the left thyroid lobe, and a hypermetabolic lesion on the left adrenal gland were found. incisional biopsy performed on the fifth rib of the patient was evaluated as urothelial carcinoma metastasis and radiotherapy was applied to the patient for these lesions (fig. a fixed, 3-cm mass was palpated on the left lobe of the thyroid. then, in the neck computed tomography (ct) with contrast, a 40 mm34 mm nodular mass was found on the left lobe of the thyroid gland, which caused left - sided destruction of the thyroid cartilage. malignant tumor cells were observed in the fine - needle aspiration sample taken from the hypermetabolic lesion on the left lobe of the thyroid. owing to the patient 's clinical history, primary thyroid carcinomas were considered in the differential diagnosis, because metastatic carcinoma and anaplastic and medullar carcinomas are of first priority. the immunohistochemical panel included thyroid transcription factor-1 (ttf-1) and thyroglobulin in terms of thyroid tumors ; calcitonin, carcinoembryonic antigen (cea), chromogranin a, and synaptophysin in terms of medullar carcinoma ; and cytokeratins (ck) 7 and ck 20 antibodies in terms of urothelial carcinoma. no reaction was observed with neuroendocrine markers (synaptophysin and chromogranin), calcitonin, thyroglobulin, or ttf-1 in tumoral cells. the tumor was evaluated as a urothelial carcinoma metastasis owing to the clinical history of the patient, the existence of other metastases, the morphological similarity of the tumor cells with the primary tumor, and ck 20 positivity in some cases immunohistochemically (fig. because the general condition of the patient worsened while planning chemotherapy, no systematic chemotherapy was applied. the prognosis and survival of patients with bladder cancer are associated with stage and grade during diagnosis. survival rates for 5 years were reported as 94% for grade 1 non - muscle - invasive disease, 40% for grade 3 non - muscle - invasive disease, 72% for stage t2, and 33% for stage t4 muscle - invasive disease. contrary to muscle - invasive disease, many superficial bladder cancers can be treated by regular monitoring cystoscopy, urinary cytology, and re - tur together or not with protective therapy, transurethral resection, and adjuvant intravesical treatment. despite these measures, about 5% to 20% of non - muscle - invasive tumors will progress to muscle - invasive disease. high - grade non - muscle - invasive tumors are related to a high risk of muscular invasion and metastasis and end up with a worse prognosis. also, it is known that metastasis can appear without muscular invasion ; these metastases are found in regional lymph nodes during cystectomy. cases of distant metastasis of low - grade non - muscle - invasive urothelial carcinoma without regional lymph node metastasis have been reported. errors in clinical staging of bladder tumors are well known. in some pathological staging cases, it is estimated that low staging was done at a rate as high as 30% to 40%. the european organisation for research and treatment of cancer (eortc) developed a scoring system and a risk table to estimate short- and long - term risks of recurrence and progression separately. in this scoring system, clinical and pathological parameters are tumor number, tumor size, previous recurrence rate, t phase, carcinoma in situ presence, and tumor grade. in our patients, possible mean progression rates according to the score are reported as 5% in the first year and 17% in the fifth year. in the eortc study, no results are reported for new concepts such as re - tur, early single - dose chemotherapy, and maintenance bacillus calmette guerin treatment, and this has been criticized by authors. in our patient, early single - dose chemotherapy and re - tur were applied and both pathology reports suggested a high - grade non - muscle - invasive bladder tumor. although the progression rate of our patient for 1 year was estimated as 5% according to the eortc study, progression developed in the patient before initiating induction treatment of intravesical ct and before symptoms associated with bone metastases developed. there are publications in the current literature that report that low - grade non - muscle - invasive bladder tumors are detected clinically and the distant lung metastasis can appear without regional lymph node involvement. rare metastases of urothelial carcinoma into the skin, uterus, and orbita and vaginal and omental involvements have also been reported.. showed a small cell carcinoma that metastasized into the thyroid gland, which is a minor variant of bladder urothelial carcinoma. small - cell carcinoma is rarely originated from the bladder and comprises of 0.48% to 1% of all bladder tumors. metastases in the thyroid gland are observed in many patients by physical examination or imaging methods during routine monitoring after primary tumor resection. in these patients, fine - needle aspiration may be useful for evaluating possibilities and limiting unnecessary operation for diagnostic purposes. the most significant conflict in cytologic comment is the differentiation between primary thyroid anaplastic carcinoma and high - grade metastatic malignity. in the fine - needle aspiration material of the patient, differential thyroid carcinomas and medullar carcinomas can be excluded morphologically and immunohistochemically. in the present case, no reaction was observed with neuroendocrine markers (synaptophysin and chromogranin a), calcitonin, thyroglobulin, or ttf-1 in tumoral cells. although a positive reaction immunohistochemically with thyroglobulin supports a primary thyroid malignancy, only 20% to 30% of anaplastic carcinomas display thyroglobulin positivity and negative results do not have meaning. because 20% to 30% of anaplastic carcinomas display thyroglobulin positivity and negative results do not have meaning the patient 's clinical history is the most essential part in the differential diagnosis of these lesions, and it is emphasized that a thyroid nodule found in a patient with a known cancer should be accepted as metastasis unless proven otherwise. because our patient had a known urothelial carcinoma history, other metastases, a morphological similarity between the tumor cells and the primary tumor, and ck 20 positivity immunohistochemically in some cells, the tumor was evaluated as a urothelial carcinoma metastasis. in conclusion, non - muscle - invasive bladder tumors may present with multiple distant metastases even without muscle invasion. this presented case showed that bladder tumors can metastasize into the thyroid gland in addition to known distant metastasis areas. metastasis of a bladder tumor should also be kept in mind in the differential diagnosis in patients who have a bladder tumor history and are found to have a mass on the thyroid gland. | bladder cancer is the most prevalent malignancy of the urinary tract. about 90% of bladder cancers are urothelial carcinomas. seventy percent of cases newly diagnosed are superficial diseases ; roughly 30% of newly diagnosed cases are muscle - invasive metastatic diseases. bladder urothelial carcinoma primarily metastasizes into regional lymph nodes and then into liver, lung, mediastinum, bone, and adrenal gland. in our case, non - muscle - invasive bladder cancer metastasized into the bone, mediastinum, iliac lymph node, and adrenal and thyroid glands. this is the first reported case in the current literature in which urothelial carcinoma metastasized into the thyroid gland. |
trauma is the leading cause of death world wide and traumatic brain injury (tbi) is one of the commonest injuries associated with it. not only the mortality is considerable, but also, the degree of morbidity and the overall social outcome are severely affected. the need for urgent resuscitation is warranted for prevention of secondary insult to brain. however, the choice of fluid in such cases is still a matter of debate. the literature does not provide enough evidence pertaining to role of colloids in tbi patients. in addition, the existing information is still scanty regarding the use of colloids in patients with different tbi subgroups including isolated head injury, head injury with extra - cranial bleed and among varying severity of head injury. these areas need extensive research and future trials. in this article, we have tried to explore the present role of colloid resuscitation in patient with head injury with special reference to underlying pathophysiological processes. severe tbi is associated with approximately two - third of all the tbi cases associated with polytrauma. the incidence of hypotension in tbi patients is much more common due to extra - cranial injury rather than the isolated head injury. these extra - cranial injuries are mainly associated with bleeding and if not managed appropriately, develop into shock. hence the role of fluid resuscitation becomes crucial to overcome the hypotensive effects of the extra - cranial injury on the cerebral hemodynamics. on the other hand, patients with tbi who had no apparent signs of hypovolemia, revealed inadequate tissue perfusion and responded well to fluids and iontropes. thus patients with isolated tbi (without extra - cranial bleed) still require fluid resuscitation to preserve optimum cerebral perfusion pressure (cpp)., in their retrospective study revealed that a negative balance of approximately 600 ml or more of the fluid was found to be independent determinant of poor neurological outcome in severe tbi patients. however, the retrospective chart review of 776,734 trauma patients showed that half of the patients who received prehospital fluid resuscitation had increased overall mortality. tbi patients (one of the subgroups) were associated with higher mortality in this study. this study develops new insight in the fluid resuscitation especially in tbi patients and in fact challenges the current practice. however, this observation requires further clinical investigations and future trials. at least for now, the goal of fluid resuscitation in such cases would include improvement in oxygen delivery, maintaining cpp and stabilization of icp. the two most important physiological factors that stabilize internal milieu of brain are cerebral auto regulation and blood brain barrier (bbb), both are frequently found to be altered in severe tbi. it is noteworthy that in post - head injury patients many inflammatory cascades get stimulated which in turn cause many local and systemic changes including exaggeration of cerebral edema, disruption of bbb, secondary brain injury, and finally multiorgan dysfunction. the roles of different fluids on these inflammatory mediators have been investigated and were found to be variable. in general, hypotonic or isotonic crystalloids often aggravate the neuro - inflammatory responses and produce worsening of the cerebral edema and confer no neuroprotection. however, fluids like hypertonic saline - dextran combination have been shown to attenuate the inflammatory cascades. in one study, no penetration of hyrdroxyl ethyl starch (he s) into cerebrospinal fluid was observed despite disrupted bbb ; however, this study involved only the small group of patients. thus this observation needs further validation in animal experiments on tbi as well as hemorrhagic shock models, fresh frozen plasma infusion was found to be superior to both artificial colloid and normal saline solutions in reducing the brain edema and lesion size. thus the type of fluids (crystalloids versus colloids) would have some differential effects on tbi patients depending upon the variable interactions with various biochemical mediators. among different subtypes of colloids, however current literature reflects the emerging role of the osmolality of an infusion solution rather than the colloid osmotic pressure per se as a key determinant in the pathogenesis of cerebral edema formation. movement of water across the bbb depends on osmotic gradient and the integrity of this barrier. bbb may be disrupted in some areas of brain while intact in other, depending upon the severity of tbi, hence the osmotic effect would also be variable. initial animal experiments on tbi models favored the use of colloids. in comparison to colloids, crystalloids were shown to produce more cerebral edema in tbi patients. it is likely that in severe head injury patients (with disrupted bbb), edema formation would be even worse with the further use of crystalloids. the high oncotic pressure of colloids decreases the cerebral edema formation and is also associated with improvement in the mean arterial blood pressure (mabp), having low infused volume, and decreased neuronal death. in a multicenter trial, all hyperosmotic solutions including 15% mannitol, 10% sodium chloride (nacl), and hyper he s (7.2% nacl combined with hydroxyethyl starch) were found to decrease the intracranial pressure in acute tbi patients. however, among all the solutions, hyper he s had a significantly prolonged effect on reduction in icp with favorable effects on both cerebral as well as hemodynamic parameters. thus the majority of beneficial effects of colloid were attributed to its unique oncotic property which reduces the formation of cerebral edema and hemodynamic property which keeps the mabp in optimal range. on the contrary, few studies report that the oncotic effect generated by colloids do not decrease the cerebral edema formation after tbi. in addition, the colloids were not associated with either increase in the cerebral oxygen delivery nor decrease in the raised icp. in fact some of the synthetic colloids increase the blood viscosity and cause decrease in systemic rheological property. however investigators of yet another study did not observe any significant improvement in rheological functions in tbi patients with different concentrations of dextran 40. this observation has been attributed to the early metabolic suppression rather the global ischemia which predominates in the early phase of head injury and thus could not be benefitted by the use of dextran solution. similarly, the normovolemic hemodilution in both adults and pediatric brain injury models, were found to be ineffective as these tend to cause hyperemia and increased transfer of water content across bbb that produced further cell swelling. the popular the saline versus albumin fluid evaluation (safe) study has been shown to cause increase in mortality among tbi patients who were treated with albumin. this study did not explain the cause of increased mortality in this sub group ; however, a dilution coagulopathy was proposed as a probable cause by other investigators. kawamata., showed that high colloid osmotic pressure produced by metabolites or idiogenic osmoles can increase the chances of post concussion edema, and possibility led to the higher mortality in albumin group. all synthetic colloids including dextran, gelatin, and he s have dose - related side effects like coagulopathy, renal failure, and tissue storage. there is altered coagulation homeostasis in severe head injury patients and the administration of synthetic colloids especially dextrans, gelatins, and high molecular weight starches have been shown to cause worsening of hemostasis. many studies revealed that use of these synthetic colloids decreased the levels of coagulation factors (vii, v111, von willibrand factor and fibrinogen), caused defect in platelet and erythrocyte aggregation. on the other hand, natural colloids like albumin, bind to nitric oxide (no) and forms nitrosothiols. even use of 6% he s was associated with more pronounced von willibrand factor dysfunctions in patients with blood group o few studies showed that isovolemic replacement of blood loss with either 6% he s or 5% human albumin did not affect the overall coagulation. however, depending upon the severity of head injury, minimal to severe coagulative dysfunctions may exist as part of the bidirectional interaction between brain and whole organism to which the use of colloids may further add disturbances in overall hemostasis. a large clinical trial would be needed to explore the clinical incidence as well as the relevance of this dysfunction in the coagulation cascade produced by the colloids. these reactions can manifest as minor (pruritis, fever, rash) to severe life - threatening symptoms (arterial hypotension, broncospasm). a series of 19,593 patients showed that there was a chance of one anaphylactic reaction for every 456 patients. the risk factors highlighted in this study were use of gelatins, dextrans, prior drug allergy, and male gender. use of colloids would certainly impose added risks of anaphylaxis in this subgroup of patients. tbi is associated with acute kidney injury in a considerable number of patients (9 - 23%) and often presents with consecutive higher mortality. colloids are also found to be associated with increased chances of acute kidney injury and increased use of renal replacement therapy in critical ill patients. thus it is likely that colloids would add more adverse effects on renal functions in patients with tbi and may affect the overall mortality. however, the role of colloids in worsening the pre existing renal dysfunctions or producing newer kidney injury in tbi patients is still to be elucidated and warrants further research. the recent cochrane reviews demonstrated that the use of colloid were not superior to crystalloids in respect to overall mortality especially in patients with trauma, burns, and post - surgery. moreover, there was no significant difference in various types of colloids in view of fluid resuscitation choices. thus, the existing data does not favor the use of colloids in certain subgroup of patients. in present scenario, considering the cerebral and other systemic adverse effects coupled with existing higher mortality data, colloids does not seem to be fluid of choice for resuscitation in patients with tbi. further substantial evidence for this requires a better understanding of the cranial and extra cranial effects of tbi. until that time local regulations being bedded into a tbi concept may be the solution. | trauma is a leading cause of death worldwide and traumatic brain injury is one of the commonest injuries associated with it. the need for urgent resuscitation is warranted for prevention of secondary insult to brain. however, the choice of fluid in such cases is still a matter of conflict. the literature does not provide enough data pertaining to role of colloids in head injury patients. in this article, we have tried to explore the present role of colloid resuscitation in patient with head injury. |
over the last three decades, there have been advances in the diagnostic techniques and treatment modalities available for cancer patients, which have led to earlier detection of cancer, improved disease management, and increased survival. such improvement has caused a surge in the number of surviving patients with secondary multiple neoplasms, posing an additional threat in terms of morbidity and mortality. such an entity of multiple neoplasms is not rare or new, being described by billroth as far back as 1889.1 in 1932, warren and gates published the first study of 1,259 patients with multiple neoplasms.1 ever since, there have been numerous reports addressing the occurrence of second primary neoplasms. nowadays, it is accepted that the prevalence of multiple neoplasms varies between 0.7% and 11.7% in different populations1 and this number is increasing.2 even though there is an adequate body of published data describing the epidemiology and clinical characteristics of patients with single and multiple primaries, there are few reports addressing patient survival with different results.3,4 in the present study, we attempt to determine the incidence of multiple primaries and possible risk factors, and to assess the impact of multiple neoplasms on patient survival. the medical records of cancer patients initially seen and/or closely followed by the author at a regional cancer center were retrospectively reviewed, with special emphasis on the incidence of secondary neoplasms, risk factors for multiple malignancies, and their impact on survival. the study included all consecutive adult patients with histologically confirmed malignancies, with the exception of nonmelanoma skin cancer and carcinoma in situ of the uterine cervix. all data collected were stripped of any patient identification in compliance with the health information protection act. the study was approved by the institutional review board at st rita s medical center. ethnic background was classified as caucasian, native american indian (from both parents), african american, and others. body mass index (bmi, kg / m) was calculated and grouped into those who were underweight (bmi 40 kg / m). multiple cancers were defined as two or more primary cancers occurring in an individual that are not an extension, recurrence, or metastasis. based on the chronology of presentation, they were categorized according to warren and gates.5 an index tumor was defined as the first diagnosed malignant tumor in patients with multiple malignancies. second primaries discovered at workup of the index primary were termed simultaneous ; within 6 months of the index primary were called synchronous ; and after 6 months, termed metachronous primaries. the latter were further defined as metachronous 5 years (table 2 and figure 3). however, there were no significant differences in survival between single, simultaneous, and synchronous primaries (figure 3). this survival advantage for patients with multiple primaries persisted even if the analysis was limited to patients with invasive cancer by excluding all 53 patients initially presenting with carcinoma in situ (stage 0, p=0.0000, figure 4) or the study was limited to a specific interval of initial cancer diagnosis, such as patients initially diagnosed as having cancer between 2005 and 2012 (p=0.00236, figure 5). further, there was no difference in survival between single and multiple primaries when survival with multiple primaries was calculated from the date of diagnosis of the second primary rather than from the date of the initial cancer diagnosis (p=0.05956, figure 6). both single and metachronous primaries fared worse compared with the control group representing the life expectancy of the us population (figure 7). between january 2005 and december 2012, of 1,873 consecutive cancer patients, 1,551 (82.8%) had a single primary neoplasm and 322 (17.2%) had multiple neoplasms. patients with multiple primaries included 284 (15.2%) patients with two primaries, 30 (1.2%) with three, six (0.3%) with four, and two (0.1%) with five neoplasms. the mean interval between the index primary and the second primary was 117124.0 months, with a median of 76.5 months. twenty - four (7.5%) patients presented with their second primary within the first month (simultaneous primary), 23 (7.1%) presented within the subsequent 5 months (synchronous), 88 (27.3%) presented within 5 years (metachronous 5 years (table 2 and figure 3). however, there were no significant differences in survival between single, simultaneous, and synchronous primaries (figure 3). this survival advantage for patients with multiple primaries persisted even if the analysis was limited to patients with invasive cancer by excluding all 53 patients initially presenting with carcinoma in situ (stage 0, p=0.0000, figure 4) or the study was limited to a specific interval of initial cancer diagnosis, such as patients initially diagnosed as having cancer between 2005 and 2012 (p=0.00236, figure 5). further, there was no difference in survival between single and multiple primaries when survival with multiple primaries was calculated from the date of diagnosis of the second primary rather than from the date of the initial cancer diagnosis (p=0.05956, figure 6). both single and metachronous primaries fared worse compared with the control group representing the life expectancy of the us population (figure 7). cancer survivors have a 14% increased risk of developing a second cancer primary compared with the general population.3,7,8 in the usa, the number of cancer survivors has tripled since 1971 and is growing by 2% each year.9 as a consequence, the number of cancer survivors presenting with multiple primaries has increased over time. multiple primaries now account for 16% of the newly diagnosed malignancies reported in the us national cancer institute s surveillance, epidemiology, and end results (seer) program.9,10 the overall incidence of multiple primary neoplasms among cancer survivors varies widely in the literature from 1% to 37%, depending on the type of analysis used, timing of data collection, and ethnicity of the subjects studied. in the usa, the incidence has varied from 3.5% in new mexico s triethnic population (1977),11 to 5.3% in an autopsy series (1968),12 to 8% according to seer data (19752001).2 in western europe, multiple primaries accounted for 0.7% in a hospital registry in scotland (1972),13 6.3% in multiple european cancer registries (19951999),14 and 11.7% in an autopsy series from sweden (1969).15 in the present study, the incidence of multiple primaries was 6.1% for patients initially diagnosed between 2005 and 2012. the numbers and types of multiple primaries relative to the total number of cancer patients also varies in the literature. the incidence of three or more primaries varied from 0.2% in a cancer center in jordan (20062011),16 1.1% in an autopsy series from japan (19621981),17 2.0% in a cancer center in france (19802009),10 to 0.5% in the present study (20052012). metachronous primaries accounted for 1.0% in an autopsy study from the usa (1982),18 5.3% in a study from switzerland (19751983),19 and 3.1% in the present study (20052012). the incidence of multiple primary cancers has recently increased. during 19952008, the percentage of multiple primary cancers at all sites increased in the usa by 25.4% using seer rules (from 14.6% to 18.4%).20 in finland, the risk of second malignancies increased by 50% between the 1950s and the 1980s.21,22 the cumulative incidence of multiple primaries also increases with increasing time since initial primary diagnosis.23 in the present study, the cumulative incidence of second primaries for all cancer patients has increased from 7.2% at 5 years to 17.2% after 20 years. it has been suggested that, in patients with familial cancer syndrome, the risk of a second primary is approximately 3% for each year of survival after the first cancer occurrence. when the figure is projected over an 18-year span of survival, the cumulative risk of a second primary exceeds 40%.24 more than one quarter (28%) of the population surviving childhood cancer experienced additional cancer primaries, and within 20 years from diagnosis of the index primary, the estimated cumulative incidence of multiple primaries was reported to reach 47%.23 the higher incidence of multiple primaries among all cancer patients reported in this study as well as in other publications may be related to the increased awareness of the possibility of second malignancies, the frequent use and increased sensitivity of screening procedures during follow - up, and better treatment delivered to the initial malignancy, with improved survival.22 this was evident in the present study, where proactive screening for second primaries led to detection of 24 (1.3%) patients with asymptomatic stage 0 second malignancies. development of multiple primaries in cancer survivors may be related to increased awareness or be secondary to previous therapies, and may also be due to shared risk factors, including environment, lifestyle, and inherited genes.14 age is considered to be a risk factor. the prevalence of multiple neoplasms was reported to be 5%12% for patients aged 5064 years, compared with 12%26% for those aged over 80 years.22 in the present study, the highest incidence was seen among those aged 6069 years. however, the differences were not statistically significant. different societies have more prevalent tumors than others. for example, the high incidence of gastric cancer but low incidence of prostate cancer in japan is in marked contrast with the us population.25 in the present study, patients of caucasian ancestry had higher incidences of multiple primaries compared with other ethnic groups, even though the differences were relatively small, being about 10% above the overall cohort. in a previous report on the same group of patients, those with caucasian ancestry were more liable to develop vascular thrombosis, with a higher cumulative incidence of multiple thrombophilic gene mutations26 (table 5). hence the possibility of other genetic factors may contribute to the development of multiple malignancies in the caucasian population. obesity has been linked with the occurrence of cancer in the general population, particularly that of the breast, female reproductive organs, and the gastrointestinal tract.22 obesity has also been reported to increase the risk of second primary cancer, particularly endometrial and colon cancers.27,28 this was not detected in the present study, with obesity being seen more frequently in patients with single primaries than in those with multiple primaries. this lack of correlation may possibly be due to the small number of patients studied. smoking - related second malignancies account for 35% of the subsequent cancer cases observed in us cancer survivors.22 a consistent excess of primary smoking - related cancers such as those of the oral cavity, pharynx, larynx, lung, and bladder have been reported in patients previously diagnosed with similar malignancies.22 it has been reported that interindividual variation in dna repair capacity may lead to increased predisposition to cancer.29 abnormal mutagen sensitivity found in studies that indirectly assessed dna repair capacity has been found in patients with head and neck, colorectal, and non - small - cell lung cancers, as well as in patients with multiple primaries of similar malignancies.30 in the present analysis, cancer survivors who were smokers had a higher risk of multiple primaries compared with nonsmokers, particularly for lung, colorectal, and prostate cancers. women whose first primary is the breast account for 25% of survivors with multiple cancers, followed by colorectal cancer (15%) as the first primary in both men and women, according to seer data.2 in the present study, the most common initial primary among cancer survivors with multiple primaries for both men and women was breast, prostate, and colorectal. patients who initially presented with thyroid, urinary bladder, prostate, cervical, and uterine cancers were more liable to develop a second malignancy, while those with hepatic, biliary tract, and pancreatic cancers as well as those with acute leukemia rarely developed a second malignancy. this may be related to the poor outcome in these patients, who did not survive long enough to develop second primaries. patients with multiple primaries usually present initially with tumors at lower stages than secondary primaries.22,3133 in the present study, initial index primaries were predominantly at an earlier stage (78.8%), compared with second primaries (47%). considering all cancer patients, the risk of developing multiple primaries was much higher for those presenting initially with stage 0 or stage i disease. this finding may be also related to the shorter survival time of patients initially presenting with advanced disease. the etiologic role of cancer therapy in the development of second malignancies has been described extensively by others.34 second primaries following chemotherapy usually arise within a few months to 9 years, while after radiation therapy or hormonal treatment, chronic sequelae usually develop after a longer latency period of 510 years.22 however, many believe that a second primary may not be attributed solely to prior treatment. it is most likely a combination of risk factors, such as genetic predisposition, lifestyle, and environmental influence, and could even be a behavioral effect.7,9,22 family history is an important risk factor for multiple primary neoplasms. almost all cancer sites showed an excess of familial clustering, and most of the evidence has been based on excess of close relationship among cases related to families of of 3 to 15 generations deep.35 practically all types of hereditary cancers showed an excess of multiple primaries as well as an early age of onset.22,24,27,28,3133,36 it has been suggested that abnormal genes or gene variants might be responsible for the clustering observed with some tumors. multiple cancer predisposition genes have already been identified in high - risk pedigrees such as brca1, brca2, and p16/cdkn2a.22,35 individuals with hereditary nonpolyposis colorectal cancer with multiple primaries have an increased frequency of mismatch repair genes, such as hmsh2 and hmlh1, leading to microsatellite instability.33 mutagen sensitivity, found in studies that indirectly assessed dna repair capacity, tends to occur in patients with head and neck, hereditary nonpolyposis colorectal, and non - small - cell lung cancers.30 in the present study, a strong family history of cancer reflecting a close relationship between first - degree relatives was statistically more common in patients with multiple primaries, especially those with three or more primaries, and in those with metachronous primaries, highly suggestive of inherited cancer predisposition gene mutations, especially among caucasian patients. survival of patients with multiple primaries varies widely in the literature, depending on the method of calculation, primary tumor site, cancer stage, year of initial cancer diagnosis, and time interval between index and second primaries. overall, patients with multiple primaries have a far better survival rate compared with those having a single primary.37 in the present analysis, a mean survival of 166 months versus 60 months was documented. in a study on oral and maxillofacial cancers at different sites, the mean survival was 127 months for patients with multiple primaries compared with 48 months for cases with a single primary (p=0.001).5 however, when the survival of patients with the same cancer site was compared, calculated from the date of their respective primary diagnosis, there were no statistically significant differences in survival between those who had one primary and those who had more than one primary.5 calculating survival from the date of the second primary has been used in several reports comparing multiple primaries with single ones, with no differences in survival37,38 or an even worse prognosis for patients with multiple primaries.3,4 in the present analysis, using a similar method of calculation, the survival of patients who developed second malignancies was relatively similar to single primary. thus, the improved survival of patients with multiple primaries may be a reflection of different patient populations having a dissimilar pattern of initial tumor sites (predominantly favorable ones) and/or stages of disease (mainly earlier ones), rather than tumor multiplicity. as a rule, the more time that elapses between the first primary malignancy and a second malignancy, the better the prognosis.5 most studies have shown that patients with synchronous primaries have a poor prognosis compared with those having metachronous primaries.5,39 in one study, the 5-year survival rate was 18% compared with 55%, respectively.19 in the present analysis, the 5-year survival of patients with a single primary was 59%, compared with those having simultaneous (61%), synchronous (59%), and metachronous primaries (95%). overall, patients initially presenting with two primaries, either simultaneously or within 6 months of each other, have a similar survival to those with a single primary, and the more time that elapses from the first index primary to the second primary, the better the survival, especially if the interval exceeds 5 years. survival of patients with multiple primaries also depends on the site of the second primary. for breast cancer, the best survival was noted for metachronous contralateral breast cancer when the second cancer was diagnosed at least 5 years after the original primary, with 5-year and 10-year survival rates of 100% and 96%, respectively.37 for lung cancer as a second primary, the 5-year survival for those with a metachronous primary was 44% compared with 10% for those with a synchronous primary.39 with hepatocellular cancer, there were no significant differences in survival, whether presenting as a single primary or as a second primary following another malignancy,38 but not as a synchronous primary concomitantly diagnosed with another cancer, which carries the worst prognosis.40 most reports on patients with multiple primaries include patients at an earlier stage 0 (carcinoma in situ), while others limit the study to invasive cancers (stage i and above).41 inclusion of stage 0 in survival analysis usually results in a favorable survival outcome. in the present analysis, the improved survival of patients with multiple malignancies persists regardless of whether or not stage 0 patients are included. most studies on multiple primaries report on patients seen at certain intervals based on the year of initial cancer diagnosis, while others report on groups of patients seen at a specific period of time, irrespective of the date of initial cancer diagnosis. the latter method has a risk of bias, by including patients with favorable cancers who live long enough to be included in the analysis. the present analysis included all consecutive patients seen during a specific period, irrespective of the date of initial cancer diagnosis. however, analysis of patients initially diagnosed between 2005 and 2012 confirmed the same findings, ie, improved survival for cases with multiple primaries, and especially for metachronous ones. this study, being a retrospective analysis of a limited number of patients, has several limitations. it explores outcomes in a heterogeneous group of patients at different stages of malignancy who have received various types of therapies. the reported incidence of multiple primaries may be underestimated because the duration of follow - up for some patients was relatively short, and a longer follow - up, logically, will reveal development of more second primaries. further, exclusion of patients with skin cancers as well as those with carcinoma in situ of the uterine cervix may also have underestimated the overall incidence of multiple neoplasms. in addition, considering the variation seen in the incidence of multiple primaries between patients of different ethnic background, our findings may not necessarily be applicable to other populations of different ethnic composition. its main strength is the inclusion of all patients seen and followed closely in a single - practice, real - world situation that provides a representative picture of cancer patients in general. it defines the relative incidence and subtypes of multiple primaries among cancer patients of various ethnic groups as well as their impact on survival. it showed relatively consistent results, whether cancer was inclusive of all subtypes or limited to a certain stage of disease, and whether the study population was initially diagnosed over several decades or limited to a defined period of time. cancer survivors have a 14% increased risk of developing a second cancer primary compared with the general population.3,7,8 in the usa, the number of cancer survivors has tripled since 1971 and is growing by 2% each year.9 as a consequence, the number of cancer survivors presenting with multiple primaries has increased over time. multiple primaries now account for 16% of the newly diagnosed malignancies reported in the us national cancer institute s surveillance, epidemiology, and end results (seer) program.9,10 the overall incidence of multiple primary neoplasms among cancer survivors varies widely in the literature from 1% to 37%, depending on the type of analysis used, timing of data collection, and ethnicity of the subjects studied. in the usa, the incidence has varied from 3.5% in new mexico s triethnic population (1977),11 to 5.3% in an autopsy series (1968),12 to 8% according to seer data (19752001).2 in western europe, multiple primaries accounted for 0.7% in a hospital registry in scotland (1972),13 6.3% in multiple european cancer registries (19951999),14 and 11.7% in an autopsy series from sweden (1969).15 in the present study, the incidence of multiple primaries was 6.1% for patients initially diagnosed between 2005 and 2012. the numbers and types of multiple primaries relative to the total number of cancer patients also varies in the literature. the incidence of three or more primaries varied from 0.2% in a cancer center in jordan (20062011),16 1.1% in an autopsy series from japan (19621981),17 2.0% in a cancer center in france (19802009),10 to 0.5% in the present study (20052012). metachronous primaries accounted for 1.0% in an autopsy study from the usa (1982),18 5.3% in a study from switzerland (19751983),19 and 3.1% in the present study (20052012). the incidence of multiple primary cancers has recently increased. during 19952008, the percentage of multiple primary cancers at all sites increased in the usa by 25.4% using seer rules (from 14.6% to 18.4%).20 in finland, the risk of second malignancies increased by 50% between the 1950s and the 1980s.21,22 the cumulative incidence of multiple primaries also increases with increasing time since initial primary diagnosis.23 in the present study, the cumulative incidence of second primaries for all cancer patients has increased from 7.2% at 5 years to 17.2% after 20 years. it has been suggested that, in patients with familial cancer syndrome, the risk of a second primary is approximately 3% for each year of survival after the first cancer occurrence. when the figure is projected over an 18-year span of survival, the cumulative risk of a second primary exceeds 40%.24 more than one quarter (28%) of the population surviving childhood cancer experienced additional cancer primaries, and within 20 years from diagnosis of the index primary, the estimated cumulative incidence of multiple primaries was reported to reach 47%.23 the higher incidence of multiple primaries among all cancer patients reported in this study as well as in other publications may be related to the increased awareness of the possibility of second malignancies, the frequent use and increased sensitivity of screening procedures during follow - up, and better treatment delivered to the initial malignancy, with improved survival.22 this was evident in the present study, where proactive screening for second primaries led to detection of 24 (1.3%) patients with asymptomatic stage 0 second malignancies. development of multiple primaries in cancer survivors may be related to increased awareness or be secondary to previous therapies, and may also be due to shared risk factors, including environment, lifestyle, and inherited genes.14 age is considered to be a risk factor. the prevalence of multiple neoplasms was reported to be 5%12% for patients aged 5064 years, compared with 12%26% for those aged over 80 years.22 in the present study, the highest incidence was seen among those aged 6069 years. different societies have more prevalent tumors than others. for example, the high incidence of gastric cancer but low incidence of prostate cancer in japan is in marked contrast with the us population.25 in the present study, patients of caucasian ancestry had higher incidences of multiple primaries compared with other ethnic groups, even though the differences were relatively small, being about 10% above the overall cohort. in a previous report on the same group of patients, those with caucasian ancestry were more liable to develop vascular thrombosis, with a higher cumulative incidence of multiple thrombophilic gene mutations26 (table 5). hence the possibility of other genetic factors may contribute to the development of multiple malignancies in the caucasian population. obesity has been linked with the occurrence of cancer in the general population, particularly that of the breast, female reproductive organs, and the gastrointestinal tract.22 obesity has also been reported to increase the risk of second primary cancer, particularly endometrial and colon cancers.27,28 this was not detected in the present study, with obesity being seen more frequently in patients with single primaries than in those with multiple primaries. this lack of correlation may possibly be due to the small number of patients studied. smoking - related second malignancies account for 35% of the subsequent cancer cases observed in us cancer survivors.22 a consistent excess of primary smoking - related cancers such as those of the oral cavity, pharynx, larynx, lung, and bladder have been reported in patients previously diagnosed with similar malignancies.22 it has been reported that interindividual variation in dna repair capacity may lead to increased predisposition to cancer.29 abnormal mutagen sensitivity found in studies that indirectly assessed dna repair capacity has been found in patients with head and neck, colorectal, and non - small - cell lung cancers, as well as in patients with multiple primaries of similar malignancies.30 in the present analysis, cancer survivors who were smokers had a higher risk of multiple primaries compared with nonsmokers, particularly for lung, colorectal, and prostate cancers. women whose first primary is the breast account for 25% of survivors with multiple cancers, followed by colorectal cancer (15%) as the first primary in both men and women, according to seer data.2 in the present study, the most common initial primary among cancer survivors with multiple primaries for both men and women was breast, prostate, and colorectal. patients who initially presented with thyroid, urinary bladder, prostate, cervical, and uterine cancers were more liable to develop a second malignancy, while those with hepatic, biliary tract, and pancreatic cancers as well as those with acute leukemia rarely developed a second malignancy. this may be related to the poor outcome in these patients, who did not survive long enough to develop second primaries. patients with multiple primaries usually present initially with tumors at lower stages than secondary primaries.22,3133 in the present study, initial index primaries were predominantly at an earlier stage (78.8%), compared with second primaries (47%). considering all cancer patients, the risk of developing multiple primaries was much higher for those presenting initially with stage 0 or stage i disease. this finding may be also related to the shorter survival time of patients initially presenting with advanced disease. the etiologic role of cancer therapy in the development of second malignancies has been described extensively by others.34 second primaries following chemotherapy usually arise within a few months to 9 years, while after radiation therapy or hormonal treatment, chronic sequelae usually develop after a longer latency period of 510 years.22 however, many believe that a second primary may not be attributed solely to prior treatment. it is most likely a combination of risk factors, such as genetic predisposition, lifestyle, and environmental influence, and could even be a behavioral effect.7,9,22 family history is an important risk factor for multiple primary neoplasms. almost all cancer sites showed an excess of familial clustering, and most of the evidence has been based on excess of close relationship among cases related to families of of 3 to 15 generations deep.35 practically all types of hereditary cancers showed an excess of multiple primaries as well as an early age of onset.22,24,27,28,3133,36 it has been suggested that abnormal genes or gene variants might be responsible for the clustering observed with some tumors. multiple cancer predisposition genes have already been identified in high - risk pedigrees such as brca1, brca2, and p16/cdkn2a.22,35 individuals with hereditary nonpolyposis colorectal cancer with multiple primaries have an increased frequency of mismatch repair genes, such as hmsh2 and hmlh1, leading to microsatellite instability.33 mutagen sensitivity, found in studies that indirectly assessed dna repair capacity, tends to occur in patients with head and neck, hereditary nonpolyposis colorectal, and non - small - cell lung cancers.30 in the present study, a strong family history of cancer reflecting a close relationship between first - degree relatives was statistically more common in patients with multiple primaries, especially those with three or more primaries, and in those with metachronous primaries, highly suggestive of inherited cancer predisposition gene mutations, especially among caucasian patients. survival of patients with multiple primaries varies widely in the literature, depending on the method of calculation, primary tumor site, cancer stage, year of initial cancer diagnosis, and time interval between index and second primaries. overall, patients with multiple primaries have a far better survival rate compared with those having a single primary.37 in the present analysis, a mean survival of 166 months versus 60 months was documented. in a study on oral and maxillofacial cancers at different sites, the mean survival was 127 months for patients with multiple primaries compared with 48 months for cases with a single primary (p=0.001).5 however, when the survival of patients with the same cancer site was compared, calculated from the date of their respective primary diagnosis, there were no statistically significant differences in survival between those who had one primary and those who had more than one primary.5 calculating survival from the date of the second primary has been used in several reports comparing multiple primaries with single ones, with no differences in survival37,38 or an even worse prognosis for patients with multiple primaries.3,4 in the present analysis, using a similar method of calculation, the survival of patients who developed second malignancies was relatively similar to single primary. thus, the improved survival of patients with multiple primaries may be a reflection of different patient populations having a dissimilar pattern of initial tumor sites (predominantly favorable ones) and/or stages of disease (mainly earlier ones), rather than tumor multiplicity. as a rule, the more time that elapses between the first primary malignancy and a second malignancy, the better the prognosis.5 most studies have shown that patients with synchronous primaries have a poor prognosis compared with those having metachronous primaries.5,39 in one study, the 5-year survival rate was 18% compared with 55%, respectively.19 in the present analysis, the 5-year survival of patients with a single primary was 59%, compared with those having simultaneous (61%), synchronous (59%), and metachronous primaries (95%). overall, patients initially presenting with two primaries, either simultaneously or within 6 months of each other, have a similar survival to those with a single primary, and the more time that elapses from the first index primary to the second primary, the better the survival, especially if the interval exceeds 5 years. survival of patients with multiple primaries also depends on the site of the second primary. for breast cancer, the best survival was noted for metachronous contralateral breast cancer when the second cancer was diagnosed at least 5 years after the original primary, with 5-year and 10-year survival rates of 100% and 96%, respectively.37 for lung cancer as a second primary, the 5-year survival for those with a metachronous primary was 44% compared with 10% for those with a synchronous primary.39 with hepatocellular cancer, there were no significant differences in survival, whether presenting as a single primary or as a second primary following another malignancy,38 but not as a synchronous primary concomitantly diagnosed with another cancer, which carries the worst prognosis.40 most reports on patients with multiple primaries include patients at an earlier stage 0 (carcinoma in situ), while others limit the study to invasive cancers (stage i and above).41 inclusion of stage 0 in survival analysis usually results in a favorable survival outcome. in the present analysis, the improved survival of patients with multiple malignancies persists regardless of whether or not stage 0 patients are included. most studies on multiple primaries report on patients seen at certain intervals based on the year of initial cancer diagnosis, while others report on groups of patients seen at a specific period of time, irrespective of the date of initial cancer diagnosis. the latter method has a risk of bias, by including patients with favorable cancers who live long enough to be included in the analysis. the present analysis included all consecutive patients seen during a specific period, irrespective of the date of initial cancer diagnosis. however, analysis of patients initially diagnosed between 2005 and 2012 confirmed the same findings, ie, improved survival for cases with multiple primaries, and especially for metachronous ones. this study, being a retrospective analysis of a limited number of patients, has several limitations. it explores outcomes in a heterogeneous group of patients at different stages of malignancy who have received various types of therapies. the reported incidence of multiple primaries may be underestimated because the duration of follow - up for some patients was relatively short, and a longer follow - up, logically, will reveal development of more second primaries. further, exclusion of patients with skin cancers as well as those with carcinoma in situ of the uterine cervix may also have underestimated the overall incidence of multiple neoplasms. in addition, considering the variation seen in the incidence of multiple primaries between patients of different ethnic background, our findings may not necessarily be applicable to other populations of different ethnic composition. its main strength is the inclusion of all patients seen and followed closely in a single - practice, real - world situation that provides a representative picture of cancer patients in general. it defines the relative incidence and subtypes of multiple primaries among cancer patients of various ethnic groups as well as their impact on survival. it showed relatively consistent results, whether cancer was inclusive of all subtypes or limited to a certain stage of disease, and whether the study population was initially diagnosed over several decades or limited to a defined period of time. in conclusion, patients with multiple primaries are usually of caucasian ancestry, have less aggressive malignancies, present at earlier stages of disease, and frequently have a strong family history of similar malignancies. they tend to have cancers with indolent clinical behavior and longer overall survival, especially in those developing second malignancies more than 5 years after the initial primary diagnosis. for patients with three or more primaries, their survival has been similar to the projected life expectancy of the age - matched and sex matched normal population, even though they were treated less aggressively. the development of multiple primaries may possibly be related to genetic disorders of known or an unidentified nature. the higher frequency of cancer predisposition genes in caucasians may well explain this increased frequency of multiple primaries. the possibility of multiple primary malignancies should always be considered during the treatment and follow - up of cancer patients, especially those of caucasian ancestry and those having a strong family history of cancer. due to the potential for long - term survival, more aggressive treatment may be warranted. for elderly and relatively asymptomatic cases having more than three primaries, a less aggressive approach to therapy may be adequate. | backgroundmultiple primary neoplasms in surviving cancer patients are relatively common, with an increasing incidence. their impact on survival has not been clearly defined.methodsthis was a retrospective review of clinical data for all consecutive patients with histologically confirmed cancer, with emphasis on single versus multiple primary neoplasms. second primaries discovered at the workup of the index (first) primary were termed simultaneous, if discovered within 6 months of the index primary were called synchronous, and if discovered after 6 months were termed metachronous.resultsbetween 2005 and 2012, of 1,873 cancer patients, 322 developed second malignancies ; these included two primaries (n=284), and three or more primaries (n=38). forty - seven patients had synchronous primaries and 275 had metachronous primaries. patients with multiple primaries were predominantly of caucasian ancestry (91.0%), with a tendency to develop thrombosis (20.2%), had a strong family history of similar cancer (22.3%), and usually presented with earlier stage 0 through stage ii disease (78.9%). when compared with 1,551 patients with a single primary, these figures were 8.9%, 15.6%, 18.3%, and 50.9%, respectively (p0.001). five - year survival rates were higher for metachronous cancers (95%) than for synchronous primaries (59%) and single primaries (59%). the worst survival rate was for simultaneous concomitant multiple primaries, being a median of 1.9 years. the best survival was for patients with three or more primaries (median 10.9 years) and was similar to the expected survival for the age - matched and sex - matched general population (p=0.06991).conclusionpatients with multiple primaries are usually of caucasian ancestry, have less aggressive malignancies, present at earlier stages, frequently have a strong family history of similar cancer, and their cancers tend to have indolent clinical behavior with longer survival rates, possibly related to genetic predisposition. |
it is defined as respiratory distress thought to arise from a delay in fetal lung fluid absorption ; it appears within the first six hours after delivery and resolves spontaneously with supportive therapy within a couple of days. the pathophysiology of transient tachypnea of the newborn (ttn) has not been fully explained. potential factors include lack of exposure to the increased effect of catecholamines and other hormones in deliveries, which are initiated before the labor begins and insufficient activity of amiloride - sensitive epithelial sodium channels (enacs) [27 ]. other potential risk factors such as negative amniotic fluid phosphatidylglycerol, apgar score < 7, male gender, and failure to evacuate fetal lung fluid due to insufficient pressure in the thorax related to cesarean section (c / s) delivery have been reported for ttn. it was first used in 700 bc during the roman era in order to remove a baby from the womb of a mother who died during childbirth during advanced stages of pregnancy. and while the safety and optimal conditions for one person are the prime concerns in a regular surgical intervention, the safety of the mother, the fetus, and the newborn should also be ensured in c / s. and it is already a common knowledge that the incidence of ttn increases in c / s, but there is only a limited number of studies covering the effects of whether general or combined epidural - spinal anesthesia (ces) used in elective cesarean deliveries on the incidence of ttn. the aim of the study was to determine whether the type of anesthesia used during cesarean delivery is related to ttn. a total of 1447 c / s were performed at the sema research and training hospital, fatih university, between january 2008 and december 2011. the international classification of diseases, tenth revision code of p22.1, inclusion criteria were the following : a respiratory rate higher than 60 per minute within six hours after delivery, tachypnea lasting for at least 12 hours, increased aeration on chest x - ray, flattening of the ribs, fluid accumulation in the fissures and costophrenic angle, and vascular congestion and exclusion of either known respiratory (meconium aspiration, respiratory distress syndrome, pneumonitis, and congenital cardiac diseases) or nonrespiratory disorders (hypocalcaemia, persistent hypoglycemia, and polycythemia). cesarean cases who were admitted to the newborn intensive care unit with a diagnosis of ttn were classified into two groups based on anesthesia techniques. the first group consisted of cases who received general anesthesia, whereas the second group consisted of cases who received ces anesthesia. ineffective cases, plural pregnancies, preterm pregnancies, and newborns with fetal anomalies and fetal growth restriction were excluded from the study. pregnant women with diabetes mellitus, hypertensive diseases, and asthma bronchioles that could affect acid - base balance were also excluded from the study. three hundred sixty parturients who underwent elective cesarean section under ces anesthesia and were enrolled in this retrospective study received the same anesthesia and surgical care. two hours before the implementation of the ces anesthesia technique, 1000 ml isotonic saline was infused via a venous catheter to all parturients in one hour. all women received aspiration prophylaxis with intravenous ranitidine 50 mg and metoclopramide 10 mg half an hour before the operation. routine monitorization included heart rate, ecg, mean arterial pressure (map), and peripheral oxygen saturation. ces anesthesia was performed in the sitting position according to the general rules of asepsis and antisepsis. spinal anesthesia was performed with a 25 g pencil point spinal needle using hyperbaric bupivacaine 10 mg + fentanyl 10 mg in the sitting position by midline approach. hypotension which had been defined by more than 20% decrease from baseline map was treated with 10 mg dose of intravenous bolus ephedrine. in general anesthesia, propofol of 2 mg / once the patient was intubated, a mixture of 50% no2 and 50% o2 was administered by inhalation. the ncss (number cruncher statistical system), 2007, and pass (power analysis and sample size), 2008 (utah, usa), statistical software were used to analyze the findings of this study for statistical analyses. when analyzing the study data in addition to descriptive statistical methods (average, standard deviation, median, frequency, and ratio), student 's t - test was used in normally distributed parameters, and mann - whitney u test was used in non - normally distributed parameters to compare quantitative data between the two groups. and the pearson 's chi - square test and the yates ' corrected chi - square test (yates continuity correction) were used to compare qualitative data. the results were evaluated in a confidence interval of 95% and at a significance level of p < 0.05. general anesthesia was performed in 1078 (74.5%) of the cesarean cases (group 1), and ces anesthesia was performed in 369 cases (25.5%) (group 2). we examined a total of 85 (5.87%) term infants treated in the nicu for ttn. the maternal ages of the 85 cases being followed up at the nicu for ttn ranged between 20 and 44, and the average age was 32.25 4.39 years in group 1 and 32.18 5.27 in group 2 ; in group 1, 73.7% were male and 26.3% were female, and, in group 2, 67.9% were male and 32.1% were female ; average hospitalization at the nicu was 4.04 2.74 days in group 1 and 3.57 2.51 days in group 2 with no statistically significant differences between both groups. average systolic and diastolic blood pressure was 115.13 18, 76.3 7.9 mmhg in group 1 and 104.09 12, 68.4 8,7 mmhg in group 2 ; the average pulse rate was 78.3 5.1 pulses / min in group 1 and 76.2 7.1 pulses / min in group 2 with no statistically significant differences between both groups (table 1). one - hour postnatal ph value in blood gases was 7.3 0.02 in group 1 and 7.3 0.04 in group 2 ; pco2 value was 49.0 4.3 in group 1 and 47.8 3.7 in group 2 ; po2 value was 56.4 2.0 in group 1 and 57.0 3.6 in group 2 ; apgar scores at 1 minute were 6.1 1.0 for group 1 and 6.5 0.9 for group 2 ; apgar scores at 5 minutes were 8.3 0.7 for group 1 and 8.5 0.8 for group 2 with no statistically significant differences between both groups. the mean time interval from spinal block to skin incision was 8.2 2.93 minutes. the mean time interval from skin incision to umbilical cord clamping was 2.69 1.8 minutes in group 1 and 2.48 1.3 minutes in group 2. no statistically significant differences were found between both groups (p < 0.1) (table 1). general anesthesia was performed in 57 (5.3%), and cse anesthesia was performed in 28 (7.6%) of the ttn cases (table 2). although this rate was found higher in cases who had cse anesthesia, since the number of cases was 3.9 times more in those cases who had general anesthesia, no statistical differences were found between ttn incidence rates by the type of anesthesia used (p < 0.105). in the group receiving spinal anesthesia, 6% of the cases had nausea, 5% vomiting, 4% sedation, 53% trembling, and 7.6% ttn. the normal spontaneous vaginal delivery (nsvd) should always be the primary choice since it is more physiological. however, in special cases where nsvd may not be applicable, the choice of anesthesia depends on the cause of the intervention, its degree of emergency, the patient 's demand, obstetric requirements, and the expertise of the anesthetist. the anesthetist must select a method that is the safest and most comfortable for the mother and that ensures the least depressing working conditions for the newborn and the optimal working conditions for the surgery. general anesthesia offers advantages in situations where there is a race against time including fetal distress, prolapse of cord, placenta previa, or the baby 's arm coming out first because of the fast induction and in situations where regional anesthesia is contraindicated including coagulopathy, infections, and bleeding as well as where the patient who has no other contraindications disapproves regional methods. it may also be considered advantageous due to lower risks of hypotension, better maintenance of cardiovascular stability, and improved control of the airway and ventilation. and the risks of general anesthesia are the pulmonary aspiration of gastric contents and difficulty with intubation [24 ]. regional anesthesia is described as the removal of neural conduction and the pain sensation in particular regions of the body without causing any loss of consciousness. regional anesthesia is more frequently preferred in recent years due to its advantages including the patient 's demand, conscious patient, no risks of aspiration, no respiratory depression in newborns, and no uterine atony. its side effects in the mother include neurotoxicity, hypotension, nausea, vomiting, respiratory depression, urinary retention, delayed gastric emptying, postdural headache, total spinal block, anterior spinal artery syndrome, back and belly aches, and hypothermia. and fetal side effects develop depending on the systemic absorption of epidural opioids or their side effects on the mother. ces anesthesia may reduce some of the disadvantages of spinal and epidural anesthesia while maintaining its advantages. ces anesthesia ensures that the period of anesthesia provided by epidural anesthesia can be extended by the rapid onset, efficiency, and minimal toxic effect produced by the spinal block. the most important cause of fetal distress in both anesthetic methods is the reduction in the amount of o2 available to the fetus as a result of the reduction of uteroplacental blood flow. when the induction - delivery interval exceeds 10 minutes in general anesthesia, fetal tissues are saturated with no2. as a result, a mild depression and, if oxygenation is not sufficient, diffusion hypoxia can occur in the newborns during the first minutes. about 80% of the cesarean section deliveries were performed by the same gynecologist at our hospital. and this resulted in standardized induction - delivery intervals. bupivacaine is a commonly used agent during deliveries as it has a long - lasting effect, it is more markedly selective over sensory neural fibers than to motor fibers, and it has a lower fetal - maternal blood ratio. when continuously infused epidurally, epidural opioids rarely cause respiratory depression. there are no significant differences between the apgar score and sensory behavioral scores and the scores of the babies of mothers who took no medications. this is generally the temporary and may be associated with hypotension. at our hospital, 1000 ml physiological saline was infused within one hour preoperatively to all cases who received ces anesthesia in order to make sure no hypotension occurs in the mother. the use of 10 mg bupivacaine + 10 mg fentanyl was administered in all cases at our hospital during ces anesthesia. the performance of all spinal anesthesia procedures by the same anesthesia provider ensured a standardized mean time interval from spinal block to skin incision. this is because some studies in the literature reported that reducing the time interval from spinal block to the onset of surgery might be one of the important measures to decrease the incidence of ttn after elective cesarean section under spinal anesthesia. it is already a common knowledge that cesarean surgeries are an important risk factor in the etiology of ttn. in our study, we planned to investigate the relationship between the type of anesthesia during the surgery and ttn independent from cesarean application. although our study found higher rates in cases who received ces anesthesia, since the number of cases was 3.9 times more in those cases who had general anesthesia, no statistical differences were found between ttn incidence rates by the type of anesthesia used. in conclusion, the incidence of ttn was found related to c / s but independent from the type of anesthesia. however, studies with a wider spectrum of patients and a lower quantitative difference between the groups are needed in order to be able to underline this conclusion. | aim. to demonstrate whether transient tachypnea of the newborn (ttn) is found more frequently in women undergoing general or combined epidural - spinal (ces) anesthesia during cesarean section. methods. this study was done retrospectively. a total of 1447 cesarean sections (c / s) were performed in our clinic between january 2008 and december 2011. general anesthesia was performed in 1078 (74.5%) of the cesarean cases. ces anesthesia was performed in 369 cases (25.5%). the international classification of diseases, tenth revision code of p22.1, was used to identify the infants with ttn. stratified multivariate analysis was undertaken on subgroups to assess the effect modification by factors known to influence the incidence of ttn : maternal age, maternal systolic - diastolic artery pressure, heart rate, apgar score at 1 and 5 minutes, sex, time interval from spinal block to skin incision, and time interval from skin incision to umbilical cord clamping. results. the rate of ttn diagnosis was found to be higher in parturients who had a cesarean section with combined epidural - spinal anesthesia, but no statistical differences were found. (p < 0.05) (odds ratio = 1.471 and 95%ci : 0.922.35). conclusions. the incidence of ttn was found related to c / s but independent from the type of anesthesia. however, studies with a wider spectrum of patients and a lower quantitative difference between the groups are needed in order to draw firm this conclusions. |
peri - implant disease may be defined as a pathologic condition including inflammatory and other kinds of lesions affecting the soft and/or hard tissues surrounding a dental implant. peri - implantitis is characterized by a severe inflammatory process involving both mucosa and bone around the implant. bone destruction, peri - implant pockets, bleeding on probing, the possible presence of exudate, and loss of supporting tissue are involved in peri - implantitis. peri - implantitis is due to bacterial contamination or technical problems, related to the implant surface itself or to implant support placement and the subsequent osseointegration process. osseointegration may be influenced by mistakes or complications occurring in the surgical phase or masticatory overload. the bacterial biofilm on the implant surfaces is similar to the one in periodontal disease. the microflora includes microorganisms such as aggregatibacter actinomycetemcomitans, peptostreptococcus micros, campylobacter rectus, capnocytophaga spp. however, it should be stressed that the residual teeth could influence the composition of microflora. bacterial species observed in edentulous patients differ from those of partially edentulous subjects. on this basis, the idea that the presence of bacteria involved in periodontal disease could contribute to development of peri - implantitis seems to be plausible. during the surgical stage, the treatment in the initial stage included elimination and of plaque and calculus, decontamination of the implant surface, and maintenance of healthy conditions. cleaning the surfaces can be through mechanical (dental curettes, ultrasonic scalers, and air - powder abrasive) and chemical (citric acid, h2o2, chlorhexidine digluconate, and edta) procedures, in association with local or systemic antibiotics [8, 9 ]. the most frequently used include diode, erbium lasers, and co2 due to their hemostatic properties, selective calculus ablation, and bactericidal effects. an alternative approach to dental implant decontamination is the association of the conventional treatment with photodynamic therapy (pdt). photodynamic therapy includes the use of a low - power diode laser in combination with photosensitizing compounds. these components are linked to the bacterial membrane and, when excited, react with the substrate. the photosensitizer binds to the target cells and when it is irradiated with light of specific wavelength, in the presence of oxygen, it undergoes a transition from a low - energy ground state to an excited singlet state ; then singlet oxygen and other very reactive agents are produced, which are toxic to these target cells. the application of photosensitive dyes into pockets and their activation with light promote killing of periodontal pathogens. outcomes of clinical studies in subjects with chronic periodontitis show beneficial effects of pdt on the reduction in gingival inflammation. the effects of pdt on the treatment of ligature - induced peri - implantitis were investigated in dogs. the results revealed a reduction in bacterial counts of prevotella intermedia / nigrescens, fusobacterium spp., and beta - haemolytic streptococcus. several studies have demonstrated bacteria destruction can be achieved without any damage to the treated titanium surfaces. the aim of this experimental study is to demonstrate the efficacy of antimicrobial photodynamic therapy in addition to the traditional approach. 40 subjects were involved in the study ranging in age from 34 to 68 years, referred to the periodontology department of the dentistry unit at bari university hospital. the study was conducted following the declaration of helsinki, according to the local ethical committee. the patients were selected with these inclusion criteria : overall plaque index (pli) 40% and at least one implant site with the following characteristics : probing depth (pd) 4 mm, bleeding on probing (bop), and presence of suppuration. exclusion criteria included decompensated systemic disease, degenerative bone disease, chronic immune - based mucomembranous disorders (e.g., lichen planus, pemphigoid, pemphigus, and systemic lupus erythematosus), chemotherapy or radiotherapy to the head and neck area, pregnancy, presence of teeth with periodontitis adjacent to sites affected by peri - implantitis, implants placed in fresh extraction sockets, smoking > 10 cigarettes daily, and alcoholism. the null hypothesis was that nonstatistically significant differences are observed with respect to the clinical parameters (e.g., ppd, bop, and pli) between the two treatment modalities (i.e., adjunctive pdt test group versus control group). the primary outcome variable was the reduction of pd in peri - implant sites with probing depth 4 mm. secondary outcome variables were the changes in bop and pli. the ratio of this study was based on the capacity of photodynamic therapy to promote bacterial inactivation by light and not by heat. 360 light irradiation is obtained by means of special probes ensuring optimal light beam diffusion. the patients were randomly assigned to two groups, that is, a test group (63 implants) and a control group (59 implants), using a software to create a randomization list (https://www.random.org/) and assigning a code to each patient. for both groups of patients the following indices were measured by means of a plastic probe : the plaque index (pli), based on the plaque control record (pcr,), bleeding on probing (bop) with or without suppuration, and probing depth (pd). mechanical and manual decontamination of the oral cavity was performed using air polishing with micronized glycine powder to remove plaque and discolorations and expose the underlying calculus (figure 1). the latter was removed with a piezoelectric ablator in combination with a universal tip for the scaling of natural teeth and a special nonmetal tip for implant scaling. root debridement at sites with pd 4 mm was performed with a periodontal ultrasonic unit and implant debridement at sites with pd 4 mm was done with carbon - fiber - reinforced plastic curettes. at the end of the procedure, according to the code of the envelope, the dental hygienist considered in the test group the addition of laser - assisted antimicrobial photodynamic therapy based on the helbo protocol at implant sites with pd 4 mm. the treatment of pdt was performed using helbo theralite (bredent medical), diode laser battery powered with a wavelength of 670 nm and output of 75 mw / cm, with a spot size of 0.06 cm in diameter. helbo blue photosensitizer was used, a liquid containing methylene blue (methylthioninium hydrochloride, also known as 3,7-bis phenothiazine-5-ium chloride). the concentration of photosensitizer was 10 mg / ml with absorbance peak at 670 nm. its use as a chromophore in photodynamic therapy is justified by its relative stability in the light, which makes it an important generator of singlet oxygen (et = 142.1 kj / mol with = 0.60 in water). the photosensitizer was applied inside the peri - implant pocket starting from the bottom and moving in apical - coronal direction (figure 2). care was taken to avoid the formation of air bubbles, allowing the fluid to dye all bacteria by leaving it in situ for 60 seconds. after rinsing the fluid off the pocket and suctioning excess liquid (figure 3), the previously dyed implant surface was exposed to helbo theralite diode laser for 1 minute (figure 4). the fluence was 25.54 j / cm, while the total energy applied was 1592 j / cm. this type of movement promotes the best activation of the dye molecules with the laser light and transfers their energy to local oxygen. they were advised to brush their teeth for two minutes, twice a day, using an oscillating - rotating electric toothbrush with little toothpaste and a special brush for interproximal hygiene. the same clinical measurements were taken as those at baseline and home oral hygiene instruction was provided again. the same clinical measurements were taken as those at baseline and home oral hygiene instruction was provided again. t3, end of the study (24 weeks). the same clinical measurements were taken as those at baseline (figure 5). a weighted arithmetic mean was taken to calculate average values for each group in terms of pd, bop, and pli at 6, 12, and 24 weeks using a computer software (graph pad prism 5). as early as at the 6th week of the study, reductions in clinical parameters were observed in both groups compared with baseline values. the reduction was first seen as early as at 6 weeks, to be confirmed at 12 weeks, when the values further declined. test group showed a better value of pd, with an average value of 2 mm if compared with control group (3 mm). with regard to the plaque index, average value was calculated for each group. in this case, a significant score reduction was recorded as early as at the 6th week. despite improving of daily oral hygiene practices, regarding bop, at baseline, all patients had bleeding on probing and suppuration at the peri - implant sites under investigation. in the test group patients, these signs of inflammation had gradually improved to disappear completely by the 24th week. in the control group, however, some improvements were recorded, but not all of the patients achieved complete remission (table 3). peri - implantitis has been defined as an inflammatory process that affects the soft tissues surrounding an osseointegrated implant in function with concomitant loss of supporting marginal bone. peri - implant mucositis, in contrast, is a reversible inflammatory reaction of the mucosa adjacent to an implant without bone loss. colonization of oral implant surfaces with bacterial biofilms occurs rapidly and the biofilm development seems to play an important role in altering the biocompatibility of the implant surface and, thus, enhancing peri - implant disease development. since photodynamic therapy has been introduced in dentistry, several advantages of laser and pdt in the many fields of dentistry have been described in the literature. an increasing interest is recently growing regarding pdt in implant dentistry and as a coadjuvant treatment for peri - implantitis. it employs visible light (laser) and a dye (photosensitizer), the combination of which leads to the release of free oxygen radicals, which in turn can selectively destroy bacteria and their products. although pdt has been used in the field of medicine since 1904 for light - induced inactivation of cells, microorganisms, and molecules, branemark 's discovery of osseointegration in 1965 was extremely important to restorative treatments and, particularly, functional oral rehabilitation. a large number of patients have been rehabilitated with dental implants, and, consequently, more cases of success and failure have appeared over the years. it is well known that laser has the ability to modify dentin so as to obtain the exposition of collagen fibers. this, in turn, may favor a speedy healing and the obtainment of a new collagen attachment in spite of long junctional epithelium. this fact could explain the faster and greater healing of the wound and the results in the test group. it is clear that further histological analysis should be carried out to demonstrate this idea. thus, photodynamic therapy (pdt) may be one such treatment alternative. only in the last 10 years or the current data show that treating chronic periodontitis with pdt alone versus conventional srp treatment has no additional benefit. in contrast, combining pdt and srp does provide an additional benefit, particularly in lesions with unfavorable anatomic conditions. a clinical controlled study compared the effect of pdt alone (without subgingival srp) with srp in the treatment of aggressive periodontitis [20, 21 ]. in addition to this, during peri - implantitis treatment, helbo technology offers the advantage of a noninvasive, painful, rapid bacterial inactivation thanks to liberation of oxygen. oxygen allows the destruction of bacteria membrane, and on the other hand its sparkling effect permits dangerous enzymes and collagenosis to be quickly removed from the pocket, for a better bacterial removal and, as a consequence, could facilitate healing. the improvement of values analyzed was more marked in the test group (table 1). test group showed a better value of pd, with an average value of 2 mm if compared with control group (3 mm). regarding pli the significant reduction recorded at the 6th week was followed by a slight increase at 12 weeks, with values remaining constant up to the 24th week. however, the plaque index score for each patient at 24 weeks was anyway lower than at baseline. finally, a comparison between baseline and final average bleeding on probing (bop) and suppuration values also shows substantial improvement. thus, the results obtained in this study suggest that photodynamic therapy could be considered an effective method for bacterial reduction on implant surfaces [1722 ]. our study also confirms its effectiveness in reducing clinical indices and the bacterial load at sites affected by peri - implantitis, with significant bacterial detoxification being achieved. photodynamic therapy should, however, be considered a coadjuvant in the treatment of peri - implantitis and associated with mechanical (scaling) and surgical (grafts) treatments in order to control peri - implant disease. | introduction. the aim of this study is to demonstrate the effectiveness of addition of the antimicrobial photodynamic therapy to the conventional approach in the treatment of peri - implantitis. materials and methods. forty patients were randomly assigned to test or control groups. patients were assessed at baseline and at six (t1), twelve (t2), and twenty - four (t3) weeks recording plaque index (pli), probing pocket depth (ppd), and bleeding on probing (bop) ; control group received conventional periodontal therapy, while test group received photodynamic therapy in addition to it. result. test group showed a 70% reduction in the plaque index values and a 60% reduction in pd values compared to the baseline. bop and suppuration were not detectable. control group showed a significative reduction in plaque index and pd. discussion. laser therapy has some advantages in comparison to traditional therapy, with faster and greater healing of the wound. conclusion. test group showed after 24 weeks a better value in terms of ppd, bop, and pli, with an average pocket depth value of 2 mm, if compared with control group (3 mm). our results suggest that antimicrobial photodynamic therapy with diode laser and phenothiazine chloride represents a reliable adjunctive treatment to conventional therapy. photodynamic therapy should, however, be considered a coadjuvant in the treatment of peri - implantitis associated with mechanical (scaling) and surgical (grafts) treatments. |
a 39-year - old man was transferred to seoul national university hospital with complaints of cough, chest discomfort, and a weight loss of 12 kg in a 3-month period. initial laboratory findings before referral revealed that the white blood cell (wbc) and eosinophil counts were 48,000/l and 14,880/l (31% of the wbc count), respectively. the possible etiologies of eosinophilia were excluded on the basis of the patient s history and laboratory findings, such as liver enzyme, serum electrolytes, parasite - specific immunoglobulin g, and toxocariasis antibodies. the patient was diagnosed with the hypereosinophilic syndrome (he s), and methyl prednisolone (mpd) therapy with a dose of 2 mg / kg / day was started. an echocardiographic evaluation was also performed due to increased cardiac enzyme levels (creatine kinase 138 iu / l, creatine kinase - mb 23.63 ng / ml, and troponin i 9.919 ng / ml). massive thrombus filling the left ventricular cavity with hypermobile portion and mitral regurgitation of a moderate degree were found (fig. the mpd dose was increased to 500 mg / day due to the increasing eosinophil counts. bone marrow biopsy and gene assay including bcr - abl, fip1l1/pdgfra1, and pdgfrb/ 5q32 were also performed. after confirming the pdgfrb/ 5q32 gene re - arrangement, a combination therapy of hydroxyurea and imatinib (glivec, basel, switzerland) was added to the treatment protocol. the patient was then transferred to our hospital with a recommendation of urgent surgical left ventricular thrombectomy. the initial laboratory test after referral demonstrated elevated levels of wbc and eosinophils with absolute counts of 85,600/l and 4,280/l (5% of the wbc count), respectively. follow - up echocardiography conducted 5 and 8 days after the initial evaluation showed a progressively decreasing left ventricular mass and the disappearance of the mobile mass at the inferobasal area (fig. fourteen days after the initial diagnosis (5 days after referral), acute right leg pain with a diminished right dorsalis pedis pulse developed. computed tomography angiogram (cta) of the lower extremities revealed arterial occlusions at multiple levels including the infrarenal abdominal aorta, right common iliac artery, left iliac bifurcation, and right popliteal artery (fig. an emergency operation was performed to resolve the thromboembolic complications. to prevent recurrent embolic events, first, thromboembolectomy of the infrarenal abdominal aorta and bilateral iliac and right popliteal arteries was performed via femoral incisions by using a fogarty catheter. after the completion of this thromboembolectomy, left ventricular thrombectomy followed via a median sternotomy and under aor - to - bicaval cannulation with cold cardioplegic arrest. further, the left ventricular thrombus was more solid than the usual fresh thrombus, and the margin between the thrombus and the endocardium was unclear. all visible thrombus was removed with care so as to not injure the endocardium and the myocardium (fig. although there was a moderate degree of mitral regurgitation at the preoperative echocardiography, intraoperative findings after thrombectomy revealed an intact mitral annulus without abnormal thickening or elongation of the subvalvular apparatus. the saline test also demonstrated only a minimal leakage. due to these findings and the concern of a recurrent thrombosis around the prosthetic mitral ring, the mitral valve procedure was not performed. cardiopulmonary bypass and aortic cross - clamp times were 68 and 32 minutes, respectively. postoperative cta performed at postoperative day 2 and echocardiography at postoperative day 15 revealed the disappearance of the abdominal and iliac thromboembolism, and left ventricular thrombus, respectively (fig. mitral regurgitation was also improved to less than a mild degree. although we performed a pathologic evaluation of the removed thrombus, there was no definite finding of eosinophil infiltration since we tried to avoid any endomyocardial resection. the patient was discharged at 18 days after the surgery with the wbc and eosinophil counts of 7,280/l and 349/l (4.8% of the wbc count), respectively. he has undergone follow - up at an outpatient clinic for 2 months without any evidence of disease relapse and recurrence of thromboembolism. further medical management plans included oral anticoagulation for 6 months after the surgery and adjustment of the immunosuppressant by tapering off methyl prednisolone and continuing the imatinib medication. he s was defined as a persistent, unexplained elevation of peripheral blood eosinophils (1,500/l) lasting at least 6 months with eosinophil - related end - organ damage. however, in 2010, simon. suggested that a 6-month duration is not necessary if unexplained eosinophilia is documented on more than one occasion. the revised definition of he s is as follows : 1) blood eosinophilia (1,500/l) on at least two occasions or evidence of prominent tissue eosinophilia associated with symptoms and marked blood eosinophilia ; 2) exclusion of secondary causes of eosinophilia, such as parasitic or viral infection, allergic diseases, drug - induced or chemical - induced eosinophilia, hypoadrenalism, and neoplasms. recently, he s has been considered a myeloproliferative disorder, and an evaluation of the genetic mutation is helpful in selecting a treatment regimen. cardiac involvement of the he s occurs in more than 50% of the patients with various symptoms, such as dyspnea, chest pain, cardiac murmur, and congestive heart failure. cardiac involvement of he s is classified in three stages : first is the acute necrotic stage, which is characterized by myocardial necrosis with eosinophilic infiltration into the myocardium. second is the thrombotic stage, which is triggered by the damaged endocardial surface in any cardiac chamber. the last is the fibrotic stage, which occurs with the replacement of thrombus by endomyocardial fibrosis. as a result when the pdgfra or various pdgfrb fusion genes are identified, imatinib shows good long - term efficacy with a low incidence of grade iii / iv toxicity. in addition, myeloid neoplasms associated with the rearrangement of pdgfrb are responsive to imatinib. therefore, in the present case, the patient harbored pdgfrb/5q32 gene re - arrangement and imatinib was included in the treatment regimen. most reported cases of he s with cardiac involvement underwent cardiac surgery due to restrictive cardiomyopathy and mitral regurgitation at the chronic stage. in addition, although previous reports presented he s patients with the left ventricular thrombus, data preferring medical or surgical treatment are lacking. the patient in the present study suffered from non - specific symptoms, such as cough, weight loss, chest discomfort, and dyspnea. echocardiography was performed due to increased serum levels of creatine kinase - mb and troponin i. despite the diagnosis of the left ventricular thrombus and referral for urgent surgery, medical management was the initial choice rather than surgical correction. the reasons for this choice were the concerns of recurrent thrombosis when performing thrombectomy before the control of the disease activity and the echocardiographic findings of the decreasing mass without any signs or symptoms of systemic embolism. however, the result was a massive thromboembolism occluding the abdominal aorta and the lower extremity arteries. concomitant surgeries including the thromboembolectomy of the low extremities and left ventricular thrombectomy were performed on an emergency basis. intra - abdominal and low - extremity arterial thrombi were effectively removed using a transfemoral approach without residual thrombus, because the thrombus was solid in nature, in contrast to the usual fresh thrombus. in addition, although the margin between the left ventricular thrombus and the endocardium was difficult to identify, perhaps due to the inflammatory involvement of the endocardium, left ventricular thrombectomy was successfully performed without residual thrombus on follow - up echocardiography. the postoperative course was uneventful without any signs and symptoms of recurrent left ventricular thrombus and systemic embolization. although there has been no guideline for anticoagulation in such patients, we planned to continue oral anticoagulation until 6 months after the initial treatment, which might be sufficiently long for the stabilization of the disease activity. | a 39-year - old man presented with cough, chest discomfort, and weight loss. on the basis of the patient history and laboratory findings, he was diagnosed with the hypereosinophilic syndrome. transthoracic echocardiography revealed a large thrombus in the left ventricle. medical treatment with anticoagulation and immunosuppression was commenced immediately. fourteen days after the initial diagnosis, the patient presented with acute pain in his right leg. computed tomographic angiogram showed embolic occlusion of the infrarenal abdominal aorta and bilateral iliac (including common, external, and internal iliac) arteries. emergent thromboembolectomy and left ventricular thrombectomy were performed. the postoperative course was uneventful, and the patient has undergone follow - up for 2 months without any evidence of recurrence of thromboembolism. |
fibrodysplasia ossificans progressiva (fop) is a rare autosomal dominant genetic disorder and characterized by postnatal progressive heterotopic ossification of the connective tissue. there are difficulties in diagnosing fop, thus delayed or misdiagnosis and mismanagement is common. 3d printers have now become widely available and inexpensive, and can be used to rapidly produce life - size models based on ct scans of an individual patient. the availability of patient specific, accurate and detailed models of complex acetabular fractures can aid planning of surgical management on a patient specific basis. we present the diagnosis and surgical management of a 9-year old indonesian girl with fop. total excision of occipito - cervico - lumbar and paravertebral ossification and also exostoses at bilateral shoulder was done. at three years follow up, she had local recurrence with similar range of movement of the shoulder and cervical spine. it is difficult to diagnose and manage fop, therefore delayed or misdiagnosis and also inappropriate management is common. fibrodysplasia ossificans progressiva (fop), once called myositis ossificans progressiva, is a rare autosomal disorder that involves connective tissue. one of the famous case of fop is harry eastlack (1933 - 1973), who died because of pneumonia and fully ossified body, his lips were the only body part that could be used properly. ectopic ossification is preceded by a soft tissue enlargement that may spontaneously resolve or gradually form an ectopic bone. delayed or missed we report a rare case of fop arising at occipito - cervical spine, paravertebrae, and bilateral shoulders focusing on diagnostic procedure and surgical management. a 9-year - old girl presented to our orthopaedic clinic, with main complaint of a mass which was getting bigger on her upper back. the initial mass size was around 1 cm in diameter (like a marble) located just below the tip of both scapulae. it was accompanied by difficulty in moving both shoulders and neck since 4 years. because of these complains, her family tried various traditional medicines without improvement. six months prior to admission, she fell down on her back, and since then the masses grew progressively. she was brought to a district hospital in other province and then referred to our hospital to get more appropriate treatment. there was no history of similar disease in her family. on physical examination, general condition was good and no abnormality was found in other organ. we found multiple bony prominences measuring about 2 cm in diameter at paravertebral region from cervical to lumbar and posterior thoracic wall. they made bone bridges starting from inferior occipital bone to cervical spine and extending to paravertebral lumbar region. the shoulder, cervical, and thoracal spine ranges of movement were limited (fig 1). there were multiple bony prominence measuring 2 cm in diameter at the back from cervical to lumbar and posterior thoracic region (arrows), they made bony bridges from inferior occipital bone, cervical spine, extending to paravertebral lumbar region. (b) slight valgus deformity of right great toe (arrow) radiograph of the cervical spine showed new bone formation starting from occipital bone, cervical, until upper thoracal spine. chest and shoulder radiographs also showed new bone formation at both humerus bridging to the tip of scapulae, with subluxation of both humeral head superiorly. the radiologist saw it as mustiple exostoses (osteochondroma lesion) with the differential diagnosis progressive myositis ossificans. hla - b27 was negative, it was investigated since we also thought ankylosing spondylitis as a differential diagnosis. the 3d computerized tomography (ct) reconstruction revealed multifocal ossification on soft tissue including right pectoral muscle, bilateral latissimus dorsi, and left longisimus thoracis muscle. magnetic resonance imaging (mri) showed similar result with ct scan and no other abnormality detected at the spine. radiograph of the cervical spine lateral view showed bone formation starting from occiput, cervical, and upper thoracic spine (arrows). (b) chest and shoulder radiographs also showed bone formation at the bilateral humerus bridging to the tip of scapula (arrows). (c) ct reconstruction revealed multifocal ossification which supported radiographic finding, paravertebral bony bridge (arrow head), bony bridges from bilateral humerus to tip of scapula (arrows) because of the problem was multiple exostoses and stiffness, total excision of occipito - cervico - lumbar and paravertebral ossification and also exostoses at bilateral shoulder was done. histopathological findings showed tissue consisting of cartilaginous component, irregular underlying bony trabeculae, and bone marrow between trabeculae. after review of all patient s data in one of the clinicopathological conference, we concluded that it was fibrodysplasia ossificans progressiva (fop). four months after surgery local recurrences were detected, which was confirmed with radiograph (fig 4). at three years follow up, she had similar signs and symptoms as before surgery. (a)histopathology showed formation of endochondral ossification (arrow) (h&e, 100x). (b) skeletal muscle and formation of endochondral ossification (arrow) (h&e, 100x). (c) formation of endochondral ossification (arrow head), bone trabeculae and marrow (arrow) (h&e, 100x). (a) clinical picture 4 months after surgery, local recurrences were detected (arrow). (b) cervical radiograph 4 months after surgery, there was local recurrence of bony bridge from occiput, cervical, and upper thoracic spine (arrow). (c) thoracic radiograph 4 months after surgery, there was recurrence of bony bridge from bilateral humerus to the tip of scapula (arrow head). fop is an autosomal dominant disorder and has a variable expression that is the most catastrophic disorder of heterotopic ossification in humans. primary genetic defects in fop are currently unknown. events which may precipitate formation of lesion are trauma, invasive medical procedure, muscle fatigue, influenza like infection, and immunization. however, it is still unclear how clinical events which initiate the formation of lesion ultimately lead to extraskeletal ossification. a study in japan involving 19 fop subjects has successfully identified gene mutation that could induce ectopic bone formation in fop. fop lesion is usually triggered by soft tissue injury, predominantly in muscle, causing release of inflammatory cytokines and migratory factors. potential soluble mediators include prostaglandins (pgs), smad1 and smad5, stromal cell derived factor 1, and bmp-4. pge2 has an anabolic effect on bone marrow stromal cells and may enhance osteoblastic differentiation in - vitro, presumably through the recruitment of nonadherent mesenchymal progenitor cells into adherent osteoblast precursors. elevated levels of a low - molecular - weight pg - like molecule have been detected in the plasma of patients with fop. stromal cell derived factor 1 is a mediator of lesion formation in a mouse model of pulmonary fibrosis and may signal circulating fibrocytes to homing regions that eventually become fibrotic. heterotopic bone formation may occur in pulmonary fibrosis and the circulating fibrocytes are abundant in patient with fop with active exacerbations. taken together, these observations suggest a plausible mechanism for extraskeletal ossification in fop that invokes the recruitment of circulating osteogenic cells. whereas bmp-4 may induce ectopic ossification in animal models, it is known to be dysregulated in fop. children who have fop appear normal at birth except for congenital malformations of the great toes. typically, during the first decade of life, sporadic episodes of painful soft tissue swellings (flare - ups) occur and are commonly mistaken for tumors. however, in our patient, no malformations of the great toes were clearly detected except slight valgus of right great toe. the ossification started from occipital bone and posterior paravertebral soft tissue, which caused limitation of neck movement, whereas ossification on both humerus and scapula caused limitation of the shoulder joint movement. eventually, the ossification process forms bridges between axial bones. most of literature stated that ectopic ossification usually started from the cervical area, then extended down paravertebrally to lower back and progressively formed bony projections in our case were similar to multiple exostoses, whereas the location of lesions was uncommon for that kind of tumor. based on our clinical diagnosis, we performed excision of all multiple exostoses which were located from occipital bone until lumbar spine and the bridging bones between scapula and both bilaterally. histopathology showed fragmented tissue consisting of cartilaginous component and irregular underlying bony trabeculae (endochondral ossification). there was also bone marrow between trabeculae, a histopathologic feature commonly found in exostosis. at discussion in our routine fop, we re - evaluated the slides and processed the whole specimen. microscopically the specimen also showed fibrous tissue, skeletal muscle mixed with endochondral ossification, and foci of calcification. although histologically, a very early fop lesions look identical to the fibroproliferative lesions of aggressive juvenile fibromatosis, in aggressive juvenile fibromatosis, the lesions do not progress beyond the connective tissue growth phase, whereas in fop, they mature through an endochondral process to form cartilage and bone one study mentioned that nearly 90% of patients with fop worldwide are misdiagnosed and 67% undergo dangerous and unnecessary diagnostic procedures. tumor, fibromatosis, bunion, myositis ossificans, calcified hematoma, exostoses, and also ankylosing spondylitis are considered in the differential diagnosis. surgical therapy which aims to improve range of movement in this patient has proven to trigger further ossification. in this patient, follow up, she presented with the same complaint and similar range of movement of shoulder and cervical spine as before surgery. radiation therapy may be considered after surgery to prevent recurrence, but there is no clear evidence that radiotherapy is effective in patients with fop, except in small number of patients. medications that have theoretical application to fop such as leukotriene inhibitor and investigational new drugs such as signal transduction inhibitor and monoclonal targeting acvr1 may show a promising result in fop patient. research in fop has reached genetic level, but not yet in molecular level that it might produce a gene therapy. so, we have to make early clinical diagnosis and the mainstay of therapy is education to the patient and family to prevent further trauma. it is difficult to diagnose and manage fop, therefore delayed or misdiagnosis and also inappropriate management is common. although rare, we should consider fop as a differential diagnosis in patient complaining of multiple masses with soft tissue ossification. we should avoid dangerous and unnecessary diagnostic procedure as well as inappropriate management which may lead to futher exacerbations. | introduction : fibrodysplasia ossificans progressiva (fop) is a rare autosomal dominant genetic disorder and characterized by postnatal progressive heterotopic ossification of the connective tissue. there are difficulties in diagnosing fop, thus delayed or misdiagnosis and mismanagement is common.3d printers have now become widely available and inexpensive, and can be used to rapidly produce life - size models based on ct scans of an individual patient. the availability of patient specific, accurate and detailed models of complex acetabular fractures can aid planning of surgical management on a patient specific basis.case report : we present the diagnosis and surgical management of a 9-year old indonesian girl with fop. she presented with extensive involvement of cervical spine and both shoulders. total excision of occipito - cervico - lumbar and paravertebral ossification and also exostoses at bilateral shoulder was done. at three years follow up, she had local recurrence with similar range of movement of the shoulder and cervical spine.conclusion:fop is an extremely rare case. it is difficult to diagnose and manage fop, therefore delayed or misdiagnosis and also inappropriate management is common. |
it has a great developing technique that is used for fixation and fusion in spine surgery. it was firstly introduced by harrington and tullos in 1969 and then in late 1980s developed by roy camille., louis, and steffe. it could be applied in degenerative, trauma, neoplastic, infectious and malformation cases that had a problem with axial instability. despite its usefullness, nerve root, spinal cord injury, vascular injury, cerebrospinal fluid leak, visceral injury, pedicle fracture were some complications that mostly related to pedicle screw malpositioning. among those complications, the nerve injury due to pedicle screw malpostioning was a common complication that was faced by spine surgeons,. there were a various neurophysiologic techniques used to assess functional integrity of the nervous system during surgical procedures. it was useful by providing real time evaluation and immediate feedback to the surgeon at a point where intervention with any doubt taken. this real time feedback would be a guide for surgeon to determine the precise decision in preventing irreversible neural damage,. once warning signal was recognized by ionm, surgeon had to make a precise decision to overcome that problem. establishing the cause, mechanism and the pathology of injury associated with nerve injury during spinal surgery were mandatory tools to surgeon as consideration to make an appropriate decision. in this review, a case of nerve entrapment problem after screw insertion on thoracolumbar compression fracture that recognized by intraoperative neurophysiologic monitoring at ciptomangunkusumo hospital was presented and correct management was discussed. a 47 year old male fell from his motorcycle following a traffic accident eleven years ago during which he landed on the asphalt with his buttocks. seven month before hospital admission, patient had the pain worse (vas 45), but he could still move his both leg. mri examination showed a compression fracture at the level vertebrae thoracal t12 and lumbar l1. it showed also there was small fragment of bone come into the canal and compressed the anterior segment of spinal cord (fig. laboratory examination showed there is an increase in parameter of erythrocyte sedimentation rate (52 mm / h), c - reactive protein (50 mg / l) and leucocyte count (12.200/l). surgery was performed with general anesthesia and under continuous neurophysiology monitoring of mep (motor evoked potentials), ssep (somatosensory evoked potentials) and free - running emg (electromyography). ssep of bilateral tibial nerves was performed with the stimulation at the ankle and scalp recording at cz - fz and c3-c4/c4-c3. mep was performed with stimulatioan at m3-m4 or m1-m2 (best response) with single train stimulation of 7 pulses with 200.000 hz frequency, 0.5 ms duration and 0100 ma intensity and recorded at abductor hallucis brevis muscle and tibialis anterior bilateral muscle. free run emg was recorded with the settting of 50 uv / division gain and 100 ms / division sweep at rectus femoris muscle and tibialis anterior muscle bilateral to prevent radix iritation during instrumentation. it was performed the facet joint release and posterior stabilization at the level vertebrae thoracal t11, t12 and lumbar l2, l3. intra - operative accident was happened when the operator inserted the pedicle screw at level left thoracal t12. the surgeon then decided to pullout the pedicle screw and change the location of pedicle insertion to one level above, vertebrae thoracal t11 (fig. after changing the screw position, ssep monitor showed that the nerve amplitude was corrected to the acceptable value (fig. final screw insertion was at t10, t11 and l2, l3 on both side. post - operative evaluation showed that the pain was decreased (vas 12) and there was no deficit in motoric and sensoric function (fig. a 47 year old male fell from his motorcycle following a traffic accident eleven years ago during which he landed on the asphalt with his buttocks. seven month before hospital admission, patient had the pain worse (vas 45), but he could still move his both leg. mri examination showed a compression fracture at the level vertebrae thoracal t12 and lumbar l1. it showed also there was small fragment of bone come into the canal and compressed the anterior segment of spinal cord (fig. laboratory examination showed there is an increase in parameter of erythrocyte sedimentation rate (52 mm / h), c - reactive protein (50 mg / l) and leucocyte count (12.200/l). surgery was performed with general anesthesia and under continuous neurophysiology monitoring of mep (motor evoked potentials), ssep (somatosensory evoked potentials) and free - running emg (electromyography). ssep of bilateral tibial nerves was performed with the stimulation at the ankle and scalp recording at cz - fz and c3-c4/c4-c3. mep was performed with stimulatioan at m3-m4 or m1-m2 (best response) with single train stimulation of 7 pulses with 200.000 hz frequency, 0.5 ms duration and 0100 ma intensity and recorded at abductor hallucis brevis muscle and tibialis anterior bilateral muscle. free run emg was recorded with the settting of 50 uv / division gain and 100 ms / division sweep at rectus femoris muscle and tibialis anterior muscle bilateral to prevent radix iritation during instrumentation. it was performed the facet joint release and posterior stabilization at the level vertebrae thoracal t11, t12 and lumbar l2, l3. intra - operative accident was happened when the operator inserted the pedicle screw at level left thoracal t12. the surgeon then decided to pullout the pedicle screw and change the location of pedicle insertion to one level above, vertebrae thoracal t11 (fig. 3). after changing the screw position, ssep monitor showed that the nerve amplitude was corrected to the acceptable value (fig. final screw insertion was at t10, t11 and l2, l3 on both side. post - operative evaluation showed that the pain was decreased (vas 12) and there was no deficit in motoric and sensoric function (fig. iatrogenic spinal cord injury was still a feared complication especially in deformity correction surgery such as scoliosis (idiopathic, congenital, neuromuscular, and related syndrome), exaggerated kyphosis, and lordosis. according to the scoliosis research society, the estimated incidence of neurological complications for such surgery was 1%, and it would increase to 1.87% when a combined surgical approach is used. mechanisms that could have been responsible for neurologic injury during spine procedure were : (a) direct injury due to surgical trauma, especially during spinal canal decompression or placement of spinal implant ; (b) traction and/or compression affecting neural structure, that could be occured during spinal realignment and deformity correction using spinal instrumentation or as a result of epidural hematoma following corpectomy procedure ; (c) ischemia resulting in decreased perfusion of the spinal cord and/or nerve roots, resulting in ischemic injury to neurologic structures (e.g. following ligation of critical segmental vessels supplying the spinal cord or after an episode of sustained hypotension). ischemia was the most common mechanism that was responsible for neurologic deficit during scoliosis surgery ; (d) compressive neuropathy as a result of patient positioning prior to or during surgery (e.g. brachial plexus injury). the causes of iatrogenic neurologic sequelae were implant - related damage, such as breach of a pedicle screw into the spinal canal or foramen, and injury during correction maneuvers, including distraction, compressive force to correct deformity, and the rod rotation technique to realign the vertebra. but among those causes the implant related damage was most common in clinical practice. the ideal pedicle screw should have a maximum diameter and length without breaching the pedicle s cortical layer, the vertebral body. and the direction of insertion should converge on both side of one vertebrae. lonstein., described that the most common type of perforation wass anterior cortex (2.8%), and followed by lateral cortex (1.0%), inferior cortex (0,6%), medial cortex of pedicle (0,4%) and superior cortex (0,2%). nevertheless, a satisfactory outcome could also be achieved despite sub - optimal screw placement and vice versa. for example, a screw that just barely touches the lower border of the pedicle may cause a clinically apparent radiculopathy and it may require revision. on the other hand, a screw that lies inside the spinal canal may produce no symptoms at all. therefore, the evaluation of successful fusion surgery should always include a clinical assessment in addition to an appraisal of screw position. intraoperative neuromonitoring (ionm) was a technique that now widely accepted to reduce the risk of neurologic complications in spinal surgery. various ionm modalities allowed con - tinuous functional assessment of the neuromuscular junction, peripheral nerves, spinal cord, brainstem, and cortex during spinal surgery. among the various ionm techniques available, somatosensory evoked potentials (ssep), transcranial electric muscle evoked potentials (tcemep), and spontaneous electromyography (free run emg) were most frequently used in clinical practice. a previous study reported that false negative ssep monitoring occurred during surgery in only 0.063% of patients. a large multicenter study had reported that postoperative paraplegia was reduced more than 50%60% with ssep monitoring. this was modification of the basic electroencephalography (eeg) in which a cortical or subcortical response to repetitive stimulation of a peripheral mixed nerve was recorded at sites cephalad and caudad to the operative field. data including signal amplitude (height) and latency (time of occurrence) were recorded continuously during surgery and compared with baseline data. ssep provided direct information about status of the ascending spinal cord sensory tracts (located in the dorsal medial columns of the spinal cord). there are some limitations such as : (a) ssep provided only indirect information about the status of the spinal cord motor tracts (located in the anterolateral columns of the spinal cord) ; (b) ssep data did not provide real - time data regarding neurologic function because there was a slight delay (usually,1 min) while the ssep response was averaged for extraction from background noise ; (c) sseps could be unrecordable in patients with severe myelopathy, peripheral neuropathy, or obesity. in addition, recording ssep is not a sensitive technique for monitoring individual nerve root function ; (d) sseps recording can be disturbed by operating room power equipment (due to electrical interference), halogenated anesthetic agents, nitrous oxide, hypothermia, and hypotension. the surgeon should be notified when ssep showed a persistent unilateral or bilateral loss of amplitude 50% or greater relative to baseline amplitude. changes in latency were common and less significant, and spinal cord injury was unlikely if amplitude is unchanged. transcranial electric motor - evoked potentials (tcemep) were neuroelectric impulses elicited by transcranial application of a high - voltage stimulus to electrodes placed over specific scalp regions to excite specific areas of the motor cortex. these descending impulses stimulated corticospinal tract axons and were typically recorded from electrodes placed over key upper and lower extremity peripheral muscles as a compound muscle action potential (cmap). motor - evoked potentials could also be recorded directly from the spinal cord (d- and i - waves) via electrodes placed percutaneously or through a laminotomy. the tcemep could provide information about the functional integrity of the spinal cord motor tracts that could not be obtained using ssep. they were extremely sensitive to alterations in spinal cord blood flow resulting from intraoperative hypotension or evolving vascular injury. in addition, alterations in tcemep presented earlier than changes in ssep in patients with evolving neurologic injury, which permits earlier initiation of corrective action to prevent permanent neurologic compromise. the tcemep were not a replacement for ssep but were used in combination with ssep to provide a direct measure of both spinal cord sensory and motor tract function, thereby increasing the efficacy of spinal monitoring. the surgeon should be notified when tcemep showed a persistent unilateral or bilateral loss of amplitude 65% or greater relative to baseline amplitude. spontaneous or free - running electromyography (emg) was widely applied to monitor selective nerve root function during spinal cord surgery. spontaneous emg could help to prevent postoperative radiculopathy during spinal instrumentation surgery, including pedicle screw placement. this technique did not require stimulation and could be recorded continuously from preselected muscle groups based on the nerve roots at risk. surgical manipulations such as pulling, stretching or compression of nerves provoked spikes or bursts of activity termed neurotonic discharges, resulting in activity in the corresponding innervated muscle(s). spontaneous emg was quite sensitive to irritation of the nerve root, such as retraction of spinal cord or nerve root, saline irrigation and manipulation during surgery. however, false spontaneous emg activation commonly occured during irrigation with cold water, cauterization and use of a high - speed drill because it was sensitive to temperature change. intraoperative triggered emg detected root irritation or the post injury condition of the root after medial pedicle breach. an irritated or damaged nerve root caused a decrease in electric threshold followed by sudden appearance of cmap of the specific muscles of the myotome after stimulation via the screw. each pedicle screw was electrically stimulated with an increasing intensity from 5 to 30 ma (duration, 0.2 ms ; frequency, 0.8 hz). recordings were made at the level of the lower limb muscles with or without the rectus abdominis muscles (depending on the root levels to test). the recording of muscle activity at an intensity under 10 ma (the classically accepted threshold) argued in favor of a medial breach (close proximity of the screw to the nerve root). although ionm generally referred to neurophysiological monitoring, the stagnara wakeup test, which provideed direct evaluation of the patient s motor functions without specialized equipment, was still used when necessary. when neuromonitoring alert (significant decrease or loss of neurophysiologic potentials) occured during surgery, the surgeon and anesthesiologist should remain calm and communicate with the spinal monitoring personnel as the following steps are taken : (a) check that the electrodes had not become displaced ; (b) elevate and maintain the mean arterial blood pressure between 85 and 95 mmhg to prevent spinal cord ischemia ; (c) assess if there has been a change in anesthetic technique ; (d) reverse any antecedent surgical event (e.g. strut graft / cage placement ; surgical maneuvers including distraction, compression, or translation) ; (e) inspect for an obvious source of neural compression (e.g. bone fragment, hematoma) ; (f) elevate body temperature and irrigate the wound with warm saline ; (g) send an arterial blood gas and laboratory tests to assess for an unrecognized metabolic abnormality or unrecognized low hemoglobin ; (h) if the tcemep / ssep data failed to recover, a wake - up test and awake clinical examination are considered ; (i) depending on the patient s response to the wake - up test and the specific spinal problem undergoing treatment, spinal instrumentation may require removal. the individual clinical scenario and stability of the spine must be considered in decision making ; (j) usage of steroids (spinal injury protocol) was an option. acute trauma as a result of spine instrumentation may result in significant edema, with mass effect causing neurophysiological dysfunction. immediate administration of an intravenous bolus of methylprednisolon, followed by constant infussion for following 2448 h, may help ameliorating the mass effect and improving the neurologic outcome. we were still considered for the protocol of steroid injection because the protocol was still implemented and adopted in our hospital. but we were not evaluated the benefit of the steroid injection related to the spinal cord injury. the impact of longer segment stabilization for the thoracic area were less than the lumbar area. in order to keep away the longer segment stabilization risk, we can strengthen the other side by adding one more rod with rod connector. by this technique a previous study reported that false negative ssep monitoring occurred during surgery in only 0.063% of patients. a large multicenter study had reported that postoperative paraplegia was reduced more than 50%60% with ssep monitoring. this was modification of the basic electroencephalography (eeg) in which a cortical or subcortical response to repetitive stimulation of a peripheral mixed nerve was recorded at sites cephalad and caudad to the operative field. data including signal amplitude (height) and latency (time of occurrence) were recorded continuously during surgery and compared with baseline data. ssep provided direct information about status of the ascending spinal cord sensory tracts (located in the dorsal medial columns of the spinal cord). there are some limitations such as : (a) ssep provided only indirect information about the status of the spinal cord motor tracts (located in the anterolateral columns of the spinal cord) ; (b) ssep data did not provide real - time data regarding neurologic function because there was a slight delay (usually,1 min) while the ssep response was averaged for extraction from background noise ; (c) sseps could be unrecordable in patients with severe myelopathy, peripheral neuropathy, or obesity. in addition, recording ssep is not a sensitive technique for monitoring individual nerve root function ; (d) sseps recording can be disturbed by operating room power equipment (due to electrical interference), halogenated anesthetic agents, nitrous oxide, hypothermia, and hypotension. the surgeon should be notified when ssep showed a persistent unilateral or bilateral loss of amplitude 50% or greater relative to baseline amplitude. changes in latency were common and less significant, and spinal cord injury was unlikely if amplitude is unchanged. transcranial electric motor - evoked potentials (tcemep) were neuroelectric impulses elicited by transcranial application of a high - voltage stimulus to electrodes placed over specific scalp regions to excite specific areas of the motor cortex. these descending impulses stimulated corticospinal tract axons and were typically recorded from electrodes placed over key upper and lower extremity peripheral muscles as a compound muscle action potential (cmap). motor - evoked potentials could also be recorded directly from the spinal cord (d- and i - waves) via electrodes placed percutaneously or through a laminotomy. the tcemep could provide information about the functional integrity of the spinal cord motor tracts that could not be obtained using ssep. they were extremely sensitive to alterations in spinal cord blood flow resulting from intraoperative hypotension or evolving vascular injury. in addition, alterations in tcemep presented earlier than changes in ssep in patients with evolving neurologic injury, which permits earlier initiation of corrective action to prevent permanent neurologic compromise. the tcemep were not a replacement for ssep but were used in combination with ssep to provide a direct measure of both spinal cord sensory and motor tract function, thereby increasing the efficacy of spinal monitoring. the surgeon should be notified when tcemep showed a persistent unilateral or bilateral loss of amplitude 65% or greater relative to baseline amplitude. spontaneous or free - running electromyography (emg) was widely applied to monitor selective nerve root function during spinal cord surgery. spontaneous emg could help to prevent postoperative radiculopathy during spinal instrumentation surgery, including pedicle screw placement. this technique did not require stimulation and could be recorded continuously from preselected muscle groups based on the nerve roots at risk. surgical manipulations such as pulling, stretching or compression of nerves provoked spikes or bursts of activity termed neurotonic discharges, resulting in activity in the corresponding innervated muscle(s). spontaneous emg was quite sensitive to irritation of the nerve root, such as retraction of spinal cord or nerve root, saline irrigation and manipulation during surgery. however, false spontaneous emg activation commonly occured during irrigation with cold water, cauterization and use of a high - speed drill because it was sensitive to temperature change. intraoperative triggered emg detected root irritation or the post injury condition of the root after medial pedicle breach. an irritated or damaged nerve root caused a decrease in electric threshold followed by sudden appearance of cmap of the specific muscles of the myotome after stimulation via the screw. each pedicle screw was electrically stimulated with an increasing intensity from 5 to 30 ma (duration, 0.2 ms ; frequency, 0.8 hz). recordings were made at the level of the lower limb muscles with or without the rectus abdominis muscles (depending on the root levels to test). the recording of muscle activity at an intensity under 10 ma (the classically accepted threshold) argued in favor of a medial breach (close proximity of the screw to the nerve root). although ionm generally referred to neurophysiological monitoring, the stagnara wakeup test, which provideed direct evaluation of the patient s motor functions without specialized equipment, was still used when necessary. when neuromonitoring alert (significant decrease or loss of neurophysiologic potentials) occured during surgery, the surgeon and anesthesiologist should remain calm and communicate with the spinal monitoring personnel as the following steps are taken : (a) check that the electrodes had not become displaced ; (b) elevate and maintain the mean arterial blood pressure between 85 and 95 mmhg to prevent spinal cord ischemia ; (c) assess if there has been a change in anesthetic technique ; (d) reverse any antecedent surgical event (e.g. strut graft / cage placement ; surgical maneuvers including distraction, compression, or translation) ; (e) inspect for an obvious source of neural compression (e.g. bone fragment, hematoma) ; (f) elevate body temperature and irrigate the wound with warm saline ; (g) send an arterial blood gas and laboratory tests to assess for an unrecognized metabolic abnormality or unrecognized low hemoglobin ; (h) if the tcemep / ssep data failed to recover, a wake - up test and awake clinical examination are considered ; (i) depending on the patient s response to the wake - up test and the specific spinal problem undergoing treatment, spinal instrumentation may require removal. the individual clinical scenario and stability of the spine must be considered in decision making ; (j) usage of steroids (spinal injury protocol) was an option. acute trauma as a result of spine instrumentation may result in significant edema, with mass effect causing neurophysiological dysfunction. immediate administration of an intravenous bolus of methylprednisolon, followed by constant infussion for following 2448 h, may help ameliorating the mass effect and improving the neurologic outcome. we were still considered for the protocol of steroid injection because the protocol was still implemented and adopted in our hospital. but we were not evaluated the benefit of the steroid injection related to the spinal cord injury. the impact of longer segment stabilization for the thoracic area were less than the lumbar area. in order to keep away the longer segment stabilization risk, we can strengthen the other side by adding one more rod with rod connector. by this technique insertion of pedicle screw in spinal surgery had a risk of complication that could prevent by usage of intraoperative neurophysiological monitoring. ssep, tcmep and free running emg could detect the neurophysiological reaction of the spinal cord, therefore preventing undesired neurological disturbance post - operatively. the authors declare that there is no conflict of interests regarding the publication of this paper | introductionintraoperative neurophysiologic monitoring (ionm) had important role related to the complications in spinal surgery. somatosensory evoked potential (ssep), transcranial electric muscle evoked potentials (tcemeps), and free run emg are parameters used to asses functional integrity of the nervous system during surgical procedures. once warning signal was recognized, surgeon have to make a precise decision to overcome that problem.presentation of casewe present a 47-year old male with back pain due to compression fracture of thoracic vertebra t12 and lumbar vertebrae l1. while stabilizing through the posterior approach on the t11 and 12 as well as l2 and l3, the ssep monitor showed 50% reduction in the waveform as the pedicle screw was inserted at the left side of t12. the instrumentation was changed into vertebra thoracal t10, t11, and vertebrae lumbar l2, l3. the ssep normalized and post operatively pain decreased. after surgery there was no neurological deficit.discussionacute trauma as a result of spine instrumentation may provoke significant edema, with mass effect causing neurophysiological dysfunction. administration of intravenous steroid would do at this stage, followed by constant infusion for following 2448 h, may help ameliorating the mass effect and improving the neurologic outcome. alternatively, immediate pedicle screw changing policy showed absolute recovery of nerve injury.conclusioninsertion of pedicle screw in spinal surgery has a risk of complication that could be treated by pedicle screw changing policy. |
research on rhesus macaques (macaca mulatta) followed the guidelines of the arvo statement for the use of animals in ophthalmic and vision research, complied with the national institutes of health (nih) guide for the care and use of laboratory animals, and were approved by the university of california - davis institutional animal care and use committee. the cnprc is accredited by the association for the accreditation and assessment of laboratory animal care (aaalac) international. adult animals were selected from the rhesus macaque colony at cnprc that were undergoing routine semiannual physical examinations. the animals were sedated with intramuscular injection of ketamine hydrochloride, midazolam, and dexmedetomidine, followed by pupillary dilation with tropicamide. all subjects underwent complete ophthalmic examination including slit - lamp biomicroscopy, dilated fundus biomicroscopy, streak retinoscopy, a - scan biometry, and rebound tonometry, as well as spectral - domain oct (sd - oct) of the macula in edi mode. animal eyes that showed any retinal or choroidal lesions on exam, or demonstrated refractive error greater than 6 diopters (d) spherical equivalent based on streak retinoscopy, were excluded from this study. research in humans was approved by the institutional review board of the university of california - davis and was performed in accordance with the tenets of the declaration of helsinki. age - matched human subjects were selected retrospectively from a database search of patients seen in the vitreoretinal service at the university of california - davis eye center who had a normal ophthalmologic examination and underwent edi - oct macular imaging. these subjects underwent imaging for eyes that were evaluated for possible ocular pathology but in which no abnormalities were found (n = 9) ; or were contralateral eyes in patients with unilateral pathologies, such as epiretinal membrane or vitreomacular traction (n = 7), retinal tear or retinal detachment (n = 6), posterior vitreous detachment (n = 5), choroidal nevus (n = 1), white without pressure (n = 1), or open globe injury (n = 1). eyes that had any retinal or choroidal diseases, history of vitreoretinal surgery, or myopia greater than 6 d were excluded. effort was made to age - match human subjects to rhesus macaques (approximately 3 human years for every year in macaques), and to select a similar proportion of patients who identified themselves as white or black race. for both rhesus macaques and humans, only one eye from each subject was selected for analysis. if both eyes qualified based on inclusion and exclusion criteria, the right eye was selected for patients with an even - numbered birth month, and the left eye selected for an odd - numbered birth month. demographic data, including age and sex, were collected for all subjects. for macaques, cycloplegic refraction (d), intraocular pressure (mm hg) measured by rebound tonometry, and axial length (mm) on a - scan biometry were also recorded. for human subjects, race, lens status (phakic, pseudophakic, or aphakic), best - corrected visual acuity (bcva ; logmar), manifest refraction (d), and intraocular pressure (mm hg) measured by applanation tonometry were collected. enhanced - depth imaging oct imaging for both humans and rhesus macaques was performed using the spectralis sd - oct device (heidelberg engineering, heidelberg, germany). oct was performed using a single 30 horizontal line scan with 1536 a - scans per b - scan, centered on the fovea, in high - resolution edi mode. up to 100 scans (range, 50100) were averaged for each b - scan, using the heidelberg eye tracking automatic real - time (art) software. all imaging on rhesus macaques (n = 25) was performed by the first author (gy) at cnprc. for nonhuman primate imaging, the head and chin rests of the imaging device were removed and replaced with a custom metal bar to allow the animal 's head to rest comfortably. animals were monitored by a trained technician and a cnprc veterinarian (lg) at all times. the mean age of the animals was 20.3 5.9 years (range, 1030 years), with 14 females and 11 male primates. study eyes included 14 right eyes (56%) and 11 left eyes (44%). mean cycloplegic refraction was + 0.3 2.0 d spherical equivalent, mean intraocular pressure was 18.0 4.1 mm hg, and mean axial length was 19.7 1.4 mm. for humans (n = 30), edi - oct was performed by trained ophthalmic photographers at the uc davis eye center. the mean age of the subjects was 62.8 17.3 (range, 3188 years), with 15 males and 15 female subjects. eighteen were self - reported as white or caucasian, while 12 were black or african american. of the study eyes, 17 were right eyes (56.7%) and 13 were left eyes (43.3%) ; 27 were phakic (90%), and the remainder were pseudophakic. mean logmar bcva was 0.11 0.13 (snellen equivalent 20/25.8), mean refractive error was + 0.19 2.0 d, and mean intraocular pressure was 15.1 3.4 mm hg. images were exported from heidelberg explorer software (version 1.8.6.0, heidelberg engineering) to imagej software (version 1.49v ; http://imagej.nih.gov/ij/ ; provided in the public domain by the national institutes of health, bethesda, md, usa) to generate vertical reflectivity profiles at the foveal center. for each eye, reflectivity values were averaged over 10 adjacent a - scans, centered at the fovea, and presented on a scale from 0 to 1.00 grayscale units after normalization to the peak of the hyperreflective band corresponding to the rpe / bruch 's membrane, which was assigned to a value of 1.0. reflectivity profiles from different eyes were aligned along the posterior border of this hyperreflective rpe / bruch 's membrane band for averaging. visualization of the choriocapillaris layer and the csj was qualitatively graded as percent of the linear distance over which the layer boundaries were visible, and classified as clear (75% visibility), intermediate (25% and < 75% visibility), or unclear (< 25% visibility) by two experienced graders (gy and vv) who were masked to species and subject demographic. choriocapillary and choroidal thicknesses were measured after semiautomated segmentation of the central 3 mm of the macula (1.5 mm nasal1.5 mm temporal to the fovea) using duke optical coherence tomography retinal analysis program (doctrap, version 62.0 ; durham, nc, usa), a custom image segmentation software designed using matlab (mathworks, natick, ma, usa). for thickness measurements, the inner boundary was automatically determined by doctrap as the outer border of the hyperreflective rpe / bruch 's complex ; and two masked graders manually traced the outer border of the hyporeflective choriocapillaris layer to measure the choriocapillary thickness, and the outer border of the choroid stroma, corresponding to the csj, to measure the choroidal thickness. all thickness measurements were determined from the mean of the two graders ' measurements, and reproducibility was determined by measuring intergrader reliability using intraclass correlations (icc) by the bland - altman method. histologic sections from rhesus macaque eyes (n = 6, mean age 19.2 7.9 years) were taken from age - matched animals at cnprc undergoing necropsy for nonocular health issues. eyes were fixed in 2% paraformaldehyde and 0.5% glutaraldehyde for 24 to 48 hours, transferred to 4% formalin, then embedded in paraffin and stained with standard hematoxylin - eosin. histologic sections from age - matched adult postmortem human eyes (n = 10, mean age 52.8 23.5 years) were selected from an archive of cadaveric eyes with no known ocular diseases at the duke university medical center (courtesy of allan proia). all sections also underwent standard fixation in formalin, paraffin embedding, and staining with hematoxylin - eosin. among the eyes analyzed, five were from white subjects (mean age 63.2 18.9 years, range, 3571 years, three males / two females), and five were from black subjects (mean age 42.4 24.7 years, range, 2583 years, three males / two females). to quantify the cell density, size, and pigmentation of choroidal melanocytes in histologic sections, three 100 100-m representative regions of choroid stroma were selected for each study eye from images of histologic sections captured at 20 using an eclipse te 200 (nikon, tokyo, japan) microscope. we then performed automated color - based segmentation using the lab color space and k - means clustering in matlab (mathworks) to isolate the pigmented melanocytes. the number of pigmented cells in each field was manually counted to determine melanocyte density (cells per 10,000 m). average melanocyte size was calculated by dividing the total segmented area of melanin pigment by the cell number in each field. finally, uveal pigmentation was evaluated by measuring the average luminosity (l - component of the lab color space) of the segmented areas from each image as an estimate of optical density (supplementary fig. the relationship of choriocapillaris or csj visibility to species or race was determined using the kruskal - wallis test. multivariate ordinal logistic regression was used to determine the independent association of age, race, and refractive error with choriocapillaris or csj visibility. choriocapillary and choroidal thickness measurements, as well as differences in cell density, size, and pigmentation of melanocytes, were compared using student 's t - tests. all statistical analyses were performed using spss statistics (version 22 ; ibm, armonk, ny, usa). research on rhesus macaques (macaca mulatta) followed the guidelines of the arvo statement for the use of animals in ophthalmic and vision research, complied with the national institutes of health (nih) guide for the care and use of laboratory animals, and were approved by the university of california - davis institutional animal care and use committee. the cnprc is accredited by the association for the accreditation and assessment of laboratory animal care (aaalac) international. adult animals were selected from the rhesus macaque colony at cnprc that were undergoing routine semiannual physical examinations. the animals were sedated with intramuscular injection of ketamine hydrochloride, midazolam, and dexmedetomidine, followed by pupillary dilation with tropicamide. all subjects underwent complete ophthalmic examination including slit - lamp biomicroscopy, dilated fundus biomicroscopy, streak retinoscopy, a - scan biometry, and rebound tonometry, as well as spectral - domain oct (sd - oct) of the macula in edi mode. animal eyes that showed any retinal or choroidal lesions on exam, or demonstrated refractive error greater than 6 diopters (d) spherical equivalent based on streak retinoscopy, were excluded from this study. research in humans was approved by the institutional review board of the university of california - davis and was performed in accordance with the tenets of the declaration of helsinki. age - matched human subjects were selected retrospectively from a database search of patients seen in the vitreoretinal service at the university of california - davis eye center who had a normal ophthalmologic examination and underwent edi - oct macular imaging. these subjects underwent imaging for eyes that were evaluated for possible ocular pathology but in which no abnormalities were found (n = 9) ; or were contralateral eyes in patients with unilateral pathologies, such as epiretinal membrane or vitreomacular traction (n = 7), retinal tear or retinal detachment (n = 6), posterior vitreous detachment (n = 5), choroidal nevus (n = 1), white without pressure (n = 1), or open globe injury (n = 1). eyes that had any retinal or choroidal diseases, history of vitreoretinal surgery, or myopia greater than 6 d were excluded. effort was made to age - match human subjects to rhesus macaques (approximately 3 human years for every year in macaques), and to select a similar proportion of patients who identified themselves as white or black race. for both rhesus macaques and humans, only one eye from each subject was selected for analysis. if both eyes qualified based on inclusion and exclusion criteria, the right eye was selected for patients with an even - numbered birth month, and the left eye selected for an odd - numbered birth month. demographic data, including age and sex, were collected for all subjects. for macaques, cycloplegic refraction (d), intraocular pressure (mm hg) measured by rebound tonometry, and axial length (mm) on a - scan biometry were also recorded. for human subjects, race, lens status (phakic, pseudophakic, or aphakic), best - corrected visual acuity (bcva ; logmar), manifest refraction (d), and intraocular pressure (mm hg) measured by applanation tonometry were collected. enhanced - depth imaging oct imaging for both humans and rhesus macaques was performed using the spectralis sd - oct device (heidelberg engineering, heidelberg, germany). oct was performed using a single 30 horizontal line scan with 1536 a - scans per b - scan, centered on the fovea, in high - resolution edi mode. up to 100 scans (range, 50100) were averaged for each b - scan, using the heidelberg eye tracking automatic real - time (art) software. all imaging on rhesus macaques (n = 25) was performed by the first author (gy) at cnprc. for nonhuman primate imaging, the head and chin rests of the imaging device were removed and replaced with a custom metal bar to allow the animal 's head to rest comfortably. animals were monitored by a trained technician and a cnprc veterinarian (lg) at all times. the mean age of the animals was 20.3 5.9 years (range, 1030 years), with 14 females and 11 male primates. study eyes included 14 right eyes (56%) and 11 left eyes (44%). mean cycloplegic refraction was + 0.3 2.0 d spherical equivalent, mean intraocular pressure was 18.0 4.1 mm hg, and mean axial length was 19.7 1.4 mm. for humans (n = 30), edi - oct was performed by trained ophthalmic photographers at the uc davis eye center. the mean age of the subjects was 62.8 17.3 (range, 3188 years), with 15 males and 15 female subjects. eighteen were self - reported as white or caucasian, while 12 were black or african american. of the study eyes, 17 were right eyes (56.7%) and 13 were left eyes (43.3%) ; 27 were phakic (90%), and the remainder were pseudophakic. mean logmar bcva was 0.11 0.13 (snellen equivalent 20/25.8), mean refractive error was + 0.19 2.0 d, and mean intraocular pressure was 15.1 3.4 mm hg. images were exported from heidelberg explorer software (version 1.8.6.0, heidelberg engineering) to imagej software (version 1.49v ; http://imagej.nih.gov/ij/ ; provided in the public domain by the national institutes of health, bethesda, md, usa) to generate vertical reflectivity profiles at the foveal center. for each eye, reflectivity values were averaged over 10 adjacent a - scans, centered at the fovea, and presented on a scale from 0 to 1.00 grayscale units after normalization to the peak of the hyperreflective band corresponding to the rpe / bruch 's membrane, which was assigned to a value of 1.0. reflectivity profiles from different eyes were aligned along the posterior border of this hyperreflective rpe / bruch 's membrane band for averaging. visualization of the choriocapillaris layer and the csj was qualitatively graded as percent of the linear distance over which the layer boundaries were visible, and classified as clear (75% visibility), intermediate (25% and < 75% visibility), or unclear (< 25% visibility) by two experienced graders (gy and vv) who were masked to species and subject demographic. choriocapillary and choroidal thicknesses were measured after semiautomated segmentation of the central 3 mm of the macula (1.5 mm nasal1.5 mm temporal to the fovea) using duke optical coherence tomography retinal analysis program (doctrap, version 62.0 ; durham, nc, usa), a custom image segmentation software designed using matlab (mathworks, natick, ma, usa). for thickness measurements, the inner boundary was automatically determined by doctrap as the outer border of the hyperreflective rpe / bruch 's complex ; and two masked graders manually traced the outer border of the hyporeflective choriocapillaris layer to measure the choriocapillary thickness, and the outer border of the choroid stroma, corresponding to the csj, to measure the choroidal thickness. all thickness measurements were determined from the mean of the two graders ' measurements, and reproducibility was determined by measuring intergrader reliability using intraclass correlations (icc) by the bland - altman method. histologic sections from rhesus macaque eyes (n = 6, mean age 19.2 7.9 years) were taken from age - matched animals at cnprc undergoing necropsy for nonocular health issues. eyes were fixed in 2% paraformaldehyde and 0.5% glutaraldehyde for 24 to 48 hours, transferred to 4% formalin, then embedded in paraffin and stained with standard hematoxylin - eosin. histologic sections from age - matched adult postmortem human eyes (n = 10, mean age 52.8 23.5 years) were selected from an archive of cadaveric eyes with no known ocular diseases at the duke university medical center (courtesy of allan proia). all sections also underwent standard fixation in formalin, paraffin embedding, and staining with hematoxylin - eosin. among the eyes analyzed, five were from white subjects (mean age 63.2 18.9 years, range, 3571 years, three males / two females), and five were from black subjects (mean age 42.4 24.7 years, range, 2583 years, three males / two females). to quantify the cell density, size, and pigmentation of choroidal melanocytes in histologic sections, three 100 100-m representative regions of choroid stroma were selected for each study eye from images of histologic sections captured at 20 using an eclipse te 200 (nikon, tokyo, japan) microscope. we then performed automated color - based segmentation using the lab color space and k - means clustering in matlab (mathworks) to isolate the pigmented melanocytes. the number of pigmented cells in each field was manually counted to determine melanocyte density (cells per 10,000 m). average melanocyte size was calculated by dividing the total segmented area of melanin pigment by the cell number in each field. finally, uveal pigmentation was evaluated by measuring the average luminosity (l - component of the lab color space) of the segmented areas from each image as an estimate of optical density (supplementary fig. the relationship of choriocapillaris or csj visibility to species or race was determined using the kruskal - wallis test. multivariate ordinal logistic regression was used to determine the independent association of age, race, and refractive error with choriocapillaris or csj visibility. choriocapillary and choroidal thickness measurements, as well as differences in cell density, size, and pigmentation of melanocytes, were compared using student 's t - tests. all statistical analyses were performed using spss statistics (version 22 ; ibm, armonk, ny, usa). enhanced - depth oct images of rhesus macaque and human eyes showed similar appearance of the retinal layers at the foveal region. rhesus macaque eyes uniformly showed a distinct hyporeflective choriocapillaris layer immediately posterior to the rpe / bruch 's complex, and no distinct csj was seen in any of the animals (figs. by contrast, images of human eyes showed variable appearance of the choriocapillaris, and many showed a well - delineated csj (figs. 1b, 1d). all macaques showed 75% visibility of the choriocapillaris, unlike humans, where few normal eyes showed 75% visibility of this layer (p < 0.001) (table 1). in contrast, few macaques showed 25% visibility of the csj, while half of human subjects showed 25% visibility of this boundary (p = 0.003) (table 1). these results show that choroidal morphology on edi - oct is different between rhesus macaques and humans, with better visualization of the choriocapillaris in macaques and greater visualization of the csj in humans. comparison of representative edi - oct images from normal rhesus macaque eyes (a, c) and human eyes (b, d). the choriocapillaris layer (arrows) is well demarcated in rhesus macaques, but not human eyes, while the choroidal scleral junction (arrowheads) is better delineated in some human eyes (33%) in comparison to macaques. visibility of choroidal morphology in rhesus macaques and humans to further compare the differential appearance of the choroidal layers in humans and macaques on edi - oct, we obtained vertical reflectivity profiles of the choroid at the fovea from all the images. all rhesus macaque eyes showed a narrow band of hyporeflectivity just posterior to the peak intensity of the rpe band, and delineated posteriorly by another peak of reflectivity that gradually tapers toward the sclera with no clearly demarcated vessel lumen or csj (fig. 2a). compared with histologic sections of age - matched animals, the hyporeflective band corresponds well with the location of the choriocapillaris. the signal sensitivity roll - off posterior to the choriocapillaris corresponds to the presence of large, darkly pigmented uveal melanocytes that are densely distributed between the medium- to large - caliber vessels, but not in the choriocapillaris layer. this heavy melanin pigmentation appears to result in obscuration of the csj in the macaque choroid. reflectivity profiles of representative edi - oct images of a rhesus macaque (a) and human eye (b), measured from 10 adjacent a - scans centered at the fovea (magnified from area of dashed box). in macaques, a hyperreflective band corresponds to the rpe layer, while the adjacent hyporeflective band corresponds to the choriocapillaris (cc) on age - matched histologic sections. the gradual signal attenuation corresponds to large, darkly pigmented melanocytes that are densely distributed across the choroid stroma up to the posterior border of the choriocapillaris, and the choroidal scleral junction (csj) is poorly demarcated. in humans, no distinct choriocapillaris band can be seen posterior to the hyperreflective rpe band, and the variable reflectivities of the choroidal vessels correspond to the small, lightly pigmented melanocytes that are loosely distributed mostly in the posterior choroid and suprachoroidal layer. plots of mean reflectivities of rhesus macaques (c) and human subjects (d) measured from the posterior border of the rpe / bruch 's complex, which shows a clear inflection point corresponding to the choriocapillaris in macaques but not humans. mean reflectivities are measured in arbitrary grayscale units scaled from 0 to 1.00, normalized to the peak of the rpe / bruch 's membrane, which is assigned to a value of 1.00. by contrast, human eyes showed variable reflectivity levels between the rpe and sclera with no single distinct hyporeflective choriocapillaris, with some eyes (33%) showing a well - delineated reflectivity drop at the choroidal scleral interface (fig. histologic sections of age - matched human choroid can be clearly distinguished from rhesus macaques by the smaller size and lighter pigmentation of the melanocytes (table 2). the melanocytes are also less densely distributed and are distributed more posteriorly within the choroid stroma and in the suprachoroidal layer (fig. the marked difference in melanocyte pigmentation is in contrast to the rpe pigmentation, which is similar between the two species (figs. hence, melanin pigmentation in uveal melanocytes rather than rpe cells appears to contribute to the differential visualization of the choriocapillaris and csj in human and macaque eyes on edi - oct. characteristics of choroidal melanocytes in rhesus macaques and humans we averaged the choroidal reflectivity profiles from edi - oct images across all macaque animals (n = 25) and from all human subjects (n = 30) to compare differences in reflectivity patterns, and found that only the nonhuman primates exhibit a consistent clear hyporeflective band corresponding to the choriocapillaris (figs. the mean location of the hyporeflective band is 12.5 6.7 m posterior to the posterior border of the rpe / bruch 's complex, which corresponds to the location of the choriocapillaris on histology. using a semiautomated graph based oct image segmentation software previously described, we measured the thickness of the choriocapillaris and choroid along the central 3-mm segment around the fovea. the mean choriocapillary thickness was similar between rhesus macaques and humans (19.3 3.4 vs. 19.8 3.4 m), with no significant difference between the two species (p = 0.615). however, the mean choroidal thickness measured was significantly lower in macaques compared with humans (191.2 43.0 vs. 266.8 78.0 m ; p < 0.001). in both species, while the choriocapillary thickness remains relatively constant across the central macula, the choroid is thickest at the fovea and slightly thinner in the nasal macula region (fig. 3). representative edi - oct images with semiautomatic segmentation of the choriocapillaris and choroid stroma in rhesus macaque (a) and human eyes (b). the posterior border of the rpe / bruch 's complex is semiautomatically determined (red line), while the outer borders of the hyporeflective choriocapillaris (green line) and choroid stroma (blue line) are manually traced. plots of mean choroidal thickness (ct) and choriocapillary thickness (cct) measured from edi - oct images of rhesus macaque (c) and human eyes (d). while cct is similar between the two species, the measured ct is significantly thinner in macaques than in humans. the overall intergrader reliability of choroidal thickness (icc = 0.760) and choriocapillary thickness (icc = 0.607) measurements was good. consistent with the difference in choroidal morphology on edi - oct between the two species, the reliability of choriocapillary thickness measurements was greater than that of choroidal thickness measurements in macaque eyes (icc = 0.661 vs. 0.297), while the opposite was true in human eyes, where measurements of choroidal thickness were more reproducible than choriocapillary thickness (icc = 0.776 vs. 0.590). the lower reliability of choroidal thickness measurements in macaques is likely due to the poor visualization of the csj, and likely contributes to an underestimate of the actual choroidal thickness in these animals. given the differential oct appearance of the choroid between humans and macaques, which appears to be attributed to the marked difference in the distribution and pigmentation of choroidal melanocytes, we decided to assess whether differences in race may affect uveal pigmentation on histology and choroidal morphology on edi - oct. while the distribution and pigmentation of melanocytes were variable among different individuals, eyes from white subjects (figs. c) have smaller and less darkly pigmented melanocytes than those from black individuals (figs. f) (table 3), as reported in prior studies. otherwise, in all human eyes, uveal melanocytes were not only smaller and less pigmented than those in rhesus macaques (table 2), but also less densely packed and more posteriorly distributed, with the majority of the cells located in haller 's layer and the suprachoroidal layer (figs. representative histology images of human choroid from white subjects (a c) and black subjects (d f). a) a 35-year - old white female, (b) a 71-year - old white male, (c) a 54-year - old white male, (d) a 29-year - old black male, (e) a 49-year - old black male, and (f) a 25-year - old black female. characteristics of choroidal melanocytes in humans of different races consistent with our hypothesis that uveal pigmentation affects choroidal morphology on edi - oct, masked grading of images of human eyes showed greater choriocapillaris visibility in black subjects (p = 0.017) and higher visibility of the csj among white individuals (p = 0.004) (table 4). to determine if race is independently associated with these choroidal morphologic parameters, we performed a multivariate logistic regression including age and refractive error, which are both known determinants of choroidal thickness. among these factors, our data showed that choriocapillaris visibility was independently associated with race alone (p = 0.016), while csj visibility was associated with both race (p = 0.004) and age (p = 0.026) in our study population (table 5). there were no significant racial differences in both the mean choriocapillary thickness (19.0 3.2 m in whites vs. 21.0 3.3 m in blacks ; p = 0.574) and mean choroidal thickness (240.0 66.2 m in whites vs. 306.9.8 79.5 m in blacks ; p = 0.377). thus, racial differences have a greater impact on the visibility than the vascular dimensions of the choroid. visibility of choroidal morphology in humans of different races factors affecting choroidal morphology on edi - oct in humans enhanced - depth oct images of rhesus macaque and human eyes showed similar appearance of the retinal layers at the foveal region. rhesus macaque eyes uniformly showed a distinct hyporeflective choriocapillaris layer immediately posterior to the rpe / bruch 's complex, and no distinct csj was seen in any of the animals (figs. by contrast, images of human eyes showed variable appearance of the choriocapillaris, and many showed a well - delineated csj (figs. 1b, 1d). all macaques showed 75% visibility of the choriocapillaris, unlike humans, where few normal eyes showed 75% visibility of this layer (p < 0.001) (table 1). in contrast, few macaques showed 25% visibility of the csj, while half of human subjects showed 25% visibility of this boundary (p = 0.003) (table 1). these results show that choroidal morphology on edi - oct is different between rhesus macaques and humans, with better visualization of the choriocapillaris in macaques and greater visualization of the csj in humans. comparison of representative edi - oct images from normal rhesus macaque eyes (a, c) and human eyes (b, d). the choriocapillaris layer (arrows) is well demarcated in rhesus macaques, but not human eyes, while the choroidal scleral junction (arrowheads) is better delineated in some human eyes (33%) in comparison to macaques. to further compare the differential appearance of the choroidal layers in humans and macaques on edi - oct, we obtained vertical reflectivity profiles of the choroid at the fovea from all the images. all rhesus macaque eyes showed a narrow band of hyporeflectivity just posterior to the peak intensity of the rpe band, and delineated posteriorly by another peak of reflectivity that gradually tapers toward the sclera with no clearly demarcated vessel lumen or csj (fig. compared with histologic sections of age - matched animals, the hyporeflective band corresponds well with the location of the choriocapillaris. the signal sensitivity roll - off posterior to the choriocapillaris corresponds to the presence of large, darkly pigmented uveal melanocytes that are densely distributed between the medium- to large - caliber vessels, but not in the choriocapillaris layer. this heavy melanin pigmentation appears to result in obscuration of the csj in the macaque choroid. reflectivity profiles of representative edi - oct images of a rhesus macaque (a) and human eye (b), measured from 10 adjacent a - scans centered at the fovea (magnified from area of dashed box). in macaques, a hyperreflective band corresponds to the rpe layer, while the adjacent hyporeflective band corresponds to the choriocapillaris (cc) on age - matched histologic sections. the gradual signal attenuation corresponds to large, darkly pigmented melanocytes that are densely distributed across the choroid stroma up to the posterior border of the choriocapillaris, and the choroidal scleral junction (csj) is poorly demarcated. in humans, no distinct choriocapillaris band can be seen posterior to the hyperreflective rpe band, and the variable reflectivities of the choroidal vessels correspond to the small, lightly pigmented melanocytes that are loosely distributed mostly in the posterior choroid and suprachoroidal layer. plots of mean reflectivities of rhesus macaques (c) and human subjects (d) measured from the posterior border of the rpe / bruch 's complex, which shows a clear inflection point corresponding to the choriocapillaris in macaques but not humans. mean reflectivities are measured in arbitrary grayscale units scaled from 0 to 1.00, normalized to the peak of the rpe / bruch 's membrane, which is assigned to a value of 1.00. by contrast, human eyes showed variable reflectivity levels between the rpe and sclera with no single distinct hyporeflective choriocapillaris, with some eyes (33%) showing a well - delineated reflectivity drop at the choroidal histologic sections of age - matched human choroid can be clearly distinguished from rhesus macaques by the smaller size and lighter pigmentation of the melanocytes (table 2). the melanocytes are also less densely distributed and are distributed more posteriorly within the choroid stroma and in the suprachoroidal layer (fig. the marked difference in melanocyte pigmentation is in contrast to the rpe pigmentation, which is similar between the two species (figs. hence, melanin pigmentation in uveal melanocytes rather than rpe cells appears to contribute to the differential visualization of the choriocapillaris and csj in human and macaque eyes on edi - oct. characteristics of choroidal melanocytes in rhesus macaques and humans we averaged the choroidal reflectivity profiles from edi - oct images across all macaque animals (n = 25) and from all human subjects (n = 30) to compare differences in reflectivity patterns, and found that only the nonhuman primates exhibit a consistent clear hyporeflective band corresponding to the choriocapillaris (figs. the mean location of the hyporeflective band is 12.5 6.7 m posterior to the posterior border of the rpe / bruch 's complex, which corresponds to the location of the choriocapillaris on histology. using a semiautomated graph based oct image segmentation software previously described, we measured the thickness of the choriocapillaris and choroid along the central 3-mm segment around the fovea. the mean choriocapillary thickness was similar between rhesus macaques and humans (19.3 3.4 vs. 19.8 3.4 m), with no significant difference between the two species (p = 0.615). however, the mean choroidal thickness measured was significantly lower in macaques compared with humans (191.2 43.0 vs. 266.8 78.0 m ; p < 0.001). in both species, while the choriocapillary thickness remains relatively constant across the central macula, the choroid is thickest at the fovea and slightly thinner in the nasal macula region (fig. 3). representative edi - oct images with semiautomatic segmentation of the choriocapillaris and choroid stroma in rhesus macaque (a) and human eyes (b). the posterior border of the rpe / bruch 's complex is semiautomatically determined (red line), while the outer borders of the hyporeflective choriocapillaris (green line) and choroid stroma (blue line) are manually traced. plots of mean choroidal thickness (ct) and choriocapillary thickness (cct) measured from edi - oct images of rhesus macaque (c) and human eyes (d). while cct is similar between the two species, the measured ct is significantly thinner in macaques than in humans. the overall intergrader reliability of choroidal thickness (icc = 0.760) and choriocapillary thickness (icc = 0.607) measurements was good. consistent with the difference in choroidal morphology on edi - oct between the two species, the reliability of choriocapillary thickness measurements was greater than that of choroidal thickness measurements in macaque eyes (icc = 0.661 vs. 0.297), while the opposite was true in human eyes, where measurements of choroidal thickness were more reproducible than choriocapillary thickness (icc = 0.776 vs. 0.590). the lower reliability of choroidal thickness measurements in macaques is likely due to the poor visualization of the csj, and likely contributes to an underestimate of the actual choroidal thickness in these animals. given the differential oct appearance of the choroid between humans and macaques, which appears to be attributed to the marked difference in the distribution and pigmentation of choroidal melanocytes, we decided to assess whether differences in race may affect uveal pigmentation on histology and choroidal morphology on edi - oct. while the distribution and pigmentation of melanocytes were variable among different individuals, eyes from white subjects (figs. c) have smaller and less darkly pigmented melanocytes than those from black individuals (figs. f) (table 3), as reported in prior studies. otherwise, in all human eyes, uveal melanocytes were not only smaller and less pigmented than those in rhesus macaques (table 2), but also less densely packed and more posteriorly distributed, with the majority of the cells located in haller 's layer and the suprachoroidal layer (figs. representative histology images of human choroid from white subjects (a c) and black subjects (d f). histologic sections are taken from (a) a 35-year - old white female, (b) a 71-year - old white male, (c) a 54-year - old white male, (d) a 29-year - old black male, (e) a 49-year - old black male, and (f) a 25-year - old black female. characteristics of choroidal melanocytes in humans of different races consistent with our hypothesis that uveal pigmentation affects choroidal morphology on edi - oct, masked grading of images of human eyes showed greater choriocapillaris visibility in black subjects (p = 0.017) and higher visibility of the csj among white individuals (p = 0.004) (table 4). to determine if race is independently associated with these choroidal morphologic parameters, we performed a multivariate logistic regression including age and refractive error, which are both known determinants of choroidal thickness. among these factors, our data showed that choriocapillaris visibility was independently associated with race alone (p = 0.016), while csj visibility was associated with both race (p = 0.004) and age (p = 0.026) in our study population (table 5). there were no significant racial differences in both the mean choriocapillary thickness (19.0 3.2 m in whites vs. 21.0 3.3 m in blacks ; p = 0.574) and mean choroidal thickness (240.0 66.2 m in whites vs. 306.9.8 79.5 m in blacks ; p = 0.377). thus, racial differences have a greater impact on the visibility than the vascular dimensions of the choroid. visibility of choroidal morphology in humans of different races factors affecting choroidal morphology on edi - oct in humans together, our results demonstrate that the cross - sectional visualization of choroidal structures including the choriocapillaris and csj on edi - oct may be affected by pigmented uveal melanocytes in the choroid of humans and rhesus macaques. additionally, racial differences in pigmentation among human subjects may affect the morphologic appearance of the choroid on edi - oct. these results have important implications in human studies, where the ability to define a well - delineated csj for reliable choroidal thickness measurements may be affected by the subject 's race. nonhuman primates are the gold standard among animal models of human macular diseases such as amd because they possess a macula a feature absent in most other mammals. rhesus macaques exhibit amd - like drusen with a prevalence of up to 50%, and even higher in the free - ranging cayo santiago colony in puerto rico. humans and macaques also share genetic polymorphisms in genes such as htra1 (high temperature requirement factor a1) and arms2 (age - related maculopathy susceptibility 2) that are associated with drusen formation. yet, while oct imaging has been widely adopted in human clinical practice, the in vivo characterization of the retina and choroid using oct in rhesus macaques has not been pursued at length. early work by toth. demonstrated the feasibility of using oct in primates, while a few later studies employed oct in macaques to visualize laser - induced choroidal neovascularization. the ocular anatomy of rhesus macaques has remarkable resemblance to human eyes, allowing ocular imaging technologies in clinical use to be readily adapted for primate research. as such, our comparative analysis of choroidal morphology in humans and macaques establishes a critical basis for future translational research involving choroidal imaging in these animals. in this study, we found that the distribution and pigmentation of uveal melanocytes are major determinants of choroidal morphology on edi - oct. in macaques, the melanocytes are bigger, more darkly pigmented, and densely dispersed across the choroid stroma up to the posterior border of the choriocapillaris, while in humans they are smaller, less pigmented, and more loosely distributed in the posterior choroid, as described previously. this difference accounts for the more distinctly hyporeflective choriocapillaris band seen in macaques and better delineation of the csj in humans on edi - oct. interestingly, the retinal layers and rpe appear similar on oct, and the rpe pigmentation is also similar on histology between the two species. thus, the melanin pigment in the uvea may be a more important contributor to choroidal morphology on oct imaging than rpe pigment. oct imaging of patients with ocular albinism shows better visualization of choroidal morphology without edi due to their reduced pigmentation. compared to amelanotic nevi, pigmented choroidal nevi on edi - oct appear as a highly reflective band with posterior shadowing. patients with choroidal melanocytosis also show an apparent increase in choroidal thickness and perivascular stromal tissue. in animal studies, comparisons of oct reflectivities from pigmented and albino mice showed differential appearance of the choroid and sclera due to the signal attenuation from choroidal melanin in pigmented mice. light scattered by tissues containing melanin is also depolarized, as shown in comparisons between pigmented and albino rats, supporting the use of polarization - sensitive oct technology for imaging pigmented structures in the eye. we note in our study that race may be an important factor in determining choroidal morphology on edi - oct, presumably due to differences in choroidal pigmentation. however, a person 's self - identified race is a poor surrogate for pigmentation, and skin color may not correlate well with uveal pigmentation. other ocular features such as iris color may be a better marker for uveal pigmentation and oct morphology. nevertheless, both race and iris color are factors associated with amd risk, and a better understanding of melanin pigmentation in the choroid may provide important insights into the pathophysiology of these retinal diseases. importantly, the reduced visibility of the csj and lower reliability of choroidal thickness measurements in blacks should be taken into account in clinical studies involving multiethnic populations. the limitations of our study include the small sample size and the retrospective selection of age - matched human subjects for comparative analyses. many of the normal human eyes were contralateral eyes of subjects with unilateral pathologies that are not known to affect the macular choroid or fellow eye, but an association can not be completely excluded. also, the variable appearance of the choriocapillaris in humans may reduce segmentation reliability, while the poor visualization of the csj in macaques may lead to an artifactually lower choroidal thickness measurement. future research using oct systems such as swept - source oct devices with longer - wavelength light sources to better visualize deeper ocular structures may help circumvent such limitations. while newer technologies such as oct angiography provide better en face visualization of the choriocapillaris, edi - oct remains more widely accessible by taking advantage of the existing generation of sd - oct devices. however, despite some attempts at segmentation of the choriocapillaris or csj, none have yet to become widely accepted owing to their variable and often ambiguous appearance in human subjects. our study of choroidal morphology in rhesus macaques showed a very clearly defined choriocapillaris and poor visualization of the csj, likely due to the abundance and darker pigmentation of uveal melanocytes in these animals. future studies on the relationship between melanin pigment and ocular imaging may improve the development of segmentation algorithms and interpretation of oct findings in retinal and choroidal diseases. | purposeto compare cross - sectional choroidal morphology in rhesus macaque and human eyes using enhanced - depth imaging optical coherence tomography (edi - oct) and histologic analysis.methodsenhanced-depth imaging oct images from 25 rhesus macaque and 30 human eyes were evaluated for choriocapillaris and choroidal scleral junction (csj) visibility in the central macula based on oct reflectivity profiles, and compared with age - matched histologic sections. semiautomated segmentation of the choriocapillaris and csj was used to measure choriocapillary and choroidal thickness, respectively. multivariate regression was performed to determine the association of age, refractive error, and race with choriocapillaris and csj visibility.resultsrhesus macaques exhibit a distinct hyporeflective choriocapillaris layer on edi - oct, while the csj can not be visualized. in contrast, humans show variable reflectivities of the choriocapillaris, with a distinct csj seen in many subjects. histologic sections demonstrate large, darkly pigmented melanocytes that are densely distributed in the macaque choroid, while melanocytes in humans are smaller, less pigmented, and variably distributed. optical coherence tomography reflectivity patterns of the choroid appear to correspond to the density, size, and pigmentation of choroidal melanocytes. mean choriocapillary thickness was similar between the two species (19.3 3.4 vs. 19.8 3.4 m, p = 0.615), but choroidal thickness may be lower in macaques than in humans (191.2 43.0 vs. 266.8 78.0 m, p < 0.001). racial differences in uveal pigmentation also appear to affect the visibility of the choriocapillaris and csj on edi-oct.conclusionspigmented uveal melanocytes affect choroidal morphology on edi - oct in rhesus macaque and human eyes. racial differences in pigmentation may affect choriocapillaris and csj visibility, and may influence the accuracy of choroidal thickness measurements. |
clinical assessment of the range of the eye movements is important in diagnosis and management of strabismus. the direction of the movements from primary position defines these movements as dextroversion, levoversion, supraversion, and infraversion. careful examination of versions helps to detect the underaction or overaction of the extraocular muscles at various directions of gaze (1). guiber (2) first described four different subjective grades of underaction or overaction of the medial and lateral rectus muscles, but he assigned no specific features to the grades of overaction or underaction. one is the limbus test of motility of kestenbaum (3) which intended to determine the shift in relative position of certain fixed points in different gaze positions and the other is the lateral and vertical version light - reflex (lvr) test of urist (4). the normal values of this test in caucasians have been reported (4, 5) and clinical applications were also described (5 - 7), but there were limited data in koreans. it is known that asian people have different anatomical configurations of orbit and lid than caucasians (8, 9) and these racial differences might affect the version movements, but there were few reports on this relationship. most tissues in the human body undergo slow involution with aging and histological examination suggests that this also occurs in the extraocular muscle pulleys (10). in the orbit some studies have documented the age - related changes in oculomotor functions, such as smooth pursuit (11) and the visually controlled eye tracking systems (saccadic, smooth pursuit, and optokinetic) (12). vertical eye movements are also known to become impaired with advancing age (13 - 15). furthermore, little was known about the absolute values of version movements in normal korean elderly. the purpose of this study is to delineate normal values of maximum versions in healthy koreans and investigate age - associated changes in eye movement using lvr test. two hundred and forty normal healthy korean people, from 4 to 79 yr old who visited department of ophthalmology at holy family hospital, the catholic university of korea from june to november 2006 were studied. the subjects were grouped according to decade of life as 30 subjects in each decade. subjects who had history of previous ocular or periocular surgery, diabetes, and cardiovascular disease were excluded ; and none of them had amblyopia, strabismus, ptosis or neurological disorder. complete ophthalmic examinations including measurement of ocular alignment with cover - uncover test were performed to confirm orthotropia. maximum sustained values of dextroversion and levoversion using lateral version light - reflex test and supraversion and infraversion using vertical version light - reflex test were measured in each eye. to find the dimensions of palpebral fissure, palpebral fissure height (fh) and horizontal palpebral length (fw), inner intercanthal distance (icd) and interpupillary distance (ipd) at the primary position were measured with a transparent ruler. subject 's head was immobilized by an assistant and a penlight was directed exactly between the eyes of the subject from a distance of 25 cm. the examiner maintained the light at the center throughout the test and both the patient and examiner kept their heads in the same centered position with relation to each other. the subject then visually tracked a toy on the examiner 's finger into maximum sustained dextroversion, levoversion, supraversion and infraversion in each eye. subjects were asked to maximize their effort into extreme range of gaze and to maintain fixation during the examination. the light reflex on the sclera of the abducted or depressed eye and on the cornea of adducted or elevated eye was noted, measured and recorded as a hirschberg - type measurement for corneal light reflex (e.g., reference points of 20 at the pupillary margin, 35 at the mid - iris position, and 45 at the corneoscleral limbus, as originally reported by urist [1, 4 ]) and as millimeters onto the sclera for the scleral light reflex (1) (fig. 1). for a sclera light reflex in the semilunar fold, either notes it as " sf " or recorded the number of millimeters from the limbus. to minimize the measurements error, three measurements were recorded at each version of each eye. if a value was greater than 5% different from the other readings at that version, an additional measurement was taken. the same experienced examiner performed all measurements and verbal encouragement was used to ensure stability of the subject 's head and maximum effort into extremes of gaze. the measurements of the right eye were selected for analysis in each case. for statistical analysis, the measurements of the scleral light reflex were converted from millimeters into degrees. in brief, one millimeter displacement of the scleral reflections from the corneoscleral limbus onto the sclera equals to 4.8 of ocular rotation in this geometrical analysis that assuming a scleral diameter of 24.0 mm (standard globe size) (16) (e.g., a lvr measurement of 10 mm on the sclera equals 45 plus [10 mm4.8/mm ] which corresponds to 93). lvr measurements on the sclera were converted into degrees by adding 45 (the ocular rotation of the light reflex to the limbus) to the ocular rotation of the light reflex on the sclera. in addition, for direct comparison of the effect of aging between the four versions, all measurements of lvr were calculated relative to the mean normal measurements of the 3rd decade group and expressed as a percentage for each version (e.g., a lvr measurement of 30 in supraversion was expressed as 79.3% of normal [30/37.8100 ]). regression analysis from the sas program (version 8.0) was used to analyze the aging changes of each type of version. the relationship between horizontal versions and horizontal palpebral length, inner intercanthal distance, and interpupillary distance, and between vertical versions and palpebral fissure height were analyzed using multiple regression analysis. the effect of aging between versions in different gaze positions were compared using multiple comparison tests. two hundred and forty normal healthy korean people, from 4 to 79 yr old who visited department of ophthalmology at holy family hospital, the catholic university of korea from june to november 2006 were studied. the subjects were grouped according to decade of life as 30 subjects in each decade. subjects who had history of previous ocular or periocular surgery, diabetes, and cardiovascular disease were excluded ; and none of them had amblyopia, strabismus, ptosis or neurological disorder. complete ophthalmic examinations including measurement of ocular alignment with cover - uncover test were performed to confirm orthotropia. maximum sustained values of dextroversion and levoversion using lateral version light - reflex test and supraversion and infraversion using vertical version light - reflex test were measured in each eye. to find the dimensions of palpebral fissure, palpebral fissure height (fh) and horizontal palpebral length (fw), inner intercanthal distance (icd) and interpupillary distance (ipd) at the primary position were measured with a transparent ruler. subject 's head was immobilized by an assistant and a penlight was directed exactly between the eyes of the subject from a distance of 25 cm. the examiner maintained the light at the center throughout the test and both the patient and examiner kept their heads in the same centered position with relation to each other. the subject then visually tracked a toy on the examiner 's finger into maximum sustained dextroversion, levoversion, supraversion and infraversion in each eye. subjects were asked to maximize their effort into extreme range of gaze and to maintain fixation during the examination. the light reflex on the sclera of the abducted or depressed eye and on the cornea of adducted or elevated eye was noted, measured and recorded as a hirschberg - type measurement for corneal light reflex (e.g., reference points of 20 at the pupillary margin, 35 at the mid - iris position, and 45 at the corneoscleral limbus, as originally reported by urist [1, 4 ]) and as millimeters onto the sclera for the scleral light reflex (1) (fig. 1). for a sclera light reflex in the semilunar fold, either notes it as " sf " or recorded the number of millimeters from the limbus. to minimize the measurements error, three measurements were recorded at each version of each eye. if a value was greater than 5% different from the other readings at that version, an additional measurement was taken. the same experienced examiner performed all measurements and verbal encouragement was used to ensure stability of the subject 's head and maximum effort into extremes of gaze. the measurements of the right eye were selected for analysis in each case. for statistical analysis, the measurements of the scleral light reflex were converted from millimeters into degrees. in brief, one millimeter displacement of the scleral reflections from the corneoscleral limbus onto the sclera equals to 4.8 of ocular rotation in this geometrical analysis that assuming a scleral diameter of 24.0 mm (standard globe size) (16) (e.g., a lvr measurement of 10 mm on the sclera equals 45 plus [10 mm4.8/mm ] which corresponds to 93). lvr measurements on the sclera were converted into degrees by adding 45 (the ocular rotation of the light reflex to the limbus) to the ocular rotation of the light reflex on the sclera. in addition, for direct comparison of the effect of aging between the four versions, all measurements of lvr were calculated relative to the mean normal measurements of the 3rd decade group and expressed as a percentage for each version (e.g., a lvr measurement of 30 in supraversion was expressed as 79.3% of normal [30/37.8100 ]). regression analysis from the sas program (version 8.0) was used to analyze the aging changes of each type of version. the relationship between horizontal versions and horizontal palpebral length, inner intercanthal distance, and interpupillary distance, and between vertical versions and palpebral fissure height the effect of aging between versions in different gaze positions were compared using multiple comparison tests. among 240 subjects, 168 (70%) were female and 72 were male. in each decade, the preponderance of females except the 1st decade was homogeneous among the different age groups (table 1). the mean (standard deviation, sd) maximum lvr values in dextroversion, levoversion, supraversion and infraversion of the right eye were 7.71.6 mm (range 4 - 10), 41.60.9 (range 20 - 69), 33.94.9 (range 20 - 45), and 7.71.7 mm (range 3 - 11), respectively in normal koreans. 2 demonstrates the mean lvr measurements in millimeters for dextroversion and infraversion versus age of the right eye. mean lvr values in degrees for levoversion and supraversion of the right eye were shown in fig. all the values were increased until the 3rd decade, and then decreased with advancing age. in levoversion, the mean values of the 3rd decade group showed maximum in all four movements and the absolute maximum values in detroversion, levoversion, supraversion, and infraversion were 9.10.9 mm (range 8 - 10), 51.38.7 (range 35 - 64), 37.83.9 (range 35 - 45), and 8.90.9 mm (range 8 - 11) respectively. from the 6th decade up, no one showed any normal mean value of lvr in any of the version. asymmetrical versions (difference within 5 or 1 mm) in both eyes were 4.6, 7.9, 0.8, and 2.9% in dextroversion, levoversion, supraversion and infraversion, respectively. the normal mean values of horizontal palpebral fissure length (fw), palpebral fissure height (fh), inner intercanthal distance (icd), and interpupillary distance (ipd) were 25.81.8 mm (range 20 - 31), 8.21.45 mm (range 5 - 11), 33.92.81 mm (range 25 - 41), and 60.83.5 mm (range 46 - 69), respectively in normal koreans. fh and icd were increased from 1st to 3rd decade then showed a decreasing tendency but was not significant. all lvr values were converted into degrees and calculated as a percentage relative to the mean values of the 3rd decade group to assess the aging changes of versions. all four maximum versions were decreased with aging according to regression lines (p<0.001). 5 plots the calculated mean lvr values in each decade group as a percentage of mean maximum lvr values. levoversion (adductive movement) was the most decreased version (p<0.0001 for each version type), and infraversion (depressive movement) was the least affected compared to levoversion (p<0.0001) and supraversion (p=0.0002), but the decline was not significantly different from dextroversion (p=0.3036) by aging using multiple comparison test. there was no significant correlation between fw, icd, and ipd and horizontal versions (p=0.1761, 0.8214, and 0.5433 respectively in dextroversion, p=0.1566, 0.6559, and 0.4545 respectively in levoversion), and between fh and vertical versions (p=0.3093 in supraversion, p=0.6580 in infraversion) in multiple regression analysis. this study established baseline normative values of maximum versions and aging changes of four version types in a large sample of healthy koreans. this study is the first, to my knowledge, to quantify the four version movements and correlate them with age objectively in koreans. the mean maximal sustained versions were found to be 7.7 mm in dextroversion, 41.6 in levoversion, 33.9 in supraversion and 7.7 mm in infraversion in normal koreans. all the versions were maximally expressed at the 3rd decade and their absolute values for dextroversion, levoversion, supraversion, and infraversion were 9.1 mm, 51.3, 37.8, and 8.9 mm, respectively. the absolute mean normal values and even the maximum values of the koreans were very different from those of caucasians which are 10 mm in abduction and depression, and 30 in adduction and elevation (4, 5). contrary to caucasians, the mean adductive movement (levoversion in right eye) value was larger and the mean depression and abductive movements (infraversion and dextroversion) were smaller in koreans. it is unclear why the absolute magnitudes of versions were different from those of caucasians. a possible explanation may be that koreans have relatively shallow orbit and less prominent superior orbital rim compared to caucasians (9). low nasal bridge of the koreans may not obstruct the visual axis of the adducting eye permitting larger adductive movement. koh and yoo (17) studied the amounts of horizontal ocular movements in koreans using three different methods in 2003. among those three methods, they reported the mean amount of abduction and adduction was 9.85 mm and 34.9 using lvr test. their values of abduction were much larger than those of this study, but the age range of their subjects were different from this study. also, they used the hirschberg test which is used in strabismus evaluation when measuring abduction, and furthermore it was somewhat different from the original lvr test that urist had described. although the lvr test utilized the hirschberg scale, it should not to be confused with the hirschberg test where the light on which the patient fixates is moved into the fields of gaze. in the lvr test, the light is stationary (4). it is not exactly known whether the normal values of caucasians were age matched, but in this study we tried to measure the normal values of maximal sustained version angle for all age groups of healthy koreans including children. the results showed a gradual increase up to the 3rd decade and then a gradual decrease in both horizontal and vertical version types with advancing age (fig. 2, 3). to assess the possible aging changes of each version, the measurements were converted as a percentage relative to the mean values of the 3rd decade. furthermore, the extraocular muscles and related structures are fully developed and their functions are arrived to the maximum at this age group, so the mean values of the 3rd decade group were chosen as standard values. these proportional values used in this study can make all comparisons easy and simple, and also enables estimating the aging changes more consistent and rapid. all maximum version movements decreased with advancing age, a finding confirmed to be significant by regression analysis (p<0.001, respectively) (table 2). these findings may useful to differentiating age - related decreases in version from other neurological diseases, such as progressive external ophthalmoplegia and progressive supranuclear palsy. the normal values of version in accordance with age may also be useful in diagnosing elderly patients with presumed rectus muscle paresis or incomitant strabismus. asymmetry between the two eyes also provides important clinical tool for evaluating versions because 84% of healthy subjects in this study showed symmetry between two eyes in all gaze position. extreme lateral version movements appear to be reflexive in nature but these large adductive movements were constant for all subjects, with 62% of subjects showing measurements over 45. in caucasians, higher nose bridge may obstruct the visual axis of adducting eye preventing diplopia. in comparison, it is speculated that the low nasal bridges in koreans may not obstruct the vision of adducting eye, permitting larger adductive movements. the corneal light reflex is usually nasally displaced approximately 1 mm in primary gaze (19), thus approximately 7 less abduction is needed when compared to adduction before the light reflex falls onto the sclera. clark and isenberg (16) studied the range of ocular movements with aging in caucasians and reported the maximum versions decrease by average of 0.5% to 1.0% per year of life from the third to ninth decade with subjects ranged in age from 23 to 84 yr. the version most affected by age is elevation in their study, contrary to our study. they suggested that the least utilized volitional field of upgaze during normal life attributes to the most affection of elevation by aging. chamberlain (13) measured the limits of supraduction and infraduction and suggested the gradual " disuse " of upgaze with advancing age might cause the diminution of supraduction in the elderly. but he reported differences due to age were not found in downgaze. using a keratometer, yamashiro (14) measured the overall limit of uniocular extent during foveal fixation in elderly subjects, and reported decrease of maximum movements for oldest age group in both depression and elevation. in 2004, oguro. they also noted significantly smaller upgaze than downgaze and suggested the differences of aging process in vertical gaze were due to different gaze mechanisms. different neuronal pathways of upgaze and downgaze systems or functional differences between upward and downward gaze systems was suspected for the possible explanation of differences in aging process of the vertical movements. clark and demer (18) suggested inferior displacement of the horizontal rectus muscles in older patients, as shown in high - resolution orbital mri, and that there may be a peripheral mechanism for the reduction in supraduction with age. in this study, supraversion and dextroversion showed similar reduction with aging, but supraversion was relatively well preserved in koreans. comparing the two vertical versions, supraversion decreased more than infraversion (multiple comparison test, p=0.0002), a finding similar to previous duction studies. the striated muscles fibers within extraocular muscles should change and lose functions at same rate. gravity would not play a substantial role in rotation of the globe downward about its center of rotation. one hypothetical explanation involves the tight coupling of the inferior rectus muscle to the lower lid retractors (19). as the lower lid sags with age, inferior rectus muscle such an effort may increase the mechanical effectiveness of the inferior rectus to the other muscles. the current study also measured the normal mean values of horizontal palpebral fissure length (fw) and height (fh), internal intercanthal distance (icd), and interpupillary distance (ipd). the mean normal measurements of fw, fh, icd, and ipd were found to be 25.8 mm, 8.2 mm, 33.9 mm, and 60.7 mm, respectively. these findings were comparable to published results of normal values of anthropometric studies in koreans (20 - 23). in this study, fh and icd showed slight decreasing tendency by age and these findings were similar to the report of park. we tired to find out whether the dimensions of palpebral fissure could affect the versions with advancing age. there were no significant correlations between fw, icd, pd and horizontal versions with aging. the anatomic characteristics of eye - lids and related structures did not affect to versions with aging in koreans. possible limitations of this study include a sampling error related to subjects because they were patients of eye clinic and may be more prone to abnormal eye movements although none had strabismus. true orthotropia is rare and small degree of esphoria or exophoria is a common finding in straight - appearing eyes of normal population. for more accurate determination of the normal versions in koreans without any effects from heterophoric or heterotropic state, subjects with orthotropia only were strictly selected in this study using repeated cover and cover - uncover test which using adequate fixation objects, reliable cooperation, and wearing refractive correction. another potential bias was the ages of the subjects were not masked from the examiner. the digital photos documenting the light reflex on the globe may compensate for this observer bias. finally the measurements error of author may affect the results, but the present author had performed the lvr test on more than 1,000 patients prior to beginning the study. in conclusion, this study standardizes the normal maximum versions and aging changes of versions in koreans and these in turn make it possible to induce a normal template that utilized fast and easy, simple in clinical office examination for detecting incomitant or paralytic strabismus, especially in elderly patients. | the purpose of this study is to estimate normative values of maximum versions in healthy koreans and investigate age - associated changes in eye movement, using lateral and vertical version light - reflex (lvr) test. two hundred forty normal healthy subjects whose corrected visual acuity was better than 20/50 in both eyes, from 4 to 79 yr old (30 subjects in each decade) were studied. maximum sustained values of dextroversion, levoversion, supraversion, and infraversion in each eye were measured using lvr test. changes of versions according to age were analyzed. mean normal value of dextroversion, levoversion, supraversion, and infraversion in normal koreans was 7.7 mm, 41.6, 33.9, and 7.7 mm respectively. contrary to values of caucasians, levoversion (adductive movement) was more excessive and infraversion (depression) was smaller in koreans. all versions were decreased with aging (p<0.001 for all). levoversion had the largest decrease with aging compared with other versions (p<0.001, respectively) and infraversion had the least decrease with age than levoversion (p<0.001), supraversion (p<0.001). all maximal sustained versions were decreased with aging but the ranges of ocular movements in koreans were different with caucasians as version least affected and most affected by age was infraversion and levoversion in koreans. the study standardized normal maximal versions and aging changes of versions in koreans. |
myxoma is the most common benign cardiac primary tumors, accounting about 50% of cardiac tumors and is more prevalent among females. because of different size of myxomas and their location in the different chambers of heart, patients with atrial myxoma usually present with cardiovascular symptoms such as heart failure and pulmonary hypertension, secondary to mitral valve obstruction. constitutional symptoms of atrial myxoma include weight loss, fatigue, fever, clubbing, anemia, orthopnea, and acute pulmonary edema. on the other hand, myxomas may cause life - threatening cardiac symptoms, e.g., syncope, requiring emergency surgery. two - dimensional echocardiography is the primary diagnostic imaging, not only as a complement to conventional echocardiography in the assessment of left atrial myxomas but also as a method of differentiating such masses from extra - cardiac tumors compressing the atrium. echocardiography is a helpful method that can determine the tumor size, shape, and mobility. here she dropped during the second prostration in prayer and decreased her mental state, with no prior history of syncope, shortness of breath, or chest pain. she had no significant past medical history, but she had a positive family history of early cardiovascular disease. at presentation, the patient was afebrile and had normal vital signs (heart rate 80 bpm, blood pressure 110/70 mmhg, oxygen saturation 97% while breathing room air) and jugular venous pressure (jvp) was normal. a 12-lead electrocardiogram revealed normal sinus rhythm with nonspecific minor st - t abnormalities. on cardiac examination, a transthoracic echocardiogram (tte) was performed which showed the presence of a pediculated mass in the left atrium, with an appearance suggestive of atrial myxoma, that was prolapsed through the mitral valve towards the left ventricle by changing patient 's position [figure 1a c ]. (a - c) transthoracic echocardiography : chambers view showing a large mobile left atrial mass prolapsing through mitral valve the patient was referred for urgent surgery. she recovered without major complication and was discharged. at 2-year follow - up, she remains with excellent cardiac function. myxoma is the most common primary benign cardiac tumor. despite the fact that they can be discovered unexpectedly in asymptomatic patients, myxomas usually present with nonspecific signs and symptoms. depending on myxomas locations and their association with the heart structures, they can provide symptoms. tumors that origins from the left atrium, especially if mobile and large, may result in symptoms similar to mitral stenosis due to obstruction of atrio - ventricular blood flow. these symptoms include : exertional dyspnea, paroxysmal nocturnal dyspnea, orthopnea, fatigue, and syncope. in our patient, the tumor plop of myxoma is a protodiastolic murmur that is heard 80 to 150 ms after s2. tumor plop may be mistaken for an opening snap, and tumor obstruction of the valve leads to a diastolic murmur. the central nervous system (cns) symptoms usually call for computed tomography (ct) or magnetic resonance imaging (mri) scan of the brain to exclude embolization or an aneurysm formation, which is sometimes associated with myxomas. it has been suggested as prudent to perform mri scan of the brain in all cardiac myxoma cases to exclude central nervous system (cns) manifestations. in our patient, transthoracic echocardiography reported a mobile and pedunculated mass in the left atrium, which was attached to the interatrial septum. the mass prolapsed into the left ventricle across the mitral valve, resulting in a mitral valve obstruction. recently, the diagnosis of cardiac tumors has improved enormously, and echocardiography has become a choice screening tool for cardiac tumors. the location of the myxoma in the left atrium and its origination from the atrial septum are chief clues for the diagnosis. thus, it is very important to recognize the location of the tumor during echocardiography. as reported in other studies, tte is usually sufficient to make the diagnosis, but if the results are suboptimal, transesophageal echocardiogram (tee) should be employed. in conclusion, echocardiography is likely to be a valuable diagnostic tool in patients who have had a cardiac mass. besides, it also offers the cardiologist and cardiovascular surgeon the chance of more accurate pre- and intra - operative assessment of cardiac masses. | cardiac myxoma is the most frequent benign tumors of heart. a 37-year - old woman dropped during the second prostration in prayer and decreased his mental state, with no prior history of syncope. on cardiac examination, there was an early diastolic sound that was compatible with a tumor plop. a transthoracic echocardiogram was performed which showed the presence of a pediculated mass in the left atrium, with an appearance suggestive of atrial myxoma. atrial myxoma can appear with non - specific symptoms. the best diagnostic method for myxoma is echocardiography that has a high sensitivity |
three different pathways can deliver cytoplasmic components into the lumen of the lysosome for degradation. they are commonly referred to as autophagy (cell self - eating) and include chaperone - mediated autophagy (cma), microautophagy, and macroautophagy. cma involves the direct translocation of specific proteins containing the kferq pentapeptide sequence across the lysosome membrane [1, 2 ]. microautophagy, on the other hand, entails the invagination and pinching off of the lysosomal limiting membrane, which allows the sequestration and elimination of cytoplasmic components. the molecular mechanism underlying the only cellular function that so far has been indisputably assigned to microautophagy is the turnover of peroxisomes under specific conditions in fungi. recently, it has been reported the existence of a microautophagy - like process at the late endosomes, where proteins are selectively incorporated into the vesicles that bud inward at the limiting membrane of these organelles during the multivesicular bodies biogenesis. in contrast to cma and microautophagy, macroautophagy (hereafter referred to as autophagy) entails the formation of a new organelle, the autophagosome, which allows the delivery of a large number of different cargo molecules into the lysosome. autophagy is a primordial and highly conserved intracellular process that occurs in most eukaryotic cells and participates in stress management. this pathway involves the de novo formation of vesicles called autophagosomes, which can engulf entire regions of the cytoplasm, individual organelles, protein aggregates, and invading pathogens (figure 1). the autophagosomes fuse with endosomal compartments to form amphisomes prior to fusion with the lysosome, where their contents are degraded and the resulting metabolites are recycled back to the cytoplasm (figure 1). unique features of the pathway include the double - membrane structure of the autophagosomes, which were originally characterized over 50 years ago from detailed electron microscopy studies. starting in the 1990s yeast mutational studies began the genetic and molecular characterization of the key components required to initiate and build an autophagosome. subsequently, genetic and transgenic studies in plants, worms, fruit flies, mice, and humans have underscored the pathway 's conservation and have begun to unveil the intricate vital role that autophagy plays in the physiology of cells and multicellular organisms. for a long time, autophagy was considered a non - selective pathway induced as a survival mechanism in response to cellular stresses. over the past several years, however, it has become increasingly evident that autophagy also is a highly selective process involved in clearance of excess or dysfunctional organelles, protein aggregates and intracellular pathogens. in this introductory piece, we will briefly discuss the molecular mechanisms of selective types of autophagy and their emerging importance as a quality control to maintain cellular and organismal health, aspects that will be presented in deep in the reviews of this special issue of the international journal of cell biology and highlighted by the research papers. autophagosomes are formed by expansion and sealing of a small cistern known as the phagophore or isolation membrane (figure 1). once complete, they deliver their cargo into the hydrolytic lumen of lysosomes for degradation. a diverse set of components are involved in the biogenesis of autophagosomes, which primarily includes the proteins encoded by the autophagy - related genes (atg). subsequent studies in higher eukaryotes have revealed that these key factors are highly conserved. to date, 36 atg proteins have been identified and 16 are part of the core atg machinery essential for all autophagy - related pathways. upon autophagy induction, these proteins associate following a hierarchical order [8, 9 ] to first mediate the formation of the phagophore and then to expand it into an autophagosome [10, 11 ]. while their molecular functions and their precise contribution during the biogenesis of double - membrane vesicles remain largely unknown, they have been classified in 4 functional groups of genes : (1) the atg1/ulk complex, (2) the phosphatidylinositol 3-kinase (pi3k) complex, (3) the atg9 trafficking system, and (4) the two parallel ubiquitin - like conjugation systems (figure 1). the atg1/ulk complex consists of atg1, atg13, and atg17 in yeast, and ulk1/2, atg13, fip200 and atg101 in mammals [1215 ]. this complex is central in mediating the induction of autophagosome biogenesis and as a result it is the terminal target of various signaling cascades regulating autophagy, such as the tor, insulin, pka, and ampk pathways (figure 1). increased activity of the atg1/ulk kinase is the primary event that determines the acute induction and upregulation of autophagy. it is important to note that ulk1 is part of a protein family and two other members, ulk2 and ulk3, have been shown play a role in autophagy induction as well [14, 17 ]. the expansion of this gene family may reflect the complex regulation and requirements of the pathway in multicellular long - lived organisms. stimulation of the ulk kinases is achieved through an intricate network of phosphorylation and dephosphorylation modifications of the various subunits of the atg1/ulk complex. for example, atg13 is directly phosphorylated by tor and the phosphorylation state of atg13 modulates its binding to atg1 and atg17. inactivation of tor leads to a rapid dephosphorylation of atg13, which increases atg1atg13atg17 complex formation, stimulates the atg1 kinase activity and induces autophagy [18, 19 ]. the matg13 is also essential for autophagy, but seems to directly interact with ulk1, ulk2 and fip200 independently of its phosphorylation state [13, 14 ]. in addition, there are several phosphorylation events within this complex as well, including phosphorylation of matg13 by ulk1, ulk2, and tor ; phosphorylation of fip200 by ulk1 and ulk2 ; phosphorylation of ulk1 and ulk2 by tor [13, 14 ]. yeast contains a single pi3k, vps34, which is present in two different tetrameric complexes that share 3 common subunits, vps34, vps15, and atg6. complex i is required for the induction of autophagy and through its fourth component, atg14, associates to the autophagosomal membranes where the lipid kinase activity of vps34 is essential for generating the phosphatidylinositol-3-phosphate (pi3p) that permits the recruitment of other atg proteins [9, 21 ] (figure 1). complex ii contains vps38 as the fourth subunit and it is involved in endosomal trafficking and vacuole biogenesis. there are three types of pi3k in mammals : class i, ii, and iii. the functions of class ii pi3k remains largely unknown, but both classes i and iii pi3ks are involved in autophagy. while class i pi3k is principally implicated in the modulation of signalling cascades, class iii pi3k complexes regulate organelle biogenesis and, like yeast, contain three common components : hvps34, p150 (vps15 ortholog), and beclin 1 (atg6 ortholog). the counterparts of atg14 and vps38 are called atg14l / barkor and uvrag, respectively [2224 ]. the atg14l - containing complex plays a central role in autophagy and functions very similarly as the yeast complex i by directing the class iii pi3k complex i to the phagophore to produce pi3p and initiate the recruitment of the atg machinery (figure 1). atg14l is thought to be present on the er irrespective of autophagy induction. upon starvation, atg14l localizes to autophagosomal membranes. importantly, depletion of atg14l reduces pi3p production, impairs the formation of autophagosomal precursor structures, and inhibits autophagy [8, 24, 26, 27 ]. complex also regulates autophagy but it appears to act at the intersection between autophagy and the endosomal transport pathways. uvrag initially associates with the bar - domain protein bif-1, which may regulate matg9 trafficking from the trans - golgi network (tgn) [28, 29 ]. uvrag then interacts with the class c vps / hops protein complex, promoting the fusion of autophagosomes with late endosomes and/or lysosomes. finally, the uvrag - containing class iii protein complex binds to rubicon, a late endosomal and lysosomal protein that suppresses autophagosome maturation by reducing hvps34 activity [26, 31 ]. importantly, both the atg14l- and uvrag - containing complexes interact through beclin 1 with ambra1, which in turn tethers these protein complexes to the cytoskeleton via an interaction with dynein [32, 33 ]. following the induction of autophagy, ulk1 phosphorylates ambra1 thus releasing the class iii pi3k complexes from dynein and their subsequent relocalization triggers autophagosome formation. therefore, ambra1 constitutes a direct regulatory link between the atg1/ulk1 and the pi3k complexes. together with the atg1/ulk and the pi3k complexes, atg9 is one of the first factors localizing to the preautophagosomal structure or phagophore assembly site (pas), the structure believed to be the precursor of the phagophore [9, 34 ] (figure 1). it is distributed to the pas and multiple additional cytoplasmic tubulovesicular compartments derived from the golgi [3537 ]. atg9 cycles between these two locations and consequently it is thought to serve as a membrane carrier providing the lipid building blocks for the expanding phagophore. one of the established functions of atg9 is that it leads to the formation of the yeast pas when at least one of the cytoplasmic tubulovesicular compartments translocates near the vacuole. retrieval transport of yeast atg9 from the pas and/or complete autophagosome is mediated by the atg2-atg18 complex and appears to be regulated by the atg1/ulk and pi3k complexes. matg9 localizes to the tgn and late endosomes and redistributes to autophagosomal structures upon the induction of autophagy (figure 1), further promoting pathway activity [29, 4042 ]. as in yeast, cycling of matg9 between locations also requires the atg1/ulk complex and kinase activity hvps34 [39, 43 ]. the core atg machinery also entails two ubiquitin - like proteins, atg12 and atg8/microtubule - associated protein 1 (map1)-light chain 3 (lc3), and their respective, partially overlapping, conjugation systems [4446 ] (figure 1). atg12 is conjugated to atg5 through the activity of the atg7 (e1-like) and the atg10 (e2-like) enzymes. the atg12atg5 conjugate then interacts with atg16, which oligomerizes to form a large multimeric complex. atg8/lc3 is cleaved at its c terminus by the atg4 protease to generate the cytosolic lc3-i with a c - terminal glycine residue, which is then conjugated to phosphatidylethanolamine (pe) in a reaction that requires atg7 and the e2-like enzyme atg3. this lipidated form of lc3 (lc3-ii) is attached to both faces of the phagophore membrane. these two ubiquitination - like systems appear to be closely interconnected. on one hand, the multimeric atg12-atg5-atg16 complex localizes to the phagophore and acts as an e3-like enzyme, determining the site of atg8/lc3 lipidation [47, 48 ]. on the other hand, the atg8/lc3 conjugation machinery seems to be essential for the optimal functioning of the atg12 conjugation system. in atg3-deficient mice, atg12-atg5 conjugation is markedly reduced, and normal dissociation of the atg12-atg5-atg16 complex from the phagophore is delayed. some evidences suggest that these two conjugation systems also function together during the expansion and closure of the phagophore. for example, overexpression of an inactive mutant of atg4 inhibits the lipidation of lc3 and leads to the accumulation of a number of nearly complete autophagosomes. while controversial, it has been postulated that atg8/lc3 also possesses fusogenic properties, thus mediating the assembly of the autophagic membrane [51, 52 ]. it has to be noted that mammals possess at least 7 genes coding for lc3/atg8 proteins that can be grouped into three subfamilies : (1) the lc3 subfamily containing lc3a, lc3b, lc3b2 and lc3c ; (2) the gammaaminobutyrate receptor - associated protein (gabarap) subfamily comprising gabarap and gabarapl1 (also called gec-1) ; (3) the golgi - associated atpase enhancer of 16 kda (gate-16) protein (also called gabarap - l2/gef2). although in vivo studies show that they are all conjugated to pe, they appear to have evolved complex nonredundant functions. the origin of the membranes composing autophagosomes is a long - standing mystery in the field of autophagy. a major difficulty in addressing this question has been that phagophores as well as autophagosomes do not contain marker proteins of other subcellular compartments [55, 56 ]. a series of new studies has implicated several cellular organelles as the possible source for the autophagosomal lipid bilayers. the plasma membrane and elements of the trafficking machinery to the cell surface have been linked to the formation of an early autophagosomal intermediate, perhaps the phagophore [5761 ]. it is possible that early endosomal- and/or golgi - derived membranes are also key factors in the initial steps of autophagy [34, 36, 39 ]. the golgi, moreover, appears also important for autophagy by supplying at least in part the extra lipids required for the phagophore expansion [29, 6265 ]. while the relevance of the er in autophagosome biogenesis was already pointed out a long time ago [5, 55, 66, 67 ], recently two electron tomography studies have demonstrated the existence of a physical connection between the er and the forming autophagosomes [68, 69 ]. these analyses have revealed that the er is connected to the outer as well as the inner membrane of the phagophore through points of contact, supporting the notion that lipids could be supplied via direct transfer at the sites of membrane contact. in line with this view, it has been found that atg14l is associated to the er and pi3p is generated on specific subdomains of this organelle from where autophagosomes emerge under autophagy - inducing conditions [25, 70 ] (figure 1). it has also been proposed that the outer membrane of the mitochondria is the main source of the autophagosomal lipid bilayers, but while the experimental evidences appear to show that mitochondria are essential for the phagophore expansion, it remains unclear whether these organelles play a key role in the phagophore biogenesis. the discrepancy between the conclusions of the various studies has not allowed yet drawing a model about the membrane dynamics during autophagosome biogenesis. the different results could be due to the different experimental conditions and model systems used by the various laboratories. alternatively, the lipids forming the autophagosomes could have different sources depending on the cell and the conditions inducing autophagy [72, 73 ]. a third possibility is that the source of phagophore membrane could depend on the nature of the double - membrane vesicle cargo. additional investigations are required to shed light on these issues. despite the potential of curing, quite a substantial range of specific pathological conditions by inducting autophagy nevertheless, there is a variety of chemicals that by acting on signaling cascades that also regulate autophagy permit to trigger this degradative process. these agents fall into two distinct categories based on the mechanism of action ; whether they work through an mtor - dependent (rapamycin or torin) or mtor - independent pathway (e.g., lithium or resveratrol). in addition to these compounds, there are biological molecules such as interferon (ifn) and vitamin d that can be used to stimulate autophagy especially in experimental setups [75, 76 ]. inhibition of autophagy can also be beneficial in specific diseases but as for the inducers there are no compounds that exclusively block this pathway without affecting other cellular processes. the small molecules inhibiting autophagy include wortmannin and 3-methyladenine, which hamper the activity of the pi3k ; bafilomycin a and chloroquine, which impair the degradative activity of lysosomes. it is becoming increasingly evident that autophagy is a highly selective quality control mechanism whose basal levels are important to maintain cellular homeostasis (see below). a number of organelles have been found to be selectively turned over by autophagy and cargo - specific names have been given to distinguish the various selective pathways, including the er (reticulophagy or erphagy), peroxisomes (pexophagy), mitochondria (mitophagy), lipid droplets (lipophagy), secretory granules (zymophagy), and even parts of the nucleus (nucleophagy). moreover, pathogens (xenophagy), ribosomes (ribophagy), and aggregate - prone proteins (aggrephagy) are specifically targeted for degradation by autophagy. selective types of autophagy perform a cellular quality control function and therefore they must be able to distinguish their substrates, such as protein aggregates or dysfunctional mitochondria, from their functional counterparts. the molecular mechanisms underlying cargo selection and regulation of selective types of autophagy are still largely unknown. this has been an area of intense research during the last years and our understanding of the various selective types of autophagy is starting to unravel. a recent genome - wide small interfering rna screen aimed at identifying mammalian genes required for selective autophagy found 141 candidate genes to be required for viral autophagy and 96 of those genes were also required for parkin - mediated mitophagy. in general, these pathways appear to rely upon specific cargo - recognizing autophagy receptors, which connect the cargo to the autophagic membranes. the autophagy receptors might also interact with specificity adaptors, which function as scaffolding proteins that bring the cargo - receptor complex in contact with the core atg machinery to allow the specific sequestration of the substrate. the selective types of autophagy appear to rely on the same molecular core machinery as non - selective (starvation - induced) bulk autophagy. in contrast, the autophagy receptors and specificity adapters do not seem to be required for nonselective autophagy. autophagy receptors are defined as proteins being able to interact directly with both the autophagosome cargo and the atg8/lc3 family members through a specific (wxxl) sequence, commonly referred to as the lc3-interacting region (lir) motif or the lc3 recognition sequences (lrs). based on comparison of lir domains from more than 20 autophagy receptors it was found that the lir consensus motif is an eight amino acids long sequence that can be written d / e - d / e - d / e - w / f / y - x - x - l / i / v. although not an absolute requirement, usually there is at least one acidic residue upstream of the w - site. the terminal l - site is occupied by a hydrophobic residue, either l, i, or v. the lir motifs of several autophagy receptors have been found to interact both with lc3 and gabarap family members in vitro, but whether this reflects a physiological interaction remains to be clarified in most cases. it should be pointed out that not all lir - containing proteins are autophagy cargo receptors. some lir - containing proteins, like atg3 and atg4b, are recruited to autophagic membranes to perform their function in autophagosome formation [84, 85 ], whereas others like fyve and coiled - coil domain - containing protein 1 (fyco1) interact with lc3 to facilitate autophagosome transport and maturation. others might use an lir motif to become degraded, like dishevelled, an adaptor protein in the wnt signalling pathway. the adaptor proteins are less well - described, but seem to interact with autophagy receptors and work as scaffold proteins recruiting and assembling the atg machinery required to generate autophagosomes around the cargo targeted to degradation the list of specific autophagy receptors is rapidly growing and the role of several of them in different types of selective autophagy will be described in detail in the reviews of this special issue. here we will briefly discuss the best studied form of selective autophagy, the yeast cytosol to vacuole targeting (cvt) pathway, as well as the best studied mammalian autophagy receptor, p62/sequestosome 1 (sqstm1) (figure 2). the cvt pathway is a biosynthetic process mediating the transport of the three vacuolar hydrolases, aminopeptidase 1 (ape1), aminopeptidase 4 (ape4) and -mannosidase (ams1), and the ty1 transposome into the vacuole [90, 91 ]. ape1 is synthesized as a cytosolic precursor (prape1), which multimerizes into the higher order ape1 oligomer, to which ape4, ams1, and ty1 associate to form the so - called cvt complex, prior to being sequestered into a small autophagosome - like cvt vesicle. sequestration of the cvt complex into cvt vesicles is a multistep process, which requires the autophagy receptor atg19, which facilitates binding to atg8 at the pas, as well as the adaptor protein atg11 (figure 2(a)). atg11 acts as a scaffold protein by directing the cvt complex and atg9 reservoirs translocation to the pas in an actin - dependent way and then recruiting the atg1/ulk complex [40, 93 ]. the pi3p - binding proteins atg20, atg21, and atg24 are also required for the cvt pathway [94, 95 ], but their precise function remains to be elucidated. interestingly, atg11 overexpression was found to recruit more atg8 and atg9 to the pas resulting in more cvt vesicles. this observation indicates that atg11 levels could regulate the rate of selective autophagy, and maybe also the size of the cargo - containing autophagosomes in yeast [90, 96 ]. indeed, a series of studies has revealed that atg11 is also involved in other types of selective autophagy such as mitophagy and pexophagy. however, the autophagy receptors involved in the different atg11-dependent types of selective autophagy are different as atg32 is required for mitophagy [97, 98 ], whereas atg30 is essential for pexophagy. like atg19 mammalian cells appear to not possess an atg11 homologue, and further studies are necessary to delineate the molecular machinery involved in sequestration and targeting of different cargoes for degradation by autophagy in higher eukaryotes. the mechanism of the cvt pathway is reminiscent of the selective form of mammalian autophagy called aggrephagy, which involves degradation of misfolded and unwanted proteins by packing them into ubiquitinated aggregates. in both cases aggregation of the substrate (prape1 or misfolded proteins) is required prior to sequestration into cvt vesicles or autophagosomes, respectively [100102 ]. similar to cvt vesicles, aggregate - containing autophagosomes appear to be largely devoid of cytosolic components suggesting that the vesicle membrane expands tightly around its cargo. aggrephagy also depends on proteins with exclusive functions in substrate selection and targeting [81, 88, 100, 103 ]. the autophagy receptors p62 and neighbour of brca1 gene (nbr1) bind both ubiquitinated protein aggregates through an ubiquitin - associated (uba) domain and to lc3 via their lir motifs and, thereby, promote the specific autophagic degradation of ubiquitinated proteins (figure 2(b)) [81, 82, 100, 103, 104 ]. nbr1 and p62 also contain an n - terminal phox and bem1p (pb1) domain through which they can oligomerize, or interact with other pb1-containing binding partners. in addition to being a cargo receptor for protein aggregates, p62 has been implicated in autophagic degradation of other ubiquitinated substrates such as intracellular bacteria, viral capsid proteins, the midbody remnant formed after cytokinesis, peroxisomes [108, 109 ], damaged mitochondria [110, 111 ], and bacteriocidal precursor proteins. the pb1 domain was recently found to be required for p62 to localize to the autophagosome formation site adjacent to the er, suggesting that it could target ubiquitinated cargo to the site of autophagosome formation or alternatively promote the assembly of the atg machinery at this location. the large scaffolding protein autophagy - linked fyve (alfy) appears to have a similar function as the specificity adaptor atg11. alfy is recruited to aggregate - prone proteins through its interaction with p62 and through a direct interaction with atg5 and pi3p it serves to recruit the core atg machinery and allow formation of autophagic membranes around the protein aggregate (figure 2(b)). interestingly, alfy is recruited from the nucleus to cytoplasmic ubiquitin - positive structures upon cell stress suggesting that it might regulate the level of aggrephagy. in line with this, it was found that overexpression of alfy in mouse and fly models of huntington 's disease reduced the number of protein inclusions. it will be interesting to determine whether alfy, as p62, is involved in other selective types of autophagy such as the one eliminating midbody ring structures or mitochondria. it is well known that posttranslational modifications like phosphorylation and ubiquitination are involved in the regulation of the activity of proteins involved in autophagy and degradation of autophagic cargo proteins, respectively. the fact that the core atg machinery is required for both nonselective and selective types of autophagy gives raise to the question of whether these two types of autophagy may compete for the same molecular machinery. such a competition could be detrimental for the cells undergoing starvation and to avoid this, there might be a tight regulation of the expression level and/or activity of the proteins specifically involved the selective autophagy. it has recently been proposed that phosphorylation of autophagy receptors might be a general mechanism for the regulation of selective autophagy. dikic and coworkers noted that several autophagy receptors contain conserved serine residues adjacent to their lir motifs and indeed, the tank binding kinase 1 (tbk1) was found to phosphorylate a serine residue close to the lir motif of the autophagy receptor optineurin. this modification enhances the lc3 binding affinity of optineurin and promotes selective autophagy of ubiquitinated cytosolic salmonella enterica. in yeast, phosphorylation of atg32, the autophagy receptor for mitophagy, by mitogen - activated protein kinases was found to be required for mitophagy [116, 117 ]. the atg8/lc3 proteins themselves have also been found to become phosphorylated and recent works have identified specific phosphorylation sites for protein kinase a (pka) and protein kinase c (pkc) in the n - terminal region of lc3. interestingly, the n - terminal of lc3 is involved in the binding of lc3 to lir - containing proteins. it is therefore tempting to speculate that phosphorylation of the pka and pkc sites might facilitate or prevent the interaction of lc3 with lir - containing proteins such as p62. it has been found that phosphorylation of the pka site, which is conserved in all mammalian lc3 isoforms, but not in gabarap, inhibits recruitment of lc3 into autophagosomes. the role of ubiquitin in autophagy has so far been ascribed as a signal for cargo degradation. ubiquitination of aggregate prone proteins, as well as bacteria and mitochondria, has been found to serve as a signal for recognition by autophagy receptors like p62 and nbr1, which are themselves also degraded together with the cargo that they associate with. the in vivo specificity of p62 and nbr1 toward ubiquitin signals remains to be established under the different physiological conditions. interestingly, it was recently found that casein kinase 2- (ck2-) mediated phosphorylation of the p62 uba domain increases the binding affinity of this motif for polyubiquitin chains leading to more efficient targeting of polyubiquitinated proteins to autophagy. ck2 overexpression or phosphatase inhibition reduced the formation of aggregates containing the polyglutamine - expanded huntingtin exon1 fragments in a p62-dependent manner. the e3 ligases involved in ubiquitination of different autophagic cargo largely remains to be identified. however, it is known that the e3 ligases parkin and rnf185 both regulate mitophagy [122, 123 ]. smurf1 (smad - specific e3 ubiquitin protein ligase 1) was recently also implicated in mitophagy, as well as in autophagic targeting of viral particles. interestingly, the role of smurf1 in selective autophagy seems to be independent of its e3 ligase activity, but it rather depends on its membrane - targeting c2 domain, although the exact mechanism involved remains to be elucidated. it is also not clear whether ubiquitination could serve as a signal to regulate the activity or binding selectivity of proteins directly involved in autophagy, and whether this in some way could regulate selective autophagy. the role of ubiquitin - like proteins as sumo and nedd in autophagy is also unexplored. the histone de - acetylase 6 (hdac6), initially found to mediate transport of misfolded proteins to the aggresome, was lately implicated in maturation of ubiquitin - positive autophagosomes. the fact that hdac6 overproduction in fly eyes expressing expanded polyq proteins is neuroprotective further indicates that hdac6 activity stimulates aggrephagy. furthermore, the acetylation of an aggrephagy cargo protein, muntant huntingtin, the protein causing huntington 's disease, is important for its degradation by autophagy. the acetyl transferase(s) involved in these forms of selective autophagy is currently unknown, but understanding the role of acetylation in relation to various aspects of autophagy is an emerging field and it will very likely provide more mechanistic insights into these pathways. basal autophagy acts as the quality control pathway for cytoplasmic components and it is crucial to maintain the homeostasis of various postmitotic cells. while this quality control could be partially achieved by nonselective autophagy, growing lines of evidence have demonstrated that specific proteins, organelles, and invading bacteria are specifically degraded by autophagy (figure 3). mice deficient in autophagy die either in utero (e.g., beclin 1 and fip200 knockout mice) [130132 ] or within 24 hours after birth due, at least in part, to a deficiency in the mobilization of amino acids from various tissues (e.g., atg3, atg5, atg7, atg9, and atg16l knockout mice) [49, 133136 ]. as a result, to investigate the physiological roles of autophagy, conditional knockout mice for atg5, atg7, or fip200 and various tissue - specific atg knockout mice have been established and analyzed [133, 137, 138 ]. for example, the liver - specific atg7-deficient mouse displayed severe hepatomegaly accompanied by hepatocyte hypertrophy, resulting in severe liver injuries. mice lacking atg5, atg7, or fip200 in the central nervous system exhibited behavioral deficits, such as abnormal limb - clasping reflexes and reduction of coordinated movement as well as massive neuronal loss in the cerebral and cerebellar cortices [137139 ]. loss of atg5 in cardiac muscle caused cardiac hypertrophy, left ventricular dilatation, and systolic dysfunction. skeletal muscle - specific atg5 or atg7 knockout mice showed age - dependent muscle atrophy [141, 142 ]. pancreatic cell - specific atg7 knockout animals exhibited degeneration of islets and impaired glucose tolerance with reduced insulin secretion [143, 144 ]. podocyte - specific deletion of atg5 caused glomerulosclerosis in aging mice and these animals displayed increased susceptibility to proteinuric diseases caused by puromycin aminonucleoside and adriamycin. proximal tubule - specific atg5 knockout mice were susceptible to ischemia - reperfusion injury. finally, deletion of atg7 in bronchial epithelial cells resulted in hyperresponsiveness to cholinergic stimuli. all together, these results undoubtedly indicate that basal autophagy prevents numerous life - threatening diseases ultrastructural analyses of the mutant mice revealed a marked accumulation of swollen and deformed mitochondria in the mutant hepatocytes, pancreatic cells [143, 144 ], cardiac and skeletal myocytes [140, 141 ] and neurons, but also the appearance of concentric membranous structures consisting of er or sarcoplasmic reticulum in hepatocytes, neuronal axons [137, 139 ] and skeletal myocytes, as well as an increased number of peroxisomes and lipid droplets in hepatocytes [133, 148 ]. in addition to the accumulation of aberrant organelles, histological analyses of tissues with defective autophagy showed the amassment of polyubiquitylated proteins in almost all tissues (although the level varied from one region to another) forming inclusion bodies whose size and number increased with aging. consequently, basal autophagy also acts as the quality control machinery for cytoplasmic organelles (figure 3(a)). although this could be partially achieved by bulk autophagy, these observations point to the existence of selective types of autophagy, a notion that is now supported by experimental data. p62/sqstm1 is the best - characterized disease - related autophagy receptor and a ubiquitously expressed cellular protein conserved among metazoan but not in plants and fungi. besides a role of p62 as the receptor, suppression of autophagy is usually accompanied by an accumulation of p62 mostly in large aggregates also positive for ubiquitin (figure 3(a)) [104, 150 ]. ubiquitin and p62-positive inclusion bodies have been detected in numerous neurodegenerative diseases (i.e., alzheimer 's disease, parkinson 's disease, and amyotrophic lateral sclerosis), liver disorders (i.e., alcoholic hepatitis and steatohepatitis), and cancers (i.e., malignant glioma and hepatocellular carcinoma). very interestingly, the p62-positive aggregates observed in hepatocytes and neurons of liver- and brain - specific atg7 deficient mice, respectively, as well as in human hepatocellular carcinoma cells, are completely dispersed by the additional loss of p62 strongly implicating involvement of p62 in the formation of disease - related inclusion bodies [104, 152 ]. through its self - oligomerization for example, this protein functions as a signaling hub that may determine whether cells survive by activating the traf6nf-b pathway, or die by facilitating the aggregation of caspase 8 and the downstream effector caspases [153, 154 ]. on the other hand, p62 interacts with the nrf2-binding site on keap1, a component of the cullin 3-type ubiquitin ligase for nrf2, resulting in stabilization of nrf2 and transcriptional activation of nrf2 target genes including a battery of antioxidant proteins [155159 ]. it is thus plausible that excess accumulation or mutation of p62 leads to hyperactivation of these signaling pathways, resulting in a disease onset (figure 3(b)). paget 's disease of bone is a chronic and metabolic bone disorder that is characterized by an increased bone turnover within discrete lesions throughout the skeleton. mutations in the p62 gene, in particular in its uba domain, can cause this illness. a proposed model explaining how p62 mutations lead to the paget 's disease of bone is the following : mutations of the uba domain cause an impairment in the interaction between p62 and ubiquitinated traf6 and/or cyld, an enzyme deubiquitinating traf6, which in turn enhances the activation of the nf-b signaling pathway and the resulting increased osteoclastogenesis (figure 3(b)). if proven, this molecular scenario could open the possibility of using autophagy enhancers as a therapy to cure paget 's disease of bone. it is established that autophagy has a tumor - suppressor role and several autophagy gene products including beclin1 and uvrag are known to function as tumor suppressor proteins. the tumor - suppressor role of autophagy appears to be important particularly in the liver. spontaneous tumorigenesis is observed in the livers of mice with either a systemic mosaic deletion of atg5 or a hepatocyte - specific atg7 deletion [152, 162 ]. importantly, no tumors are formed in other organs in atg5 mosaically deleted mice. enlarged mitochondria, whose functions are at least partially impaired, accumulate in atg5- or atg7-deficient hepatocytes [152, 162 ]. this observation is in line with the previous data obtained in ibmk cell lines showing that both the oxidative stress and genomic damage responses are activated by loss of autophagy [163, 164 ]. again, it is clear that accumulation of p62, at least partially, contributes to tumor growth because the size of the atg7 liver tumors is reduced by the additional deletion of p62, which may cause a dysregulation of nf-b signaling and/or a persistent activation of nrf2. loss of autophagy is considered to lead to a delay in the global turnover of cytoplasmic components and/or to an impaired degradation of substrates destined for the proteasome. both observations could partially explain the accumulation of misfolded and/or unfolded proteins that is followed by the formation of inclusion bodies. as discussed above, p62 and nbr1 act as autophagy receptors for ubiquitinated cargos such as protein aggregates, mitochondria, midbody rings, bacteria, ribosomal proteins and virus capsids [83, 168 ] (figure 3). although these studies suggest the role of p62 as an ubiquitin receptor, it remains to be established whether soluble ubiquitinated proteins are also degraded one - by - one by p62 and possibly nbr1. a mass spectrometric analysis has clearly demonstrated the accumulation of all detectable topologies of ubiquitin chain in atg deficient livers and brains, indicating that specific polyubiquitin chain linkage is not the decisive signal for autophagic degradation. because the increase in ubiquitin conjugates in the atg7 deficient liver and brain is completely suppressed by additional knockout of either p62 or nrf2, accumulation of ubiquitinated proteins in tissues defective in autophagy might be attributed to p62-mediated activation of nrf2, resulting in global transcriptional changes to ubiquitin - associated genes. further studies are needed to precisely elucidate the degradation mechanism of soluble ubiquitinated proteins by autophagy. concomitant with the energy production through oxidative phosphorylation, mitochondria also generate reactive oxygen species (ros), which cause damage through the oxidation of proteins, lipids and dna often inducing cell death. therefore, the quality control of mitochondria is essential to maintain cellular homeostasis and this process appears to be achieved via autophagy. it has been postulated that mitophagy contributes to differentiation and development by participating to the intracellular remodelling that occurs for example during haematopoiesis and adipogenesis. in mammalian red blood cells, the expulsion of the nucleus followed by the removal of other organelles, such as mitochondria, are necessary differentiation steps. nix / bnip3l, an autophagy receptor whose structure resembles that of atg32, is also an outer mitochondrial membrane protein that interacts with gabarap [170, 171 ] and plays an important role in mitophagy during erythroid differentiation [172, 173 ] (figure 3(c)). although autophagosome formation probably still occurs in nix / bnip3l deficient reticulocytes, mitochondrial elimination is severely impaired. consequently, mutant reticulocytes are exposed to increased levels of ros and die, and nix / bnip3l knockout mice suffer severe anemia. depolarization of the mitochondrial membrane potential of mutant reticulocytes by treatment with an uncoupling agent results in restoration of mitophagy, emphasizing the importance of nix / binp3l for the mitochondrial depolarization and implying that mitophagy targets uncoupled mitochondria. haematopoietic - specific atg7 knockout mice also exhibited severe anaemia as well as lymphopenia, and the mutant erythrocytes markedly accumulated degenerated mitochondria but not other organelles. the mitochondrial content is regulated during the development of the t cells as well ; that is, the high mitochondrial content in thymocytes is shifted to a low mitochondrial content in mature t cells. atg5 or atg7 deleted t cells fail to reduce their mitochondrial content resulting in increased ros production as well as an imbalance in pro- and antiapoptotic protein expression [175177 ]. all together recent studies have described the molecular mechanism by which damaged mitochondria are selectively targeted for autophagy, and have suggested that the defect is implicated in the familial parkinson 's disease (pd) (figure 3(c)). pink1, a mitochondrial kinase, and parkin, an e3 ubiquitin ligase, have been genetically linked to both pd and a pathway that prevents progressive mitochondrial damage and dysfunction. when mitochondria are damaged and depolarized, pink1 becomes stabilized and recruits parkin to the damaged mitochondria [122, 179181 ]. various mitochondrial outer membrane proteins are ubiquitinated by parkin and mitophagy is then induced. of note, pd - related mutations in pink1 and parkin impair mitophagy [122, 179181 ], suggesting that there is a link between defective mitophagy and pd. although p62 has been implicated in the recognition of ubiquitinated mitochondria, elimination of the mitochondria occurs normally in p62-deficient cells [182, 183 ]. when specific bacteria invade host cells through endocytosis / phagocytosis, a selective type of autophagy termed xenophagy, engulfs them to restrict their growth (figure 3(d)). although neither the target proteins nor the e3 ligases have yet been identified, invading bacteria such as salmonella enterica, listeria monocytogenes, or shigella flexneri become positive for ubiquitin when they access the cytosol by rupturing the endosome / phagosome limiting membrane [185, 186 ]. in fact, to date, three proteins, p62 [105, 185, 187 ], ndp52, and optineurin have been proposed to be autophagy receptors linking ubiquitinated bacteria and lc3. an ubiquitin - independent mechanism has recently been revealed ; recognition of a shigella mutant that lacks the icsb gene requires the tectonin domain - containing protein 1 (tecpr1), which appears to be a new type of autophagy adaptor targeting shigella to atg5- and wipi-2-positive membranes. interestingly, the shigella icsb normally prevents autophagic sequestration of this bacterium by inhibiting the interaction of shigella virg with atg5 indicating that some bacteria have developed mechanism to inhibit or subvert autophagy to their advantage. this latter category of pathogens also includes viruses such as herpes simplex virus-1 (hsv-1), which express an inhibitor (icp34.5) of atg6/beclin1. however, it was recently shown that a mutant hsv-1 strain lacking icp34.5 becomes degraded by selective autophagy in a smurf1-dependent manner, suggesting that selective autophagy plays an important role in our immune system. recently, a different antimicrobial function has been assigned to autophagy and this function appears to be selective. during infection, ribosomal protein precursors are transported by autophagy in a p62-dependent manner into lysosomes. importantly, it has been shown that induction of autophagy during a mycobacterium tuberculosis infection leads to the fusion between phagosomes containing this bacterium and autophagosomes, and the production of the antimicrobial peptides in this compartment kills m. tuberculosis. while the molecular mechanism is largely unknown, autophagy contributes at least partially to the supply of free fatty acids in response to fasting (figure 3(e)). fasting provokes the increase of the levels of free fatty acids circulating in the blood, which are mobilized from adipose tissues. these free fatty acids are rapidly captured by various organs including hepatocytes and then transformed into triglycerides by esterification within lipid droplets. these lipid droplets appear to be turned over by a selective type of autophagy that has been named lipophagy in order to provide endogenous free fatty acids for energy production through -oxidation. indeed, liver - specific atg7 deficient mice display massive accumulation of triglycerides and cholesterol in the form of lipid droplets. agouti - related peptide- (agrp-) expressing neurons also respond to increased circulating levels of free fatty acids after fasting and then induce autophagy to degrade the lipid droplets. similar to the case in hepatocytes, autophagy in the neurons supplies endogenous free fatty acids for energy production and seems to be necessary for gene expression of agpr, which is a neuropeptide that increases appetite and decreases metabolism and energy expenditure. recent experimental evidences have demonstrated that through the use of autophagy receptors and adaptors, this pathway can be selective by exclusively degrading specific cellular constituents. the list of physiological and pathological situations where autophagy is selective is constantly growing and this fact challenges the earliest concept whether autophagy can be nonselective. it is believe that under starvation, cytoplasmic structures are randomly engulfed by autophagosomes and delivered into the lysosome to be degraded and thus generate an internal pool of nutrients. in yeast saccharomyces cerevisiae, however, the degradation of ribosomes, for example, ribophagy, as well as mitophagy and pexophagy, and the transport of the prape1 oligomer into the vacuole under the same conditions requires the presence of autophagy receptors [97, 193195 ]. as a result this is a conceivable hypothesis because this process allows the cell to survive stress conditions and the casual elimination of cytoplasmic structure in the same scenario could lead to the lethal depletion of an organelle crucial for cell survival. future studies will certainly provide more molecular insights into the regulation and mechanism of the selective types of autophagy, and this information will also be important to determine if indeed bulk autophagy exists. | autophagy is a catabolic pathway conserved among eukaryotes that allows cells to rapidly eliminate large unwanted structures such as aberrant protein aggregates, superfluous or damaged organelles, and invading pathogens. the hallmark of this transport pathway is the sequestration of the cargoes that have to be degraded in the lysosomes by double - membrane vesicles called autophagosomes. the key actors mediating the biogenesis of these carriers are the autophagy - related genes (atgs). for a long time, it was assumed that autophagy is a bulk process. recent studies, however, have highlighted the capacity of this pathway to exclusively eliminate specific structures and thus better fulfil the catabolic necessities of the cell. we are just starting to unveil the regulation and mechanism of these selective types of autophagy, but what it is already clearly emerging is that structures targeted to destruction are accurately enwrapped by autophagosomes through the action of specific receptors and adaptors. in this paper, we will briefly discuss the impact that the selective types of autophagy have had on our understanding of autophagy. |
asbestos is an important occupational and environmental toxicant that affects several cell types in the respiratory tract. in an effort to understand how asbestos causes cell injury and/or altered proliferation and differentiation of cells, this laboratory has focused on reactive oxygen species as mediators of asbestos - induced biological effects. a compendium of experimental results reported by this laboratory and others supports this hypothesis. for example, scavengers of reactive oxygen metabolites and iron chelators (i.e., desferroxamine) prevent cytotoxicity after addition of asbestos to a variety of cell lines and macrophages in vitro. dna strand breakage associated with toxicity of crocidolite asbestos in c3h10 t 1/2 cells also is ameliorated with use of desferroxamine. all types of asbestos cause lipid peroxidation in mammalian cells and artificial membranes, a phenomenon that can be prevented by removal of catalytic iron. last, asbestos causes generation of active oxygen species after interaction with leukocytes or by reduction of oxygen on the surface of the fibers.imagesfigure 1. |
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enzyme - linked immunosorbent assays (elisas) are important tools in aquatic toxicology as their low detection limits allow for the fast and relatively inexpensive measurements of many compounds in complex environmental matrices (water, effluent) and biological samples (blood, urine, homogenates). as aquatic toxicologists struggled with the subtle effects of ng / l concentrations of contaminants of emerging concerns, elisas became a central tool in assessing exposure concentrations in the aquatic environment and acute physiological responses in exposed organisms. these assays utilize the inherent properties of antibodies to recognize and selectively bind a target molecule, while largely ignoring other molecules to provide semiquantitative values. commercially available assays have been developed to measure, for example, 17 -estradiol at ng / l concentrations in water or 4-nonylphenol and carbamazepine at higher ng / l and g / l concentrations. a plethora of elisas are commercially available for vertebrate physiological endpoints such as serum cortisol concentrations and other steroid hormone concentrations. one target of particular prominence to the study of endocrine active compounds is the egg - yolk precursor protein vitellogenin, which is naturally produced in mature female oviparous vertebrates and may be produced in males in the presence of exogenous estrogens and then serves as an indicator of acute exposure. although the ability of vitellogenin induction in male fishes to predict adverse health effects of exogenous estrogen exposure is unresolved ([2, 3 ] ; however, see for predictive modeling), the analysis of plasma vitellogenin in studies of endocrine disruption has become commonplace. furthermore, vitellogenin has been suggested as a regulatory endpoint with important environmental and financial consequences if approved for such purposes. a review of the published literature reporting vitellogenin concentrations for fishes exposed in laboratories or collected during field studies reveals widely diverging absolute concentrations, sometimes varying by thousand - fold factors [59 ]. differences in reported values are not explainable solely by intraspecies variability, as a result, for example, of differing degrees of maturity and reproductive status [1, 2 ], but rather suggest assay - related methodological differences. direct elisa [10, 11 ] and competitive elisa using either fathead minnow antibody [9, 12, 13 ], or crossreactive carp antibody [3, 7, 14 ], have generated a wide range of plasma vitellogenin concentrations in similarly exposed fathead minnows. however, as elisa - derived vitellogenin data are becoming more widely applied to decision - making processes, it is important to explore possible methodological mechanisms that may result in divergent relative and absolute vitellogenin measurements. limitations of immunoassay methods are not restricted to the field of aquatic toxicology, as immunologists in diabetes research have struggled for years with immunoassay standardizations [1519 ]. from the perspective of an assay development team, discussions center on linearity, recovery, accuracy, and crossreactivity [15, 18 ]. discussions regarding the proper vitellogenin assay have appeared in the literature in recent years, owing to the large variations in reported vitellogenin concentrations [2023 ]. identical laboratory protocols produce vitellogenin data in interlaboratory comparisons with coefficients of variation approaching 40% [24, 25 ]. however, to scientists studying endocrine disruption in oviparous vertebrates needing to include vitellogenin screening in their study, an understanding of the interpretive power of the data is paramount. much has been written regarding the need for a centralized vitellogenin assay [12, 26 ]. a centralized assay for a single species is a remote possibility ; however, the number of fish species utilized in endocrine disruption research complicates the single assay notion. a need exists, therefore, to illuminate some of the interassay differences and provide aquatic toxicologists with information to assist in interpreting published data generated with elisa - based assays (figure 1). several methodological mechanisms exist that could alter the measured concentration from the absolute concentration of vitellogenin in an organism. among those, differences in binding affinity between polyclonal antiserum and monoclonal antibody are well recognized among immunologists. a polyclonal antiserum is a preparation of antibody molecules with varying specific recognition targets (epitopes) while a monoclonal antibody is a preparation of antibody molecules all possessing the identical specific epitope. in addition to antibody preparation, the matrix in which vitellogenin is assessed may affect final measured concentrations. in most studies, a standard curve is established with purified vitellogenin in a buffer solution while vitellogenin from study specimens is usually measured in blood plasma or a whole organism homogenate. when combined, these two mechanisms may account for much of the absolute intraspecies variability encountered in published studies using vitellogenin induction in male fish as a bioindicator of acute exposure to emerging concern. consequently, in this study we tested the hypothesis that measured concentrations of vitellogenin will differ between elisas using polyclonal antisera versus monoclonal antibodies for identical samples. in a competitive elisa, the concentrations garnered with polyclonal antisera were predicted to be higher relative to those values obtained with monoclonal antibody usage. the addition of plasma from unexposed fish to the standards will result in a downward shift of the standard curve, resulting in lower calculated vitellogenin concentrations when compared to a protocol employing the normal method of preparing a standard curve in a buffer. plasma was obtained from three unexposed fathead minnows and used to evaluate each methodology. six - month - old mature male fathead minnows (pimephales promelas) were obtained from a laboratory fish supplier (environmental consulting and testing, superior, wi) and maintained following established us epa guidelines (16 : 8 h light : dark, 21c water temperature). fish were fed frozen brine shrimp (artemia franciscana, san francisco bay brand, inc., newark, ca) twice daily ad libitum. animal care and use in all experiments was approved by the st. cloud state university animal care and use committee (iacuc). to collect whole blood, fish (n = 3) were deeply anaesthetized in 0.1% ms-222 and fish tails were severed to harvest blood (approximately 140 l / fish) using heparinized microhematocrit tubes. blood was immediately centrifuged to isolate plasma (5,000 g for 5 min at 20c - same temperature as the harvested fish blood), and the plasma was placed on ice and analyzed by elisa for vitellogenin. plasma samples from the three fish were maintained and analyzed separately for the three fish throughout the study providing three independent replications. both were competitive antibody - capture assays using either a polyclonal antiserum preparation or a monoclonal antibody. the polyclonal antiserum was raised against purified fathead minnow vitellogenin and used at a dilution of 1 : 10000. the monoclonal antibody was purchased from biosense (bergen, norway) and used at a dilution of 1 : 1250. the assay consists of microtiter wells coated with purified fathead minnow vitellogenin at a concentration of 600 ng / ml in carbonate coating buffer (ph 9.6) for several hours at room temperature. a preincubation step combines the antibody 1 : 1 with either purified fathead minnow vitellogenin standard or sample plasma diluted in pbs/1% bsa for 1 hour at 20c. final dilutions of antibody were 1 : 20000 (polyclonal) or 1 : 2500 (monoclonal). prior to adding this preincubation mixture to the wells, the plates were washed three times with pbs - tween using an automated plate washer. two hundred microliters of the preincubation mixture was added to the vitellogenin coated - wells and incubated at room temperature for 1 hour. wells were washed with pbs - tween three times, and detection was via horseradish - peroxidase- (hrp-) labeled secondary antibody, either anti - rabbit igg - hrp in the polyclonal version or anti - mouse igg - hrp in the monoclonal version, at 1 : 10000 for i hour at rt. louis, mo), and plates were read via a multiskan ex (thermo fisher, waltham, ma) plate reader at 630 nm. a four - parameter logistic regression was used to construct seven - point standard curves. split plasma samples from each of the three fish were used in both types of elisa. the samples were tested by elisa to verify that vitellogenin concentrations were below the lower detection limit of the assay (detection range : 3.75 g / ml to 4.8 mg / ml). each plasma sample was spiked with 300 g / ml purified vitellogenin and tested to ascertain detection by the two elisa methods. pooled estradiol - exposed male fathead minnow plasma (following a 21-day flow - through exposure to 50 ng / l 17 -estradiol) was included in both elisa methods as a positive control. the objective of the second experiment was to test the effect of standard curve preparation and included the same split, spiked plasma samples from the first experiment (section 2.2). standard curves were generated using three separate protocols : the standard procedure of diluting standards in pbs/1% bsa, and two variations of fathead minnow plasma replacing the pbs / bsa buffer (plasma substitution protocols). the normal procedure of standard preparation consists of standard vitellogenin diluted in pbs/1% bsa dilution buffer. vitellogenin stock standard (600 g / ml) was diluted 1 : 62.5 in this dilution buffer to create the working stock with a concentration of 9.6 g / ml. this working stock was then serially diluted in a seven step two - fold manner, creating a range of standards from 4.8 g / ml to 0.075 g / ml vitellogenin. the diluted plasma substitution protocol consisted of pooled plasma from numerous unexposed fish added to pbs/1% bsa at a 1 : 8 dilution. the pooled plasma was tested by elisa to verify that vitellogenin concentrations were below the lower detection limit of the assay (detection range : 1.95 g / ml to 2.4 mg / ml). the vitellogenin stock standard (600 g / ml) was added to this 1 : 8 plasma / pbs at a 1 : 62.5 dilution to create a working stock, and this working stock was then serially diluted 1 : 1 for 7 steps in pbs/1% bsa. the standard range was 4.8 g / ml 0.075 g / ml vitellogenin, while the effective plasma dilution was 1 : 16 1 : 1024. this plasma dilution range was selected to approximate the dilution of plasma in tested samples, which were diluted 1 : 50, 1 : 250 and 1 : 1000. the constant plasma substitution protocol consisted of the prepared standards from the normal method added to equal volumes of 1 : 250 pooled plasma. the effective dilution of plasma was 1 : 500, again selected to approximate the dilution of plasma in tested samples. the range of this set of standards was 2.4 g / ml 0.075 g / ml. all three standard curve methods utilized separate maximum binding, blank and bsa coated well controls, prepared according to the standard curve method. the bsa - coated wells were coated with 1% bsa instead of purified vitellogenin to demonstrate lack of bsa recognition by the antibodies. each standard was prepared and then split to test in the polyclonal and monoclonal elisa variation. given the objective of the study, the number of replicates (n = 3) and the range of calculated concentrations (table 1) only qualitative comparisons were performed. standards were prepared in phosphate dilution buffer and split into equal fractions, then used to prepare standard curves in competitive elisas using either a polyclonal or monoclonal antibody for binding (figure 2). regression analysis of the standard curves was used to calculate vitellogenin values of the spiked plasma samples. the polyclonal antiserum produced calculated amounts 156% higher on average than those calculated with the monoclonal antibody (table 1). to test the effect that plasma may have on the ability of the assay to accurately ascertain vitellogenin concentrations in fish plasma samples, plasma from unexposed fish both plasma substitution schemes resulted in visible shifts of the standard curve compared to the standards prepared in the usual manner with pbs - based assay buffer (figure 2). effects that a shift of the standard curve can have are illustrated in the calculated values of vitellogenin in the spiked plasma samples (table 1). regardless of the selection of antibody (polyclonal or monoclonal), the calculated vitellogenin concentrations were less when calculated using a plasma substituted standard curve (table 1). elisas are an important tool to determine concentrations of compounds in environmental and biological samples at very low (g / the number of compounds for which antibodies have been developed is substantial, antibody development is ongoing, and custom - antibody production through service laboratories has become inexpensive. elisa kits for many compounds of interest to aquatic toxicologists have been developed and have been optimized to allow even novices with minimal training and equipment to use elisas in their research. as a result, aquatic toxicologists in academia, government agencies, and industry routinely turn to elisas to assess the presence and effects of endocrine active compounds in the environment. however, many users of immunoassay techniques are not familiar enough with the pitfalls and limitations of this technique and may misinterpret the resultant data sets. clinical studies on diabetes involving insulin and insulin antibody measurements have been hampered for over twenty years due to discordance in results generated by different laboratories [19, 30 ]. attempts to remedy the disparities have not been overly successful and have included interlaboratory comparisons of identical plasma samples, using polyclonal antisera versus monoclonal antibodies, using identical assay kits in different labs, and use of a common reference standard among labs [15, 18 ]. through two experiments, we tested the hypotheses that (i) polyclonal antisera overestimate vitellogenin concentrations in fish plasma and that (ii) matrix effects will further diverge measured vitellogenin concentrations from actual blood plasma concentrations. a polyclonal antiserum is a preparation in which a test animal such as a rabbit or goat is injected with the immunogen, and the resulting test bleeds contain several to numerous reactive antibody molecules. the injected immunogen in most cases is prepared from fish exposed to a chemical capable of producing the molecule of interest, such as estradiol used to stimulate vitellogenin production [3234 ]. any substance contained in the injection capable of eliciting the immunogenic response will have an antibody produced against that substance. the multiple antibody molecules, of different specificities, will all be contained within the preparation. as a result, the polyclonal antiserum will also contain antibodies specific for nontarget molecules, such as plasma proteins not removed during the immunogen purification process. the vitellogenin protein of at least two oviparous vertebrates has been immunologically separated into differentially reactive polypeptides [35, 36 ]. considering the biological impact of estradiol, there are likely other proteins upregulated by estrogens, potentially resulting in antibodies not specific to vitellogenin in the polyclonal preparation. these antibodies then have the potential to disrupt the accurate quantification of vitellogenin (figure 1). monoclonal antibodies seem to be the more accurate target detector, though considerations exist for this avenue as well. in the production of monoclonal antibodies, the same initial step of immunizing a test animal, usually a mouse, with plasma of exposed fish will generate numerous antibodies of varying specificities, that is, the polyclonal response. harvesting antibodies from the immunized test animal involves the removal and homogenization of the spleen. this homogenate is then mixed with cell - cultured lymphocytes to create immortal hybrid cell cultures from which antibodies can be harvested in perpetuity. the rather crude process of the fusion of millions of spleen cells and millions of cultured cells will yield hybrids at random. through the nature of the vertebrate immune system, any single lymphocyte will produce antibodies of only a single specificity, effectively ignoring the multitude of plasma proteins present in the immunogen. it is essential to isolate a single hybrid cell via dilution techniques to guarantee the antibodies produced are of a single specificity. this single cell is then allowed to multiply to large numbers, ensuring a long - term supply of the desired antibody. performed correctly, production of a monoclonal antibody will result in antibody molecules of a single specificity. if the hybrid cell is not truly isolated before the growth phase, other antibody specificities will be present in the preparation. as with polyclonal preparations, the existence of additional, possibly contaminating antibodies will alter the accuracy of an assay utilizing a monoclonal antibody. this will be amplified, as in the case of polyclonal usage, when the standard curve is produced using purified vitellogenin in a dilution buffer. the matrix in which the standard curve is established can also alter measured vitellogenin concentrations in any equilibrium assay (figure 1). plotting known concentrations of purified vitellogenin easily yields a robust standard curve with very high r - squared values. in nearly every case, other proteins in fish plasma have the potential to disrupt the recognition and binding of the target molecule by the antibody, either by nonspecifically binding to the antibody or by simple steric hindrance. a potential amplifier of this effect is that, in normal assay protocol, the standards are prepared in some type of dilution buffer such as pbs with bsa. the purified standards are diluted in this buffer to constitute the range for the standard curve. then the samples are diluted to appropriate dilution factors and assayed to compare against the standard curve and determine a concentration. however, the animal plasma will contain numerous other proteins with the potential to be recognized by an antibody and bound, thereby factoring into the final concentration determination. this problem is addressed in elisas such as those used to determine 17 -estradiol concentrations by the suggestion that the samples are purified prior to assaying to remove any contaminating aromatic compounds if it appears that there is contamination present [39, 40 ]. applying this to fish plasma in the determination of vitellogenin is not practical, owing to the inability to separate vitellogenin from the remaining proteins, while having the original concentrations upheld. to determine if contaminating entities are present in vitellogenin assays, the second experiment evaluated the effect of plasma being added to the standard curve preparations. this addresses the validity of calculated concentrations of vitellogenin from fish plasma samples when calculated from a standard curve of purified vitellogenin in phosphate buffer. in testing the effect that a different matrix has on the reliability of a standard curve, previous analyses in this elisa showed good recovery (77117%) when purified vitellogenin spikes were prepared in a pbs dilution buffer (table 2). in this study regression analysis using a standard curve constructed in pbs dilution buffer and using a polyclonal antiserum produced calculated values over 600% higher on average than the spiked amount (table 1). using a monoclonal antibody, the calculated value was 180% higher on average than the spiked amount (table 1). regression analysis of the standard curves constructed with plasma substituted for the dilution buffer and using a polyclonal antiserum or monoclonal antibody produced calculated vitellogenin values 70% lower and 42% higher than the spiked amounts on average, respectively (table 1). since plasma samples contain plasma, and normally prepared standards do not, it is likely that vitellogenin values are being substantially overestimated. the differences are reflective of the hypothesis that different matrices will produce different effects as far as the standard curves are concerned (figures 1 and 2). again, as it is not practical to separate vitellogenin from remaining plasma factors prior to assaying, and it is not very practical to add a variable supply of plasma to the standards, which may or may not contain residual vitellogenin, assays should be conducted with the knowledge that the calculated values are not absolute. this likely explains the large variability in calculated vitellogenin levels between different laboratories employing different assays with different antibodies, on similarly exposed fish. in addition to affinity and matrix effects, dilutions may impact further the accuracy of calculated vitellogenin concentrations. a method of determining the presence of contamination in plasma samples is by preparing several dilutions of the sample. sample plasma diluted across several dilutions should produce nearly identical values when each result is multiplied by its dilution factor. if the values fall within the range of the standard curve if they fail to coincide, and instead the calculated values diverge by amounts corresponding to the difference between the dilution factors, this would constitute a strong indicator of a contaminating presence binding specifically to the antibody. matrix effects, especially at low dilutions, are assumed to be occurring when immunological methods are used [4143 ]. these effects should be reduced with dilution, as the typical matrix effect is presumed to result from concentrated nonspecific plasma proteins. unfortunately, the individual results of several dilutions of each sample are rarely reported in endocrine disruption studies. mylchreest. described plasma samples diluted ten - fold producing calculated vitellogenin values which diverged three - fold and upon changing the matrix the divergence increased to four - fold. the effect of antibodies specifically recognizing nontarget molecules in a complex matrix that differs greatly between sample and standard is magnified by correcting for the dilution factors. when considering the use of elisas in toxicology studies, a number of considerations should guide methodological decisions. most laboratory studies involve only a few fish species, in many cases just a single species. in contrast, field studies can encompass numerous and sometimes varying species of fish based on geography, ecology, and seasonal timing. the generation of polyclonal antisera specific for a single species entails a shorter timeline, low cost (< $ 1,500 in most cases), and fewer logistic hurdles than does production of a monoclonal antibody. polyclonal antiserum can be produced and be available for use in 8 weeks where monoclonal antibodies can require several months. in addition, monoclonal antibodies require a cell culture facility and a complicated supply list of chemicals. while it is advisable for laboratory and field studies alike to use species - specific antibodies and purified vitellogenin, field studies may achieve adequate data quality through the use of a polyclonal, species - specific antibody, which can be produced during the planning phase of a study. in contrast, laboratory studies in dedicated aquatic toxicology laboratories may be well advised to invest the time and resources to develop monoclonal species - specific antibodies for the assessment of plasma vitellogenin in exposure experiments. the use of standards prepared in conspecific plasma may remain an unattainable goal for all but the most stringent test standards. elisas are a powerful tool in the arsenal of aquatic toxicologists. however, like most other tools, data obtained via the use of elisas need to be placed into the context and limitations of the technique. this study focused on assay - specific sources of variability attributable to antibody preparations and sample matrices. however, user - induced sources of variability remain as potential reasons for divergent data points and need to be continually evaluated. polyclonal antisera are more prone to inflate the quantity of the measured compounds and natural matrices may further alter measured quantities. as a consequence, it is advisable to aquatic toxicologists to avoid comparing absolute calculated plasma vitellogenin concentrations among studies and instead compare either percentages or ratios derived from within the studies to be compared. for example, plasma vitellogenin concentrations in a study can be expressed as % concentration over baseline, as is already widely done when comparing gene mrna induction or may use a ratio of male vitellogenin in comparison to mean control female plasma vitellogenin, with control female fish assumed to have reproductively optimized plasma vitellogenin concentrations. the latter approach is particularly useful in field studies to compare effects across multiple species (e.g.,). with the use of elisas in aquatic toxicology likely to increase in the future and with vitellogenin likely to remain an important biomarker for the exposure of oviparous vertebrates to endocrine active compounds, aquatic toxicologists and regulators alike need to educate themselves about the potential and pitfalls of this technique to avoid type 1, false positive, errors. | enzyme - linked immunosorbent assays (elisas) are important tools in aquatic toxicology and have become crucial in assessing exposure concentrations in the aquatic environment and acute physiological responses in exposed organisms. these assays utilize the inherent properties of antibodies to recognize and selectively bind a target molecule, while largely ignoring other molecules to provide semiquantitative values. a variety of methodologies to measure plasma vitellogenin using elisas have generated widely divergent data. limitations of the elisa method are known in the wider immunology field, though aquatic toxicologists may be less familiar with these limitations. we evaluated several mechanisms contributing to the divergent vitellogenin data in the literature. antibody affinities and the matrix in which standard curves are constructed are possible error generators. these errors can be amplified by large sample dilutions necessary to fall within the standard curve. it is important for the aquatic toxicology research community to realize the limitations and understand the pitfalls of absolute plasma vitellogenin data in their studies. |
clinical effects of the shrinkage stress may include postoperative sensitivity, cuspal strain or microcracks in enamel or dentin, marginal gap formation, and microleakage.1 microleakage has been identified as a significant problem because of interfacial gap formation, which can result in tooth discoloration, recurrent caries possible pulpal involvement, and restoration replacement.2 internal adaptation means adaptation to the internal dimensions of the cavity form.3 a correlation exists between internal adaptation and the presence of total voids. there were improvements in all aspects of solutions that reduce interfacial gaps under composite restoration. the use of low - modulus lining materials such as resin - modified glass ionomers, resinous liners like flowable composite or new - generation bonding agents have been proposed. liners are relatively thin layers of materials used primarily to provide a barrier to protect the dentin from residual reactants diffusing out of the restoration or oral fluids that may penetrate leaky restoration interface.4 resin - modified glass ionomer cement (vitrebond [vt ]) placement using a sandwich technique can provide reliable chemical adhesion to dentin, micromechanical bond to the overlying resin, pulp protection, and anti - cariogenicity from fluoride release and reduction in volume of resin used, thereby reducing the degree of shrinkage stress in composite resin.5 flowable resin composites (synergy flow [sy ]) are less viscous materials when used as a liner which result in less leakage and also help in relieving stresses during polymerization shrinkage of the restorative resin.6 modern dentin adhesives (sb) are currently believed to bond to dentin by a micromechanical hybridization process. so these new dentinal bonding systems initiate formation of a hybrid layer, increase the stability and durability of adhesion, thus reducing interfacial gaps and marginal leakage.7 hence, the present study was conducted to evaluate the interfacial microgaps between different types of liners and dentin, liners and composite (filtek p60 [flp60 ]) using scanning electron microscope (sem). hypothesis of the study is dentin hybridization provides superior sealing of the dentin - restoration interface than does flowable composite or resin - modified glass ionomer. thirty freshly extracted crack, defects, caries free, human third molars were selected and stored in a solution of 0.5% chloramine at 4c. all the polished 30 third molars were randomly divided into three groups each containing 10 teeth. group ii : included teeth that were lined with sb, sy, and flp60. group iii : included teeth that were lined with sb, vt, and flp60. class v cavities were prepared on the buccal / lingual surfaces with the gingival margins located 1.0 mm below the cement enamel junction. cavity dimensions were standardized (6.0 mm diameter, 3.0 mm depth) using marked 245 carbide bur.8 depth of cavity initially limited to 2 mm later deepens 0.5 mm with acrylic stop on bur tip to stimulate differences in depth of cavity (figure 1). liner application was carried out using the material corresponding to the appropriate study group and according to the manufacturer s instructions using the following steps : cavities were etched for 15 s with 35% phosphoric acid gel (scotch bond etchant, 3 m espe) and rinsed with water for 30 s and air dried. two coats of sb (3 m espe) were applied and cured for 10 s. 0.5 mm thick sy was inserted into the preparation with a ball burnisher instrument and light - cured for 20 s. one scoop of powder was mixed with one drop of liquid on a mixing pad for 10 - 15 s. the vt was inserted into the preparation with a ball burnisher instrument and light - cured for 20 s. depending on group, the two deepest levels were filled with lining materials or uniform layer of flp60 composite resin. the remaining cavities were restored using horizontal incremental technique with each increment being 2 mm. each increment was cured for 30 s from cervical to occlusal thirds using halogen visible light curing device with intensity 900 - 1200 mw / cm (coltolux, 3 m, espe). the restored teeth were finished with sequential abrasive disks and stored in distilled water at 37c for 24 h later thermocycling done. teeth were sectioned longitudinally through the center of the restoration to achieve a sample thickness of 2 mm.8 the sections were fixed in glutaraldehyde and 0.1 m and 0.2 m sodium cacodylate, later immersed in 25%, 50%, 75%, 95%, and 100% alcohols followed by immersion in hexamethyldisilazane for drying the specimens. the specimens mounted in epoxy resin and polished for 10 s each. after polishing, the specimens were ultrasonicated in absolute ethanol for 10 min, etched with 10% of phosphoric acid for 5 s. the specimens were mounted on aluminum stubs, sputter coated with gold and palladium, and examined under sem. under sem samples were photographed using magnification of 1000 and 120 and gaps were measured using image analyzer software.8 class v cavities were prepared on the buccal / lingual surfaces with the gingival margins located 1.0 mm below the cement enamel junction. cavity dimensions were standardized (6.0 mm diameter, 3.0 mm depth) using marked 245 carbide bur.8 depth of cavity initially limited to 2 mm later deepens 0.5 mm with acrylic stop on bur tip to stimulate differences in depth of cavity (figure 1). liner application was carried out using the material corresponding to the appropriate study group and according to the manufacturer s instructions using the following steps : cavities were etched for 15 s with 35% phosphoric acid gel (scotch bond etchant, 3 m espe) and rinsed with water for 30 s and air dried. two coats of sb (3 m espe) were applied and cured for 10 s. 0.5 mm thick sy was inserted into the preparation with a ball burnisher instrument and light - cured for 20 s. one scoop of powder was mixed with one drop of liquid on a mixing pad for 10 - 15 s. the vt was inserted into the preparation with a ball burnisher instrument and light - cured for 20 s. depending on group, the two deepest levels were filled with lining materials or uniform layer of flp60 composite resin. the remaining cavities were restored using horizontal incremental technique with each increment being 2 mm. each increment was cured for 30 s from cervical to occlusal thirds using halogen visible light curing device with intensity 900 - 1200 mw / cm (coltolux, 3 m, espe). the restored teeth were finished with sequential abrasive disks and stored in distilled water at 37c for 24 h later thermocycling done. teeth were sectioned longitudinally through the center of the restoration to achieve a sample thickness of 2 mm.8 the sections were fixed in glutaraldehyde and 0.1 m and 0.2 m sodium cacodylate, later immersed in 25%, 50%, 75%, 95%, and 100% alcohols followed by immersion in hexamethyldisilazane for drying the specimens. the specimens mounted in epoxy resin and polished for 10 s each. after polishing, the specimens were ultrasonicated in absolute ethanol for 10 min, etched with 10% of phosphoric acid for 5 s. the specimens were mounted on aluminum stubs, sputter coated with gold and palladium, and examined under sem. under sem samples were photographed using magnification of 1000 and 120 and gaps were measured using image analyzer software.8 interfacial gaps between dentin and liner were subjected to statistical analysis through kruskal - wallis test. the mean interfacial gap values of group iii were the highest and were statistically significant, with group i and ii. group ii showed slightly higher values than group i. group i showed least value among the groups tested. the mean interfacial gaps and standard deviation values are presented in tables 1 and 2. pair - wise comparison of study groups between dentin and liner (dunns - test). to minimize the effects of resin shrinkage, the class v preparations, which exhibited a high configuration factor, were filled using 2.0 mm thick three horizontal layers of resin.9,10 flp60 composite was used in this study since, photo polymerizing hybrid composite resins develop higher polymerization shrinkage stresses than microfilled composite resins. in group i (sb+ fl p60), only one of 10 specimens had gap between sb and dentin. but excellent internal adaptation found between sb and flp60 (figure 2a - c). (b and c) group i specimens (interface between single bond [sb ] treated dentin and flp60) showing excellent adaptation. (d - g) group ii specimens (interfaces between sb treated dentin synergy flow [sy ], sy - flp60) showing good adaptation between sb treated dentin and sy but gap between sy and flp60. (h) interface in group iii at 120 magnification showing gaps between sb treated dentin (i and j) interface between sb treated dentin vt showing larger gap. acid etching of dentin (1) removes the smear layer (2) opens and widens the orifices of dentinal tubules (3) demineralize the intratubular dentin up to a depth of 7 m (4) exposes the collagen fibers of demineralized dentin.11 the exposed collagen may provide reactive groups that can chemically interact with bonding primers. the ethanol solvent of sb, due to its high vapor pressure facilitates the diffusion and polymerization of the monomer into the exposed collagen network.12 this serves as a framework for mechanical interlocking with etched dentin by means of resin tags, adhesive lateral branches and resin dentin interdiffusion zone or hybrid layer.13,11 this ultrastructure could probably account for the higher bond strength for sb between 17 mpa and 30 mpa.14 according to studies, shear bond strength between 17 mpa and 21 mpa is required to resist contraction forces of composite resin and prevent gap formation at dentin restoration interface.15 sb is polyalkenoic acid based adhesive so (a) assist bond strength to dentin (b) associated with moisture resistance (c) might be intrinsic stress relaxation capacity by ca - polyalkenoic acid complexes.16,17 in group ii (sb+sy+ fl p60), all specimens have shown proper internal adaptation between dentin and flowable composite (figure 2d - g). the idea behind is flowable composite materials have low elastic modulus and increased elasticity.18,19 therefore, they act as a cushion and absorb the stress of polymerization shrinkage. the wetting effect of the flowable composite and or a relatively low surface tension when applied to the tooth surface helps in proper adaptation to dentin.20 internal adaptation between flp60 and flowable composite (figure 2d - g) 70% of samples showed gaps which were smaller in dimensions compared to group iii. it may be because flowable composite lining achieved intimate cavity adaptation but resulted in no evident improvement in adaptation to flp60 probably because of its higher resin matrix content.21 contraction stress from hybrid composite might be concentrated on the interface between flowable and flp60 composite thus forming the gap.22 the results of current study are consistent with studies carried out by miguez. and eitetsu. both researchers investigated their effect on dentin bonding whereby voids were found at the interface between flowable and condensable composite.23,22 the presence of such gap may degrade the mechanical integrity of the restoration. according to some studies, the application of bonding agents improves the wettability of glass ionomer cement to adhere to composite resin thus promoting a strong shear bond between rmgic and the resin composite.24,25 hence, the present study was conducted to evaluate internal adaptation of vt to hybridized dentin and to composite resin. in group iii, 8 specimens showed gaps at the interface between sb treated dentin and vt liner (figure 2i and j). the gaps size should be considered because they were up to 5 m in width which was larger compared to other groups. the reasons probably : (1) increase bond strength between sb and dentin. (2) (3) polymerization and dehydration shrinkage of the vt.26 this confirms no adhesion between vt and sb treated dentin. gap formation was observed in all specimens between vt and flp60 interface (figure 2k and l). the reason could be vt tended to adhere more to adhesive resin than to composite during the composite polymerization and also because of the physicochemical adhesiveness of the glass ionomer to tooth structure further researches are needed.27 further studies are required to better understand the bonding mechanism of rmgics with adhesive system applied to the dentinal substrate. cavity lining materials which are used as pulp protective measures must provide effective dentinal tubule sealing and be the inherent buffer to compensate for polymerization contraction stress of the composite restoration. the results obtained in this study demonstrate that the lining materials used did not contribute to a reduction of internal gap formation. thus, results require rejection of the null hypothesis that the use of vt or flowable composite liner does not result in larger gaps than those seen using adhesive resins as liners. bonding agents are capable of coating the dentin by hybrid layer and thereby can minimize gap formation. however, the efficacy of these materials, durability of composite dentin bond, and risk of adverse biocompatibility on deep dentin usage should be evaluated further with in vivo studies before drawing definite conclusions.28 the following can be concluded from the results of this study : resin hybridization provides superior sealing of the dentin - restoration interface than does flowable composite or resin - modified glass ionomer.larger microgaps were found at the hybridized dentin - restoration interface when resin - modified glass ionomer was used as a liner.use of flowable composite resin as a liner showed good internal adaptation to hybridized dentin but smaller microgaps at the restoration interface when compared to resin - modified glass ionomer groups. resin hybridization provides superior sealing of the dentin - restoration interface than does flowable composite or resin - modified glass ionomer. larger microgaps were found at the hybridized dentin - restoration interface when resin - modified glass ionomer was used as a liner. use of flowable composite resin as a liner showed good internal adaptation to hybridized dentin but smaller microgaps at the restoration interface when compared to resin - modified glass ionomer groups. | background : the use of resin - modified glass ionomer cement in sandwich technique is widely practiced with the advent of various newer generation of composites the bond between resin - modified glass ionomer and these resins should be validated. this study is done to evaluate the interfacial microgaps between different types of liners and dentin, liners and composite (filtek p60 [flp60 ]) using scanning electron microscope (sem).materials and methods : standardized class v preparations were performed in buccal / lingual surfaces of 30 caries, crack and defect - free extracted human third molars. the prepared teeth were divided into three groups. group i : single bond (sb), group ii : sb + synergy flow, group iii : sb + vitrebond. they were restored with composite resin flp60, according to the manufacturer instructions. the sb + vitrebond, cross - sectioned through the canter of the restoration. the specimens were fixed, dehydrated, polished, and processed for sem. the internal adaptation of the materials to the axial wall was analyzed under sem with 1000 magnification.results:the data obtained were analyzed with nonparametric tests (kruskal wallis, p < 0.05). flowable composite or resin - modified glass ionomer applied in conjunction with adhesive resulted in statistically wider microgaps than occurred when the dentin was only hybridized prior to the restoration.conclusion:hybridization of dentin only provides superior sealing of the dentin - restoration interface than does flowable resin or resin - modified glass ionomer. |
spinal cord teratomas are uncommon. excluding sacrococcygeal teratoma in neonates, teratomas in the spinal canal are rare. only a few central nervous system(cns) delayed presentation of spinal teratomas has generally been associated with its slow - growing characteristics5). dermoid tumors may rupture and fatty material may escape into the subarachnoid space, and/or ventricles, resulting in variable neurological symptoms6). the symptoms are dependent on location and are due to the irritative effect on and/or compression of the adjacent structures3) aseptic meningitis may also occur15). here, we report a rare case of a mature intradural spinal teratoma in an adult with concomitant additional intracranial lipid droplet dissemination associated with neurologic deterioration after iatrogenic rupture of the cyst portion occurs. iatrogenic rupture of cystic portion during spinal teratoma surgery may worsen intracranial lesion that previously exist. a 67-year - old man was transferred to our department from the urology department of our hospital. he complained of nocturia and frequent urination. during evaluation, an approximately 3 cm mass in the spinal canal abdominal ct imaging showed a fat - containing mass with mural calcification at the l2 level (fig. the patient looked ill and had experienced aplastic anemia 2 years previously. on neurologic examination, an approximately grade 4 weakness was noted in both lower extremities. lumbar magnetic resonance (mr) imaging showed a hyperintense intradural mass on a t1-weighted image. after gadolinium enhancement, there was no significant enhancement of the mass in the conus medullaris and focal enhancement of the distal soft tissue component (fig. after surgery, his lower - extremity weakness did not improve, and he could not walk without assistance. preoperative ct imaging showed low density lesion in both lateral ventricle and cistern. at that time postoperative brain mr imaging showed hyperintense lesion on a t1-weighted image on both the lateral ventricle and as well as slightly enlarged ventricles (fig. we performed extra ventricular drainage (evd), and his mental status recovered temporarily. however, his mental status again declined ; evd was performed on the other side after 7 days but, we could not retrieve tissue from the mass. during treatment, spinal cord teratomas are uncommon. only a few cns teratomas arise from the spinal cord. the prognosis of teratoids is poor and metastases at diagnosis are common, especially in neonates and children4). teratomas are slow growing, but, often grow outside the spinal column and extend to the paraspinal and retroperitoneal spaces. mature teratomas are composed of well - differentiated elements, and immature teratomas are primitive elements derived from any or all of the three germ cell layers2). mr imaging is considered as the gold standard for the detection of teratomas. total resection is desirable ; however, it is almost impossible because of the intimal adhesion between the teratomatous cyst and the circumferential nervous parenchyma. radical surgery is not recommended as the pathology is benign and recurrence rates are low even after subtotal resections1,11). the surgical goal is subtotal resection for decompressing the cord and resecting the exophytic component5), and excised tissues should be taken as extensively as possible to establish an accurate diagnosis7). after subtotal resection, consecutive and long - term radiological follow - up is recommended16). a case of spinal mature teratoma accompanying the intracranial dissemination of fatty droplets has been reported3,6,13,15). dissemination of lipid droplets within ventricles occurs because of spontaneous, iatrogenic, or traumatic rupture. our case is similar to this previous case, although, an intracranial mass lesion was not confirmed histopathologically. the symptoms of intracranial lipid droplet dissemination were headache, chemical meningitis, seizure and mental deterioration. the cystic contents gain access to the ventricular system by way of retrograde flow through the foramen of luschka and magendie15). it is possible that the central canal can serve as a pathway for ruptured material into the brain13). ct and mr imaging are useful in diagnosing free fat in the subarachnoid space and intraventricular fat - fluid level. lipid is differentiated from cerebrospinal fluid by negative values (-20 to -130 hounsfield units)15). mr imaging is reliable in the evaluation of the presence of rupture and its extent in the subarachnoid spaces or into the central spinal canal10). we report a rare case of a mature intradural spinal teratoma and concomitant intracranial lipid droplet dissemination in an adult. additional lipid droplet dissemination to intracranial space with neurologic deterioration after a spinal teratoma surgery should be considered when iatrogenic rupture of the cyst portion occurs. it might develop the symptom and worsen hydrocephalus, although asymptomatic lipid droplets dissemination was previously existed before lumbar spine surgery. | a teratoma is a neoplasm that contains tissues originating from three germ cell layers at ectopic sites. the embryology of teratomas remains unclear. teratomas are usually composed of cystic and solid components, and they are usually associated with syringomyelia. cystic lesions of teratomas may rupture in a spontaneous, iatrogenic, or traumatic manner. lipid droplets in the ventricles and subarachnoid space are rare. we managed a case of a spinal teratoma in the lumbar region in a 67-year - old man. he complained of nocturia, frequent urination, and difficulty in walking for 2 months. radiographic imaging revealed a lumbar spinal intradural mass. intracranial lipid droplets dissemination was also existed. the patient underwent surgery, and a diagnosis of mature teratoma was confirmed histopathologically. during the operation, the cystic portion of the intradural mass ruptured. during the hospital stay, the patient 's mental status declined. on radiological examination, slightly enlarged ventricle size was observed. dissemination of lipid droplets within ventricles occurs because of spontaneous, iatrogenic, or traumatic rupture. additional lipid droplet dissemination to the intracranial space associated with neurologic deterioration after a spinal teratoma surgery should be considered when iatrogenic rupture of the cyst portion occurs. |
the implant - supported prostheses are attached to implant abutments either by cementation, or by retaining screws. because of the advantages of cement - retained implant - supported restorations, such as passive fit, reduced cost, ease of fabrication, superior esthetic, fewer components, and uncomplicated laboratory technique, these restorations have gain popularity compare to screw - retained restorations.[13 ] even though cement wash outs occur in cement - retained restorations, they do have high clinical success rates. retrievability may be a critical aspect of implant - supported restorations because of problems such as loosening or fracture of the abutment screws, mechanical failures, treatment of peri - implant tissues, and evaluation of ailing implants mobility. although using provisional cements are considered to achieve retrievability of implant - supported prostheses, retrievability of screw - retained prostheses is more secure than cement - retained ones. retention of implant - supported restorations plays an important role in success of the treatment. uncemented restorations may cause problems such as inhalation of the restorations, increased bone loss around the implant, prosthesis failure breakage, trauma to antagonistic teeth, food impaction, accumulation of microorganisms resulting in bad odor and soft tissue response. extra practitioner time and patient embarrassment have also been noted. on the other hand, luting agents that are too retentive may damage the osseointegration of the implant during removal of the restoration. behavior of permanent and provisional luting agents in cementation of implant - supported prostheses differs from cementation on natural teeth. in particular, water - based cements such as zinc phosphate, zinc polycarboxylate, and glass ionomer, have shown a wide variety of retentive values, which can sometimes be unpredictable.[61115 ] some authors suggested the use of permanent and provisional cements for luting single - unit and multi - unit implant - supported restorations, respectively. provisional cements have been recommended for restorations that may require intervention. whereas more retentive cements, such as resin cement, are more appropriate when future retrievably is not necessary. the use of different cements, protocols, and implant systems may alter the retentive strength of implant - supported restorations. in addition, different aging processes, such as thermal cycling and mechanical loading, as well as different pretreatment techniques can also affect the retentive strength.[2022 ] the aim of this study was to evaluate the retention values of cement retained implant - supported restorations with different luting agents. the null hypothesis was that there is no differenece in retentiveness of cement retained implant - supported restorations with different luting agents. twenty iti solid abutments with 5.5 mm length and 8 taper (048.541, iti dental implant system, straumann ag, basel, switzerland) and twenty iti implant analogs (048.124, iti dental implant) were used. the implant - abutment complex were embedded vertically in a block of autopolymerizing acrylic resin (meliodent, heraeus kulzer, hanau, germany) using a dental surveyor for precise alignment. a loop of wax was added to the occlusal surface of each coping for retention test. the wax patterns were invested in a phosphate - bonded investment (ceravest quick, gc, tokyo, japan) and cast in a base metal alloy (rexillium iii, pentron, wallingford, ct). after divesting and cleaning with an ultrasonic cleaner and hydrofluoric acid, the inner surface of the castings were inspected under magnification (4), and surface irregularities were removed with a small round carbide bur. the metal copings were checked for fitness using a silicon disclosing medium (fit checker, gc co, tokyo, japan), and further potential interferences of castings were evaluated and adjusted if necessary. each casting was randomly numbered and paired with an implant - abutment assembly for further procedures. eight commercially available luting agents, including provisional (temp bond, gc - free eugenol, tempspan) and definitive (panavia f2.0, fuji plus, fleck 's, poly f, and fuji i) cements were evaluated in this study [table 1 ]. each cement was mixed according to the manufacturer 's instruction and applied to intaglio surface of the copings (n=10). the copings were gently seated on the abutments and held in place under a 5 kg load for 10 minutes. after initial setting of the cement, the excess cement was removed with an explorer. the specimens were stored in a 37c incubator for 24 hours, immersed in artificial saliva for 7 days and thermocycled for 5000 cycles (5 - 55c) with a 30-s dwell time. after aging process, the dislodging force of the copings was measured using a universal testing machine (4302 instron ltd., high wycombe, uk) at a crosshead speed of 5 mm / min [figure 1 ]. the failure modes were recorded after the dislodgment of the copings. luted metal coping during pull out test by universal testing machine the same copings and abutments were used with each of the eight cements evaluated. to remove the residual cement, the castings were heated to 600c for 90 minutes and allowed to cool at room temperature. a spoon excavator was used to remove any residual cement from the intaglio surface of castings and the abutments were cleaned with a plastic explorer. the castings and abutments were immersed in ultrasonic cleaner containing cement removal agent (removalon - i, premier dental products co, norristown, pa) for 30 and 15 minutes, respectively. mann - whitney test with a bonferroni adjusted significance level of 0.001 was used for each pairwise comparison to control the overall error rate for all comparisons at an error rate of 0.05. mann - whitney test with a bonferroni adjusted significance level of 0.001 was used for each pairwise comparison to control the overall error rate for all comparisons at an error rate of 0.05. table 2 represents the mean retentive values and standard deviations in newton (n), as well as the mean rank of the studied luting agents. kruskal - wallis test revealed a significant difference between the retentive values of the studied luting agents (p<0.001). mann - whitney test showed no significant difference between fuji plus, fleck 's, ploy f, and panavia f2.0 and these cements were superior to the provisional and fuji i cements (p<0.001)which showed statistically the same retentive strength. mean (standard deviation) dislodging forces of the studied cements (n) and mann - whitney groups cement failure occurred in the cement - abutment interfaces in all of the fuji plus, panavia f2.0, and fleck 's samples. zinc polycarboxylate and zinc oxide with and without eugenol cements remained on both the copings and abutment surfaces whereas tempspan completely remained on the abutment surfaces. in this study, the null hypothesis that the retentiveness of the metal copings would not affect with the studied cements was rejected. the resin modified glass ionomer cement (fuji plus) had the highest retention, but there were no significant differences between definitive cements. some studies indicate that the retentive strength of resinous cement is superior to that of, zinc phosphate and zinc polycarboxylate cements. the higher retentive strength of the resin cements was attributed to the adhesive system which was not used in the current study. furthermore, resin modified glass ionomer cement adheres to metal by chelating metallic ions, but the retentive strength may be weakened by early water contact. in this study, the copings were incubated for 24 hours at 37c before the aging process and there was no early water contact. in a study on titanium copings over short iti solid abutments, the mean retentive values of zinc phosphate cement was comparable to panavia 21, but the retentive strength was higher than in the present study. a combination of panavia 21 and titanium alloy was reported to have higher bond strength compared to other alloys. in another study on the retention of base metal copings with dental implants, zinc phosphate and resin modified glass ionomer cements showed same retention strength. with regard to the ceraone system, zinc phosphate cement had highest retention value, and resinous cement presented statistically similar retentive strength. in a similar investigation on retention values of metal copings on iti solid abutments, interestingly, zinc phosphate and zinc oxide eugenol cements had similar retentive strength. the different results with the present study maybe due to using different cements and thermal cycles. long - term thermal cycling has been shown to reduce the retentive strength of luting agents. squier. reported a higher retentive value for panavia than zinc phosphate and resin modified glass ionomer, which differs from the current study. this discrepancy may be related to sandblasting and surface conditioning in this study. surprisingly, glass ionomer cement had the lowest retention among the definitive luting agents, which was comparable to the temporary cements. this finding is consistent with a previous study that demonstrated glass ionomer cement showed significantly lower retention than zinc phosphate and resinous cements, and similar retention to zinc oxide cement without eugenol. in the present study, no pretreatment was performed on either the copings or the abutments, and glass ionomer cement does not adhere to an inert surface. furthermore, the solubility of glass ionomer cement is more than that of other cements, and it is very susceptible to early water contact and desiccation, which can dramatically reduce the mechanical properties of the cement. the retentive strength of restorations is influenced by taper, surface area, height and surface roughness of the abutment, restoration characteristics, type of cement, marginal accuracy and the treatment method. thus a direct comparison with other studies may be difficult because of differences in the implant systems, specimens, and methodology. adhesion of the cement to abutment may be difficult to remove and damage the abutment surface. in this study, the resinous and resin modified glass ionomer cements completely adhered to the intaglio surface of the copings. the surface of implant abutment is relatively smooth, which caused remaining zinc phosphate cement in the inner surface of the copings. the primary failure mode for the polycarboxylate cement was adhesive, but there was also some cohesive failure. the temporary cements, with the exception of tempspan, adhered to both inner surface of the copings and the abutment surfaces. our results suggest that the tested definitive cements are not desirable if retrievability of single - unit metal restorations is sought. the dislodgement force for an implant with 4-mm diameter and 6 mm height directly out of the its long axis was about 290 n. in this study the snap - on mechanism of the burn - out cap was removed by a reamer from the inner surface of the castings, which resulted in a passive fit. the force that is used to retrieve the implant - supported restoration is high impact and of short duration. on the other hand, in this study the failures of the restorations result in several comparative small dynamic loadings and the axial dislodging of cemented implant crowns is a rather seldom clinical event. the results of this in vitro study should be interpreted with caution and confirmed with clinical studies. within the limitations of this study, the following conclusions can be drawn : the most retentive luting agent was resin - modified glass ionomer, but there was no significant difference between this cement and panavia 2.0, zinc phospate, and zinc polycarboxylate cements. these definitive cements are recommended for luting single - unit implant - supported metal restorations.the temporary and glass ionomer cements were the least retentive cements and might not be suitable for luting single - unit implant - supported restorations. the most retentive luting agent was resin - modified glass ionomer, but there was no significant difference between this cement and panavia 2.0, zinc phospate, and zinc polycarboxylate cements. these definitive cements are recommended for luting single - unit implant - supported metal restorations. the temporary and glass ionomer cements were the least retentive cements and might not be suitable for luting single - unit implant - supported restorations. | background : with regard to potential retrievability of cement - retained implant restorations, the retentive strength of the luting agents is critical. the aim of this study was to evaluate the retention values of implant - supported metal copings using different luting agents.materials and methods : twenty iti implant analogs and solid abutments of 5.5-mm height were embedded vertically in autopolymerizing acrylic resin blocks. metal copings with a loop on the occlusal surface were fabricated using base metal alloy (rexillium iii). the copings were luted using eight cements with different retention mechanisms (panavia f2.0, fuji plus, fleck 's, poly f, fuji i, temp bond, gc - free eugenol, and tempspan) under static load of 5 kg (n=10). all specimens were incubated at 37c for 24 hours, conditioned in artificial saliva for 7 days and thermocycled for 5000 cycles (5 - 55c). the dislodging force was measured using a universal testing machine at a crosshead speed of 5 mm / min. statistical analyses were performed using kruskal - wallis (=0.05) and mann - whitney tests with bonferroni correction (=0.001).results : fuji plus and tempspan had the highest and the least mean retentive strength, respectively (320.97161.47, 3.392.33). there was no significant difference between fuji plus, fleck 's, ploy f, and panavia f2.0. these cements were superior to provisional cements and fuji i (p<0.001) which showed statistically same retentive strength.conclusion:within the conditions of this study, the resin modified glass ionomer, zinc phosphate, zinc polycarboxylate, and panavia f2.0 had statistically the same retentive quality and are recommended for definitive cementation of single implant - supported restorations. the provisional cements and glass ionomer may allow retrievability of these restorations. |
pulmonary valve replacement (pvr) or right ventricular outflow tract reconstruction (rvotr) is the most common valve replacement / repair procedure performed in the society of thoracic surgeons (sts) congenital database, comprising 16% of procedures performed in the adult congenital population. there is a growing population of children surviving well into adulthood with repaired congenital heart disease and the number of pvr / rvotr procedures required will grow with this population. since ross performed the first rvotr using an aortic homograft in 1966, the search for the optimal conduit choice has been the subject of much debate. whilst a cryopreserved homograft is the gold standard, calcification and homograft failure remain an issue, with only 40 - 75% of homografts remaining free from structural valve deterioration (svd) at 10 years in the congenital population [2, 3 ]. an alternative is the medtronic freestyle valve, derived from a porcine aortic root, decellularised using glutaraldehyde and treated with alpha - amino oleic acid to minimize xenograft calcification. potential benefits include its stentless design, the possibility to achieve a greater effective orifice area, the availability in a range of sizes (19 - 29 mm), and the lack of requirement for lifelong anticoagulation. the aim of this study, therefore, was to systematically review all of the primary publications describing the outcomes associated with using the freestyle valve for rvotr in both the ross procedure and in congenital heart disease, primarily to determine short - term rates of structural valve dysfunction and reintervention, and functional status. a review of available literature was undertaken via online searches of the major clinical databases : medline, pubmed, cochrane database and google scholar. the search terms used were - freestyle or xenograft or stentless, and bibliographies, from included papers, were assessed for suitable references in an attempt to avoid missing potentially useful material. publications were eligible for inclusion if they presented original data on freestyle valves in the pulmonary position. review articles and opinion pieces, isolated case reports and animal studies were excluded because they lacked any outcomes of interest. all publications deemed suitable for inclusion on the above criteria were reviewed separately by two authors (bd and da) to ensure suitability and disagreements resolved by consensus. the primary outcome of interest was rates of structural valve deterioration as defined by akins. in their joint sts secondary outcomes of interest included reintervention (defined as percutaneous balloon valvuloplasty, percutaneous pvr or surgical revision) and functional status (defined according to new york heart association class or modified ross score). meta - analysis was undertaken where sufficient data existed to pool studies, using a fixed effects or random effects model depending on study heterogeneity. we defined a priori two subgroups : one group that underwent rvotr as part of a ross procedure and another group that underwent rvotr as surgical management of congenital heart disease. two statistical models, namely, fixed effects and random effects models, were used depending on study heterogeneity. fixed effects models were used if the between study variance was less than 0, whilst a random effects model was used, where the between study variance was larger than 0. a search for comparative series of alternative bioprostheses and homografts was also performed to allow for comparison with the results of our meta - analysis. a total of 435 papers were initially retrieved with the search strategy described above, of which 13 retrospective case series fulfilled the eligibility criteria and were included in the systematic review. one study presented their data only as a kaplan meier curve and so could not be analysed with the other 12 studies but was reviewed separately. the twelve papers included in the meta - analysis yielded data on 311 patients with a mean follow - up of 33.6 months. of the 12 studies, 2 reported on solely paediatric patients, 3 reported solely on adult patients and 7 reported data on both paediatric and adult patients. seven of the papers solely reviewed cohorts of patients that rvotr were performed on as part of a ross procedure, while the other 5 papers were from series of predominantly congenital cases (table 1). twelve studies (n=311) reported echocardiographic data on svd at 34 months follow up (range 10 - 98 months). the mean rate of svd was 4.8% (95% ci 0.8 - 10.6%). analyzing ross recipients separately (7 studies, n=137), the pooled rate of svd was 7.1% (95% confidence interval (ci) 0.2 - 19.3%) at 45 months follow up. for congenital recipients (5 studies, n=174 patients) the pooled rate of svd was 3.5% (95% ci 0.9 - 7.3%) at 18 months. heterogeneity between studies was high, so random effects modeling was used for this analysis. reintervention was reported in twelve studies (n=311) with a mean follow - up of 34 months. reintervention occurred in nine patients (mean 1.1%, 95% ci 0.0 - 3.3%). analyzing ross recipients separately (7 studies, n=137), the rate of reintervention was 2.1% (95% ci 0.0 - 6.4%) at 45 months. the 5 congenital series (n=174) were also analysed separately and had a mean rate of reintervention of 1.0% (95% ci 0.1 - 3.7%) at 18 months. heterogeneity between studies was low, so fixed effects modeling was used for this analysis. in the whole cohort, the mean time to reintervention was 28 months. the indications for reintervention were subvalvar stenosis due to pannus and stricture formation at the proximal suture line (n=7) and true valvular stenosis (n=2). five of these reinterventions were percutaneous (2 balloon valvuloplasties, 3 percutaneous valve replacements), and 4 were open surgical conduit replacements. in comparison, lee. reported only 31% freedom from reintervention in their cohort of 23 patients with a mean of 91 months follow up. they did not state what proportion of reinterventions were due to svd, but did describe that three cases were due to conduit compression by the overlying sternum. functional status. eight of the twelve studies (n=214) provided data on the functional status of their patients with a mean follow - up of 36 months. freedom from the symptoms of heart failure was 97.7% (95% ci 94.6 - 99.7%). in the ross population (n=53), it was 91.7% (95% ci 81.2 - 98.9%). in the congenital population (n=161) heterogeneity between studies was low, so fixed effects modeling was used for this analysis (table 2). pooled outcome data for freestyle valves. a review of all available prosthetic options was also performed to allow for comparison between the many available alternatives (see table 3). there is a shortage of truly long - term data available, however 5-year results are available for most bioprostheses. across all bioprostheses a 5-year failure rate of 10 - 40% exists, with failure rates at 10 years climbing to 25 - 60% (table 3). to our knowledge, this is the first systematic review and meta - analysis of the performance of the medtronic freestyle valve in the pulmonary position. first, our results suggest that the freestyle valve has a low incidence of svd, reintervention and symptoms of heart failure, and compares well with alternative bioprostheses at similar relatively short duration of follow - up. this is an atypical failure mode for rv - pa conduits and it may relate to the stiffness of the freestyle root wall in comparison to the mobility of the muscular rvot leading to anastomotic site tension. second, long - term follow up data for the freestyle valve in the pulmonary position is limited. only 3 studies [5, 7, 8 ] have truly long - term data on the performance of the freestyle valve and display markedly different results. dohmen and hechadi had no svd or reintervention at 60 and 98 months follow - up respectively, while lee describes a 69% rate of reintervention at 90 months. further studies are required to truly determine the long - term outcomes of this valve in the pulmonary position. third, the promising findings potentially increasing options for rvotr may stimulate competition within the industry and provide the impetus to reduce the cost of these bioprostheses ; a cost - effectiveness analysis may provide further useful data to guide clinicians. the meta - analysis did not include any randomized controlled trials of the freestyle valve, the patient numbers were low and the duration of follow up relatively short, precluding definitive conclusions in comparing valve outcomes. the included studies were also heterogeneous in nature with a mixture of pediatric and adult patients, and no data on valve sizes used was available. we did however identify a priori subgroups for analysis and did not find substantial differences upon analysis according to ross or congenital procedure, although mean length of follow - up differed between these two groups. pulmonary valve replacement or right ventricular outflow tract reconstruction using the freestyle valve is associated with satisfactory short - term echocardiographic and clinical outcomes, including functional status and freedom from reintervention at over 2 years. freestyle valves may be a reasonable alternative to homografts or other bioprostheses, particularly when homografts are not readily available. determination of the optimal valve, however, is currently limited by lack of long - term comparative data and this review has identified a significant need for long - term data on the freestyle valve in the pulmonary position. | introductionreconstruction of the right ventricular outflow tract is the most commonly performed valve repair / replacement procedure in congenital cardiac surgery. there is an ongoing shortage of homografts, and existing bioprosthetic options suffer from substantial rates of structural valve deterioration over time. the medtronic freestyle valve is used extensively in the aortic position, but little data is available on its performance in the pulmonary position.methodsa systematic review and meta - analysis of primary studies reporting echocardiographic and clinical outcomes, including reintervention and functional status, associated with the freestyle valve in the pulmonary position for both ross and congenital surgery.results13 observational studies including 334 patients with a mean follow - up of 34 months (range 10 - 98 months) fulfilled the eligibility criteria and were included in the review. structural valve deterioration occurred in 4.8% (95% confidence interval 0.8 - 10.6%) of patients. reintervention was required in 1.1% (95% confidence interval 0.0 - 3.3%). freedom from symptoms of heart failure occurred in 97.7% (94.6 - 99.7%). the results did not change substantially when analysed according to ross or congenital surgery.conclusionsthe freestyle valve performs well at short - term follow - up and provides a viable alternative when homografts are unavailable. further long - term studies are required to better assess its role in right ventricular outflow tract reconstruction. |
similar to the approach used in our previous review, we performed a computer search of the scientific literature (in english and other languages) published from january 2000 through june 2004 using the words rotavirus and the truncated stem rota-. we restricted the analysis to studies that met the following criteria : 1) were initiated after 1993 ; 2) were conducted for at least 1 full calendar year ; and 3) examined rotavirus among at least 100 children us $ 9,265) (10), and calculated the median (interquartile range [iqr ]) proportion of diarrhea hospitalizations attributable to rotavirus for each income group. we next calculated an overall median detection rate by taking a weighted average of the median detection rates for each of the income groups. the weights assigned to each income group corresponded to the proportion of deaths from childhood diarrhea among countries in that income group as determined on the basis of our previous analysis (2) : 85% in low - income countries, 13% in low - middle income countries, 2% in high - middle income countries, and 30 countries in asia, africa, the middle east, and latin america. data from these and other studies, particularly from countries such as india and china, which account for a large fraction of global rotavirus deaths, should be used to update our estimate of rotavirus - related deaths and further refine it to develop country - specific figures. these data, together with information on effects and costs of rotavirus disease, will allow policymakers to assess the magnitude of the problem of rotavirus and the value of new vaccines that may soon be available. | studies published between 1986 and 1999 indicated that rotavirus causes 22% (range 17%28%) of childhood diarrhea hospitalizations. from 2000 to 2004, this proportion increased to 39% (range 29%45%). application of this proportion to the recent world health organization estimates of diarrhea - related childhood deaths gave an estimated 611,000 (range 454,000705,000) rotavirus - related deaths. |
house dust is a repository for environmental pollutants that may accumulate indoors from both internal and external sources over long periods of time. dust and tracked - in soil accumulate most efficiently in carpets, and the pollutants associated with dust and soil may present an exposure risk to infants and toddlers, who spend significant portions of their time in contact with or in close proximity to the floor and who engage in frequent mouthing activities. the availability of carpet dust for exposure by transfer to the skin or by suspension into the air depends on particle size. in this study, a large sample of residential house dust was obtained from a commercial cleaning service whose clients were homeowners residing in the raleigh - durham - chapel hill (research triangle) area of north carolina. the composite dust was separated into seven size fractions ranging from < 4 to 500 microm in diameter, and each fraction was analyzed for 28 pesticides and 10 polycyclic aromatic hydrocarbons (pahs). over 20% of the fractionated dust sample consisted of particles < 25 microm in diameter. fourteen pesticides and all 10 of the target pahs were detected in one or more of the seven size - fractionated samples. sample concentrations reported range from 0.02 to 22 microg / g ; the synthetic pyrethroids cis- and trans - permethrin were the most abundant pesticide residue. the concentrations of nearly all of the target analytes increased gradually with decreasing particle size for the larger particles, then increased dramatically for the two smallest particle sizes (4 - 25 microm and < 4 microm).imagesfigure 1figure 2figure 3figure 4 |
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individuals with obesity are at increased risk of several health conditions including type 2 diabetes, dyslipidaemia, liver steatosis, hypertension, cardiovascular disease, osteoarthritis, as well as various forms of cancer (faulds and dahlman - wright, 2012, mauro and marelli - berg, 2012, norata., 2015). a strong link between obesity and the establishment of chronic systemic inflammation has recently emerged (bjrndal., 2011) and is often related to increased expression of adipokines (i.e., leptin, chemerin, and resistin), myokines, and pro - inflammatory mediators (i.e., tumor necrosis factor [tnf]-, interleukin [il]-1, and il-6), and decreased expression of the anti - inflammatory cytokine, adiponectin (norata., 2015). in obesity, activated immune cells (including t cells, macrophages, and dendritic cells [dc ]) infiltrate adipose tissue, promoting in turn the production of pro - inflammatory cytokines, such as tnf-, il-6, resistin, and monocyte chemotactic protein-1 (mcp-1/ccl2) (mathis and shoelson, 2011, norata., 2015). this process is thought to underlay the activation of both the innate and adaptive arms of the immune system, which results in the chronic inflammatory response observed in obese subjects (larbi., 2008, shaw., 2010). while t cells, b cells, macrophages, neutrophils, and mast cells are increased in number in the visceral adipose tissue (at) of obese individuals, specific subsets of t cells (helper t cell type [th2 ], regulatory t cells, and invariant natural killer t [inkt ] cells), as well as eosinophils are reduced (cildir., 2013). obesity - related inflammation, caused by excessive and inappropriate activation of the immune system, is pivotal to disease progression and the development of complications, such as atherosclerosis and type 2 diabetes (norata., 2015). t lymphocytes play a major role in the development of these inflammatory processes both via direct cytotoxic activities and secretion of cytokines that influence the severity and outcome of the inflammatory reaction (hamann., 2000). based on the observation that t cells in the adipose tissue display specific t cell receptor (tcr) rearrangements, it has been proposed that obesity might be associated with an autoimmune response (gerriets and rathmell, 2012). the mechanisms of enhanced t cell activation and tissue infiltration during obesity, however, remain elusive. altered antigen presentation by dc has been proposed to underlie the activation of adaptive immunity observed in dyslipidaemia. although at - derived dc in obesity display an immature phenotype with lower expression of activation markers, such as cd40, cd80, and cd86 (chen., 2014), they promote the differentiation of pro - inflammatory interferon (ifn) cd4 t cells (chen., 2014). a recent report has implicated dietary long- and medium - chain fatty acids (fa) in t cell differentiation toward th1 and th17 subsets in autoimmune responses of the cns (haghikia., 2015) ; however, whether fa can directly affect t cell activation and differentiation toward specific cell subsets during metabolic overload remains to be established. in this study, we investigated the phenotypic, functional, and migratory features of memory cd4 t cells in experimental models of saturated fa - induced metabolic stress, as well as in human cd4 t cells obtained from lean, overweight, and obese individuals in a large cohort of free - living people (progressione della lesione intimale carotidea [plic ] study : n = 2,606). mechanistic studies show that direct exposure of cd4 t cells to saturated fa ; i.e., palmitate, which is enriched in high - fat diet (hfd), induces cd4 t cells to acquire a specific cd44-ccr7-cd62l - cxcr3-lfa1 pro - inflammatory functional phenotype via enhanced engagement of a pi3k p110-akt - dependent pathway upon priming, but independent of the modality of antigen presentation by dc. given that lymphocyte infiltration of non - lymphoid tissues is a prominent feature of chronic inflammation, we first assessed whether hfd - induced metabolic stress directly promotes the generation of t cell populations with specific migratory features. to this aim, memory cd4 t cells were generated in rag2-deficient tcr transgenic marilyn female mice, which harbor monoclonal cd4 t cells specific for the male antigen h - y (lantz., 2000), by intraperitoneal (i.p.) cd4 t cells were then isolated from the lymph nodes of donors fed 8-week chow (cd) or hfd and adoptively transferred in hfd- or cd - fed (i.e. fed chow or high - fat diet for 8 weeks, respectively) c57bl/6 male recipients that had been i.p. injected with zymosan and ifn 48 hr earlier to induce localized inflammation (figures s1a s1c) (fu., 2014). in vivo - primed cd4 t cells isolated from hfd donors exited from the blood circulation of cd recipients with a kinetic 24 hr faster than their cd counterpart (figure 1a). cd4 t cells that were primed in hfd donors also trafficked preferentially to the non - lymphoid, inflamed sites (peritoneal cavity) of both hfd and cd recipients (figure 1b). in contrast, primed cd4 t cells from cd donors preferentially localized to the mesenteric lymph nodes of both cd and hfd recipients (figure 1c). no difference in cd4 t cell localization to the spleen was observed when comparing equal numbers of adoptively transferred cd and hfd donors (figure 1d). these results indicate that memory t cell populations induced in cd- or hfd - fed animals are characterized by different trafficking patterns. to establish the pathophysiological relevance of these differential trafficking patterns, we performed hy - mismatched skin grafts, whereby male skin was implanted on the upper right back of cd- or hfd - fed marilyn female recipients. we found that rejection of hy - mismatched skin was significantly accelerated in hfd as compared to cd female recipients (figure 1e) (molinero., 2016). activation of cd4 t cells and their localization to the graft are known to be instrumental to rejection in this system (chen., 2003). indeed, we observed that hy - mismatched skin graft rejection was completely abrogated in rag2ko mice (that are deficient for mature t and b cells) independent of the diet they received (figure s1d). reconstitution of hfd rag2ko mice with cd4 t cells isolated from marilyn mice resulted in a rapid rejection of the transplanted male - derived skin (figure s1d), showing the crucial role these cells play in such system. these findings support the conclusion that biased differentiation of cd4 t cells toward a pro - inflammatory effector memory phenotype takes place during hfd - induced metabolic stress. based on the distinct homing preferences of memory t cells generated in hfd- or cd - fed animals and the stronger immune response elicited in hfd animals, we asked whether a biased differentiation of activated cd4 t cells toward a pro - inflammatory phenotype and associated homing receptors takes place upon priming following hfd - induced metabolic stress. using the same immunization procedure as that used for the experiments presented in figures 1a1d, we found that hfd - fed hosts displayed an increase in the size of the cd44-ccr7-cd62l - cxcr3-lfa1 effector memory - like cd4 t cell population and no difference in the cd44-ccr7-cd62l central memory cd4 t cells as compared to cd4 t cells primed in cd - fed mice (figures 2a2d and s2a s2d). in contrast, the size of naive t cells was reduced in hfd (figures 2a, 2b, s2a, and s2b). these data suggest a preferential cd4 t cell differentiation toward an effector memory phenotype under hfd regimen in concomitance with a reciprocal reduction of the naive cell population without affecting the size of the central memory pool. no difference was observed in the expression of the activation markers cd25 and cd44 (figures 2e, 2f, s2e, and s2f) by memory cd4 t cells activated in cd or hfd hosts, indicative of similar priming efficiency in either metabolic background. in agreement with the observed phenotypes, a significantly larger fraction of in vivo - primed cd4 t cells isolated from the lymph nodes of hfd mice migrated in response to the pro - inflammatory chemokine cxcl10 (a cxcr3 ligand) in ex vivo chemotaxis assays, while most in vivo - primed cd4 t cells isolated from the lymph nodes of cd mice migrated to the homeostatic lymphoid tissue chemokines ccl19 and ccl21 (ligands of ccr7) (figure 2 g). to investigate the cellular sources of cxcl10 in vivo injection of ifn. at 48 hr later, intracellular staining of cxcl10 in a number of immune cell populations retrieved from the peritoneal cavity was performed. we chose this system because of its relevance to the peritoneal recruitment system shown in figures 1a1d. data indicate that resident (peritoneal) and to a less extent infiltrating (monocyte - derived) macrophages are main sources of cxcl10 in this model (figures s2 g and s2h). furthermore, we observed an increase in the ifn and il4 populations upon priming following hfd (figures 2h and 2i). our findings are in line with previous work showing that increased expression of cxcr3 in inflammatory conditions, such as obesity and atherosclerosis, drives accumulation of inflammatory t cells in fat sites. indeed, cxcr3- or cxcl10-deficient mice are protected from obesity- and atherosclerosis - induced inflammation in association with reduced accumulation of effector t cells and macrophages in at and atherosclerotic plaques (gupta., 1997, rocha., 2014, a cohort of 1,172 subjects was selected by probability sampling from a large survey of free - living people (plic study : n = 2,606 ; average age 66 years). the selected cohort was representative of the entire plic cohort in terms of age, gender, and clinical parameters according to the kolmogorov - smirnov test ; individuals were stratified in three groups (i.e. lean, overweight, and obese), based on bmi, and a number of biochemical and cellular markers of their peripheral blood were analyzed (table s1). in particular, this analysis revealed increased total lymphocyte counts in the blood of overweight and obese as compared to lean individuals (figure 3a ; table s1). from this cohort, a detailed immunophenotype was performed in a group of n = 187 subjects. again, these individuals were selected by probability sampling and were representative of the entire cohort according to the kolmogorov - smirnov test. specific subsets of circulating cd4 t cells were assessed by 10-parameter/8-color polychromatic flow cytometry with cd3, cd4, cd45ro, cd45ra, ccr7, ccr5, cxcr3, and hla - dr as markers. the main cd4 t cell subsets present in the blood were identified by flow cytometry based on a well - defined gating strategy (figures s3a and s3b). the percentage of total t - helper lymphocytes was equal in the three groups (figure 3b) ; however, we found a strong association of the obese condition with reduced naive t cells and increased effector memory t cells (figures 3c and 3d). to gain further insights into the relationship in humans between obesity and the effector memory phenotype of cd4 t cells, we focused on the few very obese patients in our cohort (bmi > 35 kg / m2, n = 5) and observed that the percentage of effector memory t cells was raised to 18.2% (11.221.3) as compared to 13.4% (8.818.25) of the entire obese population (figures 3d and 3e) and to 10.2% (8.412.5) of the lean population (see figure 3d). to address the potential link of this phenotype with obesity - related chronic inflammation, we also analyzed the blood levels of c reactive protein (crp) in the lean, overweight, and obese subjects. as expected, crp levels rose progressively with increasing bmi (figure s4a), corroborating the correlation between obesity and inflammation. however, no significant correlation was observed between crp levels and effector memory t cells (figure s4b). furthermore, no correlation between fasting glucose levels and effector memory t cells was observed (p = 0.117). also, effector memory t cells were not significantly increased in type 2 diabetes subjects (n = 13) as compared to the rest of the subjects in the cohort (p = 0.069). the body mass fat distribution of each individual was also analyzed by dual - energy x - ray absorptiometry (figure s4c), and the resulting android / gynoid ratio was correlated with the percent of various t cell subsets. the android / gynoid ratio correlated negatively with the naive t cell subset (figure 3f) and positively with the cxcr3 effector memory t cell subset (figures 3 g and 3h). no significant correlation between the android / gynoid ratio and hladr or ccr5 effector memory t cell subsets was observed instead (figures s4d and s4e). prompted by previous suggestions that altered antigen presentation by dc might affect t cell activation during metabolic stress (chen., 2014, shamshiev., 2007), we assessed the effect of splenic dc purified from hfd or cd animals on t cell activation and differentiation. we found that in vitro co - cultures of dc isolated from the spleen of cd - fed c57bl/6 male mice with cd4 t cells obtained from the lymph nodes of cd - fed marilyn mice induced a strong proliferative response of marilyn cd4 t cells, which was reduced when cd4 t cells were isolated from donors fed hfd (figures 4a and s5a). in contrast, dc purified from the spleen of c57bl/6 male mice fed hfd were significantly less effective in promoting cd4 t cell proliferation compared to their cd - fed counterpart, irrespective of the t cell metabolic status (i.e., cd or hfd cd4 t cell donors [figures 4a and s5a ], in line with previous reports ; shamshiev., 2007). these data suggest that the metabolic status of dc plays a role in determining their ability to promote cd4 t cell proliferation. however, it did not affect the differentiation of cd4 t cells to central versus effector memory phenotype. indeed, hfd cd4 t cells showed a higher percent of cd44-ccr7-cd62-cxcr3 cells as compared to cd cd4 t cells (consistent with figures 2a2c), independent of whether they were co - cultured with dc from hfd- or cd - fed donors (figure 4b). similar results were obtained in vivo when cd4 t cells isolated from female marilyn mice were co - injected with dc from hfd or cd c57bl/6 male donors in c57bl/6 female recipients (i.e., reduced cd4 t cell proliferation in the presence of dc from hfd - fed mice and no effect on central memory versus effector memory differentiation ; figures s5b and s5c). in line with previous reports (shamshiev., 2007), dc isolated from hfd - fed mice showed impaired activation as indicated by reduced expression of the activation markers cd80 and cd86 as compared to lean dc (figure 4c). having ruled out a possible role for altered antigen presentation by dc in biasing cd4 t cell differentiation to the cxcr3 effector memory phenotype observed during hfd - induced metabolic stress, we sought to establish whether cd4 t cell intrinsic alterations were responsible for this event by interrogating pathways controlling nutrient responses in t cells. pi3k / akt and mammalian target of rapamycin complexes (mtorc1 and mtorc2) are known to be rapidly activated upon t cell priming and control transcriptional and metabolic programs that sustain cell activation (finlay., 2012, macintyre., 2011). we found a substantial increase in the levels of akt activation (phosphorylation at serine 473, a target of mtorc2) in in vivo - primed cd4 t cells isolated from the lymph nodes of hfd- as compared to cd - fed mice (figure 5a, compare lanes 68 versus 12). similar results were obtained with cd4 t cells isolated from the peripheral blood of obese versus lean individuals selected from the plic cohort (figure 5b). in contrast, threonine 308 on akt and the mtorc1 target s6 did not undergo any significant modulation when comparing in vivo - primed cd4 t cells isolated from the lymph nodes of hfd or cd mice (figures 5a and s6a). in line with recent literature (finlay., 2012, macintyre., 2011), our data suggest that cd4 t cell priming following hfd regimen triggers a pi3k / akt pathway via mtorc2, but does not result in downstream mtorc1 activation. a recent report has implicated direct signaling induced by dietary fa in t cell differentiation in the gut, with middle- and long - chain fa supporting th1 and th17 cell differentiation and short - chain fa promoting treg cell development (haghikia.,, using lipidomic analysis, we found that hfd alters the fatty acid and phospholipid profile of t cells (figure s6b), resulting in increased membrane fluidity (figure 5c). this might underlay a reduced threshold for tcr clustering and activation in hfd (swamy.,, imagestream analysis enabled us to observe that in vitro activation of t cells isolated from hfd as compared to cd mice results in increased aggregation of cholera enterotoxin (ctx) b staining (figures 5d and s6c), indicative of enhanced formation of lipid rafts, a key signaling event downstream of tcr engagement (viola., 1999). the saturated fa palmitate has been shown to directly promote the activation of the inflammasome via nlrp3 and asc (wen., 2011) and of the toll - like receptor (tlr) 4/myd88 pathway (eguchi., 2012 we therefore reasoned that the observed effector memory phenotype and enhanced activation of the pi3k / akt pathway during cd4 t cell hfd priming could be due to a direct effect of the saturated fa palmitate, whose levels increase during hfd. mirroring the results shown in figures 2a2d, we found that palmitate enriched diet (ped)-fed animals displayed an increase in the size of the cd44- ccr7-cd62l - cxcr3-lfa1 effector memory - like cd4 t cell population and no difference in the cd44-ccr7-cd62l central memory cd4 t cells as compared to cd4 t cells primed in the corresponding palmitate control diet (pcd) fed - mice (figures 5e5h). in contrast, the size of the naive t cell subset was reduced upon ped (figures 5e5h). further, cd4 t cells were cultured overnight with il-7 in the presence or absence of either palmitic or linoleic acid at a concentration which mirrors plasma levels in obese individuals (200 m), followed by activation with anti - cd3 and anti - cd28 for the indicated time points. as shown in figure 5i, cd4 t cell activation in the presence of palmitic acid for 2 hr showed a trend of enhanced akt activation compared to linoleic acid or untreated control without any modulation of s6. in similar experimental settings, only cd4 t cell activation in the presence of palmitic, but not stearic, acid for 48 hr led to reduced levels of ccr7 and cd62l mrna (figure 5j). in line with previous reports (eguchi., 2012, wen., 2011), these data suggest the existence of specific sensing mechanisms for palmitate. to further link the activation of the pi3k / akt pathway to the observed specific cd4 t cell differentiation during hfd priming, we activated cd4 t cells via cd3/cd28 antibody ligation in the presence or absence of the akt activator sc79. as expected, such treatment led to marked akt phosphorylation (figure s6d). importantly, enhanced akt activation led to reduced levels of ccr7 and cd62l mrna, as well as ccr7 protein (figures 5k and 5l), indicating that increased akt activation during cd4 t cell priming directly promotes the pathway leading to the differentiation of effector memory - like t cells. we addressed the question as to whether enhanced pi3k / akt activation as we observed during hfd priming in cd4 t cells was instrumental to the biased t cell differentiation to the observed cxcr3 effector memory phenotype. the p110 subunit of pi3k is crucial for correct tcr signaling and t cell activation (okkenhaug., 2002). furthermore, p110 engagement during t cell priming is instrumental for akt activation and differentiation toward an effector memory phenotype, at least in cd8 t cells (finlay. we therefore took genetic and pharmacologic approaches to selectively target p110 and akt activities in vivo. first, allogeneic cd4 t cells were primed in vivo in hfd - fed p110 (expressing kinase - dead p110) or wild - type (wt) mice. genetic inhibition of p110 activity completely reversed cd4 effector memory differentiation in hfd - fed animals (figures 6a6d). this was accompanied by a reduction of both akt activation on serine 473 (figure 6e) and migration in response to the chemokine cxcl10 (figure 6f). similarly, administration of the selective p110 inhibitor ic87114 during priming prevented biased differentiation of allogeneic cd4 t cells toward an effector memory phenotype upon priming (figures 6g6j). of note, administration of ic87114 completely blocked akt activation in hfd cd4 t cells, but only partially in cd mice (figure 5a, lanes 911 versus 35). finally, to further causally link pi3k and akt, we also treated cd wt mice with the akt - selective activator sc79. similar to what we had observed in vitro (figures 5k and 5l), this treatment led to increased effector memory differentiation to levels similar to those observed in hfd - fed animals (figures 6g6j and s6e). given the important role of fa oxidation (fao) in the generation of memory t cells (osullivan., 2014), we assessed whether this metabolic pathway contributes to enhanced effector memory differentiation upon priming following saturated fatty acid - enriched diet (ped). as shown in figures 5e5h, enrichment of dietary palmitate led to enhanced differentiation of primed cd4 t cells toward an effector memory phenotype. in vitro, naive t cells increased their rate of oxidative phosphorylation (ocr : oxygen consumption rate) only in response to palmitic, but not stearic, acid (figure 7a). etomoxir is an irreversible inhibitor of carnitine palmitoyltransferase-1 (cpt-1), an upstream enzyme in the fao cascade. with etomoxir during priming in ped - fed mice and found that this treatment completely prevented biased differentiation of effector memory cd4 t cells we observed upon priming following ped (figures 7b7e). chronic systemic inflammation in obesity is characterized by the secretion of th1 cytokines, as well as aberrant t cell infiltration of non - lymphoid tissue, including adipose tissue and arterial wall. obese at contains more th1 lymphocytes and higher levels of ifn than its lean counterpart (rocha., 2008). in this study, we have tested the hypothesis that excess dietary saturated fa directly affects the differentiation and trafficking of cd4 t cells to non - lymphoid tissues. we report that differentiation of memory cd4 t cells primed in hfd - fed mice is biased toward a cd44-ccr7-cd62l - cxcr3-lfa1 effector memory - like phenotype without major changes in the size of the central memory pool. these t cells display preferential trafficking to non - lymphoid, inflammatory sites, as well as increased effector function following adoptive transfer. although metabolically imbalanced dc from obese / dyslipidaemic hosts tend to support th1/th17 responses (chen., 2014,, 2012, reynolds., 2012, shamshiev., 2007), our data show that they play no role in determining cd4 t cell commitment to effector memory - like phenotype nor homing. the cd4 t cell developmental bias is instead mediated by direct exposure of t cells to saturated fa ; i.e., palmitate, leading to enhanced activation of a pi3k p110-akt - dependent pathway upon priming. our data suggest that this pathway might involve mtorc2 leading to akt activation, but does not result in the downstream activation of mtorc1. the key role of the pi3k p110/akt axis is further confirmed by our observation that selective inactivation of p110 in hfd can reestablish homeostatic cd4 t cell responses. a similar signaling cascade has previously been shown to direct the differentiation of cd8 effector t cells (finlay., 2012, macintyre., 2011), whereby high levels of akt activation led to the downregulation of cd62l and ccr7 expression and redirected the trafficking of effector cd8 t cells away from the secondary lymphoid tissues into the sites of inflammation. specifically, it has been shown that downstream of tcr engagement, akt controls transcriptional programs directing cytotoxic t cell fate, but is dispensable for t cell metabolism (macintyre., 2011), while pdk1 directly regulates mtor and hypoxia - inducible factor 1, leading to an integrated control of metabolism and migration of cd8 t cells (finlay., 2012). in addition to showing that similar signaling events underlay the differentiation of cd4 t cells, our data expand the reach of these studies from the basic mechanisms of t cell activation and effector functions control to the biased establishment of unwanted t cell responses ; i.e., specific pro - inflammatory effector and migratory phenotypes, in a pathophysiologic context such as obesity. this is in line with a recent study showing the link between obesity and metabolic diseases via increased th17 t cell differentiation (endo., 2015). it has also been reported that obesity could cause impairment in the t cell memory response to secondary influenza infection, thereby affecting vaccine efficacy (karlsson., 2010). as we did not observe major differences in the size of the central memory population upon priming following hfd or cd, it is possible that central memory cells are functionally impaired in obesity. in line with the above study interestingly, our data shown in figure 5a suggest that in physiologic conditions (cd), different upstream kinases might control the activation of akt induced upon t cell priming, as the p110 inhibitor ic87114 did not completely block akt phosphorylation. by contrast, in hfd mice, akt phosphorylation was completely abrogated by ic87114, indicating that the enhancement of akt activation by dietary lipids is particularly dependent on p110. the molecular mechanisms of lipid exchange on the t cell membrane in physiology and during hfd remain to be fully established, as well as the question as to whether fa act by altering the cell membrane lipid balance or by binding to specific receptors, as recently suggested (eguchi., 2012, haghikia., 2015, wen., 2011). our data showing increased membrane fluidity, as well as enhanced formation of lipid rafts upon tcr engagement, might provide some clues as to how excess dietary saturated fa impact on t cell responses. similarly, our data suggest that specific mechanisms exist for palmitate sensing, which remain to be defined. in this context, we have observed that t cell exposure to fa at the time of tcr triggering in vitro is not sufficient to induce the biased effector memory differentiation we observed in vivo, suggesting that palmitate sensing and alteration of the t cell membrane must occur prior to antigen recognition. hence, the need to maintain naive t cell viability over prolonged culture with or without fa and in the absence of antigen stimulation has been a major challenge in setting up in vitro models to study t cell differentiation following prolonged exposure to fat overload. we have partially overcome this issue by adding il-7 to the culture of naive t cells during incubation with or without fa prior to tcr stimulation, and this led to the results we showed in figures 5i and 5j. defining in vitro systems of immunization following prolonged exposure to certain nutrients would be extremely useful to test pathways involved with t cell differentiation, such as palmitate sensing. the observation that etomoxir prevents biased differentiation of effector memory cd4 t cells upon priming following saturated fatty acid - enriched diet supports the concept that preferential utilization of fao during t cell activation promotes effector memory cd4 t cell differentiation following hfd. ampk - induced fao has been shown to play an important role in the generation of memory t cells (osullivan., 2014). increased fao is also a feature of hfd (cole., 2011, however, reduced ampk activity is also observed in hfd (lindholm., 2013, liu., 2006), and its reactivation by metformin is known to ameliorate glucose tolerance in obesity / type 2 diabetes. thus, during hfd, ampk - induced fao is unlikely to play a role in the biased effector memory cd4 t cell differentiation we observe. whether and how an enhanced pi3k / akt pathway leads to enhanced fao remain open questions. the biased development of pro - inflammatory effector memory cd4 t cells occurred in mice within 48 weeks from exposure to hfd, a time that precedes overt obesity and associated complications. this might imply a causative role of altered t cell differentiation in the pathology accompanying metabolic stress, and that immune challenges occurring naturally in individuals before the establishment of overt obesity could drive cardiovascular disease later in life. finally, our observation that selective pi3k p110 and fao inhibition can correct cd4 biased differentiation might provide targets for the therapeutic control of inflammation during metabolic stress. likewise, our study supports the notion that by modulating metabolism, we can affect immune responses, a relatively novel concept with substantial translational implications. in this regard, hints and hopes come from few recent studies, such as the report that cd4 t cells from systemic lupus erythematosus (sle) patients exhibited enhanced glycolysis and mitochondrial metabolism that correlated with their activation status and their excessive ifn production was significantly reduced upon in vitro treatment with metformin. the same study also showed that in a number of murine lupus models the combination of metformin and 2-deoxy - glucose led to normalization of cd4 t cell metabolism and overall reversed disease biomarkers (mehta and chandel, 2015, yin., 2015). human blood was obtained from healthy donors according to ethics approval obtained from the universit degli studi di milano (cholesterol and health : education, control and knowledge - studio check [sefap / pr.0003 ] - reference number fa-04-feb-01). all in vivo experiments were conducted under the uk home office regulation (ppl 70/7443). the plic study (a substudy of the check study) is a large survey of the general population living in the northern area of milan (n = 2,606 ; average age 66 years) (baragetti.., 2007), followed at the center for the study of atherosclerosis, bassini hospital (cinisello balsamo, milan, italy). for all participants, information on medical histories and therapies was obtained, and bmi (kg / m) and waist / hip ratio (w / h) were calculated. after an overnight fast, blood samples were collected from the antecubital vein for determination of lipid profile and glucose levels, as previously described (ammirati., 2012, information on total leukocytes and subfraction counts was available for n = 1,172 subjects, representatives of the entire population in terms of age, gender distribution, and clinical parameters according to the kolmogorov - smirnov test. this cohort was divided by bmi in accordance to the world health organization definition (lean for bmi 99%, as assessed using the molecular probes patented live / dead viability (invitrogen), according to the manufacturer instructions. in vitro : murine cd4 t cells were isolated with commercially available isolation kits (negative selection ; easysep, invitrogen) according to manufacturer s instructions from pooled lymph nodes (inguinal, mesenteric, axillary, brachial, superficial and deep cervical, lumbar, and sacral) of c57bl/6 mice and activated with plate bound anti - cd3 (1 g / ml, ebioscience), anti - cd28 (5 g / ml, ebioscience), and il-2 (10 ng / ml, roche) in the presence or absence of the akt activator sc79 (500 nm, tocris). in alternative, isolated cd4 t cells were cultured over night with il-7 (1 ng / ml, peprotech) in the presence or absence of palmitic, linoleic, or stearic acid (200 m, sigma), followed by activation with plate bound anti - cd3 (0.5 g / ml, ebioscience) and anti - cd28 (2.5 g / ml, ebioscience). in vivo : in rag2ko marilyn c57bl/6 female mice fed cd or hfd (test diet ; table s2) for 8 weeks memory antigen - specific (hy - specific) cd4 t cells were generated by i.p. immunization with wt c57bl/6 male splenocytes (1.5 10) for 14 days. t cells were then isolated from pooled lymph nodes of these mice (figures 1a1d, 2a2 g, and s2a s2f) and injected i.v. in recipients (see experimental procedures, in vivo peritoneal recruitment, below) or used for chemotaxis or flow cytometry. in p110 or wt c57bl/6 mice fed hfd (or cd as control) or ped (or pcd as control ; test diet ; table s2) for 8 weeks, memory cd4 t cells were generated by intraperitoneal immunization with cba / balbc splenocytes (1.5 10 ; allogeneic immunization) for 7 days. t cells were then isolated from mesenteric lymph nodes of these mice and analyzed by facs or western blot (figures 2h and 2i, 5a, 5e5h, 6a6e, 6g6j, and 7). in this system, the pi3k p110 inhibitor ic87114 (tocris) was administered i.p. at a dose of 50 mg / kg daily during the 7-day immunization (ying., 2012). the akt activator sc79 (tocris) was administered i.p. at a dose of 7 mg / kg daily during the 7-day immunization (moreira., 2015). etomoxir (sigma) was administered i.p. at a dose of 15 mg / kg daily during the 7-day immunization (shriver and manchester, 2011). in ot - ii p110 mice fed cd or hfd for 8 weeks, memory antigen - specific (ova - specific) cd4 t cells were generated by intraperitoneal immunization with ovalbumin peptide (10 g, cambridge research biochemicals) and poly ic (1:1, sigma) for 7 days. t cells were then isolated from mesenteric lymph nodes of these mice (figure 6f) and analyzed for chemotaxis. in vivo activated t cells from rag2ko marilyn female mice were isolated from pooled lymph nodes of hfd- or cd - fed donors, labeled with cfse (hfd ; 3.3 m, invitrogen) or pkh26 (cd ; 2 m, sigma), pooled together, and intravenously injected in hfd or cd recipient male mice (5 10 cells / mouse) that had received an intraperitoneal injection with zymosan (1 mg / mouse, sigma) and ifn (600 u / mouse, peprotech) 48 hr earlier. at 48 hr after injection, blood, peritoneal lavage, peritoneal membrane, mesenteric lymph nodes, and spleen were collected and analyzed by facs for presence of cfse- and pkh26-labeled t cells. dead cells were excluded from analysis by staining with fixable aqua dead cell stain (invitrogen). isolated lymphocytes or dc were stained for surface markers : cd3, cd4, cd8, cd25, cd44, ccr7, cd62l, cxcr3, lfa1, v6, cd40, cd80, cd86, mhcii, cd11c, cd19, cd14, and f4/80 with fluorescently conjugated primary antibodies (1:200, ebioscience / biolegend) at 4c for 30 min (37c for ccr7) in facs buffer (pbs + 2% fcs + 0.1% sodium azide) and fixed at 4c for 30 min with 1% pfa in facs buffer. for intracellular staining, isolated lymphocytes were incubated in permeabilization / fixation buffer (ebioscience) at 4c for 1 hr. samples were washed in permeabilization buffer and stained for the cytokines / chemokine ifn, il4 (1:200, ebioscience / biolegend) and cxcl10 (1:200, stratech) with fluorescently conjugated primary antibodies at 4c for 30 min in permeabilization buffer. all samples were then assessed by flow cytometry using a lsrfortessa (bd biosciences) and flowjo version 10 software. dot plots were concatenated to be representative of the independent biological replicates used in each experiment. chemotaxis assays were performed in 5 m transwell inlays (corning). in vivo primed lymphocytes were isolated from mice fed cd or hfd for 8 weeks, suspended in migration medium (rpmi 2% fcs), and seeded in the upper transwell chamber (3 10 cells / transwell). chemokines (peprotech) were added to the lower chamber, cxcl10 (300 ng / ml), or ccl19/21 (200 ng / ml of each chemokine). migrated t cells were counted with a haemocytometer 3, 6, and 24 hr after seeding, then the percent of migrated cells were calculated. protein lysates were prepared from : (1) mesenteric lymph nodes isolated from in vivo primed mice fed cd or hfd for 8 weeks, (2) cd4 t lymphocytes isolated by magnetic beads (mylteni) from lean subjects (median bmi 22.43 kg / m, minimum 17.67 kg / m, and maximum 24.54 kg / m) and obese subjects (median bmi 31.64 kg / m, minimum 30.48 kg / m, and maximum 41.79 kg / m), and (3) cd4 t lymphocytes isolated from pooled lymph nodes and activated as described above for the indicated time points. samples were lysed in nonidet p-40 lysis buffer (50 mm hepes ph 8.0, 350 mm nacl, 1% nonidet p-40, 1 mm edta, 1 mm na3vo4, 1 mm naf, 20 mm glycerol-2-phosphate, 1 mm pmsf, 1 mm dtt, 10 g / ml aprotinin, 10 g / ml leupeptin, and a protease inhibitor mixture ; roche). equivalent amounts of protein as determined by standard bradford assay (bio - rad) were separated by sds / page and transferred to a nylon membrane (ge healthcare). membranes were blocked for 2 hr at room temperature in 5% milk / tbs - t (5% bsa / tbs - t for phosphorylation antibodies), incubated overnight at 4c with primary antibodies (1:1,000), and subsequently with hrp - conjugated secondary antibody (amersham bioscience) (1:5,000). antibodies against p - akt (s473 and t308), akt, p - s6 (s235/236), s6 and -actin were purchased from cell signaling. density of bands was calculated relative to akt, s6, or -actin as shown in figures via the use of imagej software. rna was isolated using commercially available kits (qiagen) according to the manufacturer s instructions and assessed for quality and quantity using absorption measurements. reverse transcription to cdna was performed according to the manufacturer s instruction (applied biosystems). gene expression analysis was done using sybr green supermix (bio - rad) in cfx connect light cycler (bio - rad), according to the manufacturer s instructions. gene - relative expression was calculated using the ct method and normalized to a reference control (gapdh) : f 5-ggctcatgaccacagtcca-3 ; r 5-cacattgggggtaggaacac-3. ccr7 and cd62l primers sequences were the same as published in finlay. t cells were isolated from pooled lymph nodes of rag2ko marilyn female mice fed cd or hfd for 8 weeks and were labeled with cfse (3.3 m) in pbs for 10 min at room temperature. t cells (2 10/well) were then stimulated with cd11c dc (7 10/well) isolated from the spleen of c57bl/6 male mice fed cd or hfd for 8 weeks. cd11c dc were treated with mitomycin c to prevent proliferation prior to incubation with t cells. t cells were harvested 3 days later and cfse dilution was assessed by flow cytometry (figures 4a, 4b, and s5a). in vivo : t cells were isolated from pooled lymph nodes of rag2ko marilyn female mice, labeled with cfse (3.3 m) in pbs for 10 min at room temperature, and intravenously injected in c57bl/6 recipient female mice (5 10 cells / mouse). concurrently, cd11c dc were isolated from the spleen of c57bl/6 male mice fed cd or hfd for 8 weeks and intraperitoneally injected in the c57bl/6 recipient female mice (1/4 spleen derived dc / mouse). cells were harvested from the peritoneal lavage, spleen, and draining lymph nodes 5 days later and cfse dilution was assessed by flow cytometry (figures s5b and s5c). rag2ko and marilyn female mice fed cd or hfd for 8 weeks received a c57bl/6 male - derived skin graft on the upper right back. some rag2ko mice were i.v. injected with 1 10 cd4 t cells isolated from marilyn females 9 days post - transplantation. t cells isolated from pooled lymph nodes of c57bl/6 mice fed cd or hfd for 8 weeks were activated as described above for 4 hr or left inactivated. cells were stained with ctxb - fitc (8 g / ml, sigma) and cd4-pecy7 (1:200, ebioscience) at 4c for 30 min in facs buffer (pbs + 2%fcs + 0.1% sodium azide) and fixed at 4c for 30 min with 1% pfa in facs buffer. samples were assessed using imagestream flow cytometer (amnis) and ideas software. for quantitative image analysis of lipid raft aggregation, bright the bright detail intensity feature calculates the sum of intensity values from the brightest areas within a cell that are morphologically defined as the peak fluorescence distributions of 3 pixel radius or less. there were 5,000 cd4 t cells that were analyzed per condition. as described before, isolated naive cd4 t cells were cultured over night with il-7 in the presence or absence of palmitic or stearic acid. 2 10 cd4 t cells were then seeded in a 96-well microplate in xf assay modified dmem and incubated in a non - co2 incubator for 1 hr prior to the assay. statistical analysis of human data was performed using the spss v.21.0 for windows (ibm corporation) program. a kolmogorov - smirnov test was performed to test whether the studied cohort was representative of the entire plic population in terms of age, gender distribution, and clinical parameters. a shapiro - wilk test was performed to verify the normal distribution of linear variables ; median and inter - quartile range (iqr) was reported and mann - whitney u non - parametric test was performed. outliers detection (above and below 1.5 iqr) was performed by use of grubb s test. anova test was performed to compare clinical parameters and leukocyte subfraction among subjects divided by bmi and then adjusted for age, gender, and therapies by using analysis of covariances (ancova). for all analysis, statistically relevant differences were considered for p values < 0.05. for murine studies, data are expressed as mean sem. two - tailed student s t test was used to compare two groups with parametric data distribution. for multiple comparison analysis, 1-, 2-, or 3-way anova was used, unless otherwise specified (figure 1e). in all cases, g.d.n., and f.m.m.- b. performed the experiments : c.m., j.s., d. cucchi, d. coe, f.b., | summarylow - grade systemic inflammation associated to obesity leads to cardiovascular complications, caused partly by infiltration of adipose and vascular tissue by effector t cells. the signals leading to t cell differentiation and tissue infiltration during obesity are poorly understood. we tested whether saturated fatty acid - induced metabolic stress affects differentiation and trafficking patterns of cd4 + t cells. memory cd4 + t cells primed in high - fat diet - fed donors preferentially migrated to non - lymphoid, inflammatory sites, independent of the metabolic status of the hosts. this was due to biased cd4 + t cell differentiation into cd44hi - ccr7lo - cd62llo - cxcr3 + -lfa1 + effector memory - like t cells upon priming in high - fat diet - fed animals. similar phenotype was observed in obese subjects in a cohort of free - living people. this developmental bias was independent of any crosstalk between cd4 + t cells and dendritic cells and was mediated via direct exposure of cd4 + t cells to palmitate, leading to increased activation of a pi3k p110-akt - dependent pathway upon priming. |
the management of diabetes has seen changes in the past 30 years, which have improved the lives of patients afflicted with this disease. improvement in glucose measurement started with the introduction of selfmonitoring of blood glucose (smbg) in the late 1980s, which was one of those important changes. as the second device for measurement of blood glucose level, the continuous glucose monitoring system (cgm) was introduced to the market around the beginning of this century. it provides maximal information about fluctuations in blood glucose levels throughout the day and optimal treatment decisions to control diabetes. in 1999, the food and drug administration approved the cgm device in the usa. since then, the device has been improved dramatically, and the realtime cgm and insulin pumpintegrated system are already available commercially in the usa and europe. in japan, the history of cgm is relatively short. the ministry of health, labor and welfare approved the cgm device in 2009, approximately 10 years after the usa. furthermore, the secondgeneration blind cgm, the minimed gold of medtronic, has been the only device approved under the governmentsupported medical insurance in japan. a new wireless, but blind cgm, is going to be in japanese market in a very near future, at last. with this different historical background, cgm has already been studied extensively in the usa and europe. at this stage, it is necessary for japanese clinicians to know what has already been investigated. in particular, we need to understand whether the continuous glucose monitoring system really is an accurate, safe and clinically effective device, or not. in the present article, we review the literature and introduce some useful information on diabetes care in the clinical setting especially to the physicians in countries with short history of cgm. the diabetes control and complications trial (dcct) confirmed that patients treated intensively with insulin show better results with regard to the prevention of diabetic microvascular complications than those treated conventionally. however, we also know that the intensive insulin therapy enhances the risk of hypoglycemia. patients who experience frequent episodes of hypoglycemia often develop various abnormalities and lose their ability to detect hypoglycemia. hypoglycemia unawareness and it perpetrates a vicious cycle of recurrent hypoglycemia. however, there is strong evidence showing that minimization of the hypoglycemic episodes through more meticulous glucose control can actually reverse many of the counterregulatory defects. for meticulous glucose control and prevention of hypoglycemia, the cgm is a useful device, as it provides maximal information about fluctuating blood glucose levels throughout the day. the finger stick test provides only information on blood glucose at one point in time, so even if hypoglycemia unawareness occurs between the measured points, we are not able to detect it. with the cgm, we might accomplish both purposes better control with the intensive insulin therapy and prevention of hypoglycemia unawareness. the cgm is a method that provides constant checking of blood glucose level through measurement of glucose concentrations in the interstitial fluid. the minimed gold of medtronic, which is currently available and used in japan under the governmentbased health insurance system, features a sensor inserted in the subcutaneous tissue just under the skin of the abdomen. the sensor comprises a flexible, platinumplated electrode housed inside a permeable membrane, which can be used for up to 72 h. subcutaneous glucose level is measured by the glucoseoxidase method, and the interstitial glucose level measured every 10 s is sent to a monitor that shows the average blood glucose value every 5 min. the minimed gold calculates blood glucose values in the range of 40400 mg / dl. athough the user has no access to realtime glucose levels, the measured data can be downloaded into a spreadsheet. this is carried out at least four times a day in the minimed gold, using the finger stick test. it means that selfmonitoring of blood glucose is closer to real glucose levels, the venous glucose levels, at any one point in time. it should be noted that the device has a time lag between blood and interstitial glucose levels. there was no information on the performance of the sensor during sustained hypoglycemia or during recovery from hypoglycemia at that time. venous blood glucose levels were maintained at euglycemia for the first 60 min, then fell to 45 mg / dl for 60 min, but were finally restored to euglycemia. the sensor profiles paralleled blood glucose levels at each of the three plateaus with a correlation coefficient of 0.79, and mean absolute error of 7%. the drop in glucose level measured by the sensor closely matched the drop in blood glucose, but the recovery from hypoglycemia was delayed by an average of 26 min. the device was the firstgeneration minimed cgm, which was probably less accurate than the secondgeneration, minimed gold. in 2003, boyne. tried to measure a time lag between the firstgeneration minimed cgm and blood glucose, and also between the different sensors. a total of 14 patients with type 1 diabetes each had two sensors placed subcutaneously in the abdomen, acquiring data every 5 min. at the same time, blood glucose was sampled every 5 min for 8 h. the results showed that the time differences between blood and interstitial glucose levels ranged from 4 to 10 min, with the interstitial glucose lagging behind blood glucose in 81% of cases. the mean (sd) difference between the two sensors in each patient was 6.7 5.1 min, representing random variation in sensor response. the authors also showed that the lag times were statistically significant for the rise (10.1 10.1 min, p 180 mg / dl ; figure 3). as the cgega is intended for software application, most of these parameters could be user selectable. for example, the time lag between blood and interstitial glucose has a default value of 7 min, based on literature data. if a device has a longer technical lag, then the software would allow the time lag used by the pega to be changed. the continuous glucoseerror grid analysis (cgega) computes the accuracy of the combination of rateerror grid analysis (rega) plus pointerror grid analysis (pega) into three clinically relevant regions : hypoglycemia (blood glucose 70 mg / dl), euglycemia and hyperglycemia (blood glucose > 180 mg / dl). used the cgega to evaluate the continuous glucose monitoring system, therasense freestyle navigator. using the cgega, they reported that the accuracy of the navigator, measured as a percentage of accurate readings plus benign errors, was significantly different at hypoglycemia (73.5%), euglycemia (99%) and hyperglycemia (95.4%). wentholt. explored the cgega by comparing it with classical accuracy assessment methods, such as correlation, linear regression and mean absolute difference (mad), using data reported in a previous study that compared two different continuous glucose sensors in type 1 diabetic patients. they found differences in the accuracy of mad and cgega, and that the cgega was not reliable. in contrast, clarke. responded by showing that the methods used by wentholt unfortunately failed to take into account the basic structure of continuous glucose monitoring data, which represents a time series analysis. they reported that it is impossible to compare the different methods altogether, and approached the issue of accuracy by applying several different methods for each different purpose. using the different methods complementarily is the most effective way to prove the accuracy of the device. the investment in cgega, which has been widely used by scholars until now, is really meaningful for assessing the accuracy of cgm. by using multiple established assessment methods for evaluation, we can reach the true accuracy of cgm, thus ensuring the safety and clinical effectiveness of the device. in the symposium, wolpert published some important points. as we already know, there is a lag time between interstitial and blood glucose. the lag time ranges from 5 to 15 min depending on the rate of glucose change. increasing glucose levels are reflected first in capillary blood. however, detecting decreases in glucose after insulin injection or exercise might be confounded by the placement of the cgm sensor. for example, if the cgm sensor is placed next to an insulinsensitive tissue, the glucose level might decrease first in the interstitial fluid before it is reflected in capillary blood glucose. also, wolpert pointed out that the sensor needs to be calibrated when glucose is in a steady state. if the patient carried out the calibration when glucose is rising at 3 mg / dl / min and there is a 10min lag, then there will be a discrepancy of approximately 30 mg / dl between the capillary glucose measurement and the interstitial glucose measurement. another important point relates to the education of the patient. in the usa and europe, the realtime cgm are already available on the market, and many articles have been published on the device the realtime cgm wireless type is a very convenient and useful device, but it can potentially lead to excess treatment based on the measured data. if patients overcompensate for rising glucose levels using frequent bolus injections of insulin, they are at increased risk of hypoglycemia. also, patients could reduce or discontinue basal insulin therapy in order to avoid hypoglycemia. this occurs more commonly in patients who lack the understanding of the concept of the lag time between capillary and interstitial glucose, thus considering their glucose to be much lower than it actually is. we need to educate these patients about insulin pharmacodynamics, as well as the factors that affect postprandial glucose patterns. they must also be taught not to rely on interstitial glucose data alone to assess hypoglycemia. a group at the mayo clinic concluded that the device was associated with a significant reduction in mean glycated hemoglobin (hba1c). it was true for adults with type 1 diabetes as well as type 2 diabetes. there was no significant difference in hba1c reduction between studies of realtime vs nonrealtime devices, if the devices were appropriately used. the metaanalysis by mayo clinic is important, because it was newly published in 2011, and the number of the trials providing the data, 19, was the biggest among the metaanalyses about cgm. data of adverse events were available only in a few trials and comprised mainly mild reactions, such as redness and itching, at the sensor implantation site. the device errors of data storage, and alarms going off infrequently occurred. in october 2011, continuous glucose monitoring : an endocrine society clinical practice guideline was published. even if there are various conclusions in metaanalyses about cgm, the task force evaluated three potential uses of cgm : (i) realtime cgm in an adult hospital setting ; (ii) realtime cgm in children and adolescent outpatients ; and (iii) realtime cgm in adult outpatients. the authors used the available data to develop evidencebased recommendations for good control of glycemia and limiting the risk of hypoglycemia. in that guideline, an alternative for patients who can not safely and effectively take advantage of the information provided to them by realtime cgm, even though there are some studies concluding that there was no significant difference in hba1c reduction between realtime vs nonrealtime devices. however, it might be an appropriate statement in countries where the realtime cgm is the first choice cgm. by reviewing the literature on cgm, we understand that this device has the potential to improve diabetes care in the clinical setting, provided we take into consideration its pros and cons. there is a need for more knowledge about its accuracy, safety and clinical effectiveness through daily medical examination. furthermore, the introduction of the more developed devices, such as realtime cgm available in the usa and europe, under the japan governmentsponsored healthcare system should be encouraged. | abstractthe continuous glucose monitoring system (cgm) has been used for constant checking of glucose level by measuring interstitial glucose concentrations, since the early days of the 21st century. it can potentially improve diabetes care if used carefully with the understanding of the characteristics of this system. although there is a time lag of approximately 515 min between blood and interstitial glucose levels, the system is considered the most suitable device for meticulous glucose control and prevention of hypoglycemia. a large number of studies have examined its accuracy, safety and clinical effectiveness. the continuous glucoseerror grid analysis (cgega), designed by wl clarke, evaluates the clinical accuracy of cgm. it examines temporal characteristics of the data, analyzing pairs of reference and sensor readings as a process in time represented by a bidimensional time series and taking into account inherent physiological time lags. investment in cgega is clearly meaningful, even though there are other methodologies for evaluation. the use of each method complementarily is the most effective way to prove the accuracy of the device. the device has improved gradually, and realtime cgm, which allows realtime monitoring of blood glucose level, is already available commercially. the use of realtime cgm could potentially lead to over or undertreatment with insulin. patient education through proper and effective handling of the new device is essential to improve diabetes care. (j diabetes invest, doi : 10.1111/j.20401124.2012.00197.x, 2012) |
the prevalence of urinary incontinence (ui) is increased among older adults and is associated with poor quality of life, social isolation, falls, and depression(14). drug therapy for ui is associated with the potential for adverse events and, may have significant cost ; thus adherence is often maintained for only a few months(6, 7). additional treatment modalities are needed in order to maximize the impact of multicomponent therapy and reduce the potential for adverse events. recent studies have suggested a relationship between vitamin d and conditions increasing the risk of ui among both men and women. vitamin d receptors are present in the bladder and striated muscle of the pelvic floor musculature(8, 9). additionally, prostatic cells express 1-hydroxylase and can synthesize 1,25-di - hydroxyvitamin d (the active form of vitamin d)(10). low vitamin d levels are associated with increased prevalence of pelvic floor disorders among women and moderate - to - severe ui in men(1113). however, these studies are based on cross - sectional data. one randomized controlled trial demonstrated treatment with a vitamin d analog reduced prostate enlargement in the setting of benign prostatic hyperplasia(14). evaluation of data assessing the longitudinal relationship between vitamin d and incident ui would allow for additional insights and provide next steps in determining the need for interventional studies. thus, our aim was to evaluate the association of low vitamin d with incident ui in a cohort of community - dwelling older adults. the university of alabama at birmingham study of aging (soa) is a longitudinal cohort study of community - dwelling older adults designed to assess factors associated with changes in mobility. between 1999 and 2001 the study recruited 1,000 individuals 65 years of age and older from five counties in west central alabama, including 3 rural counties and 2 urban counties. individuals were randomly selected from medicare beneficiary lists stratified by race and gender so that the cohort included equal numbers of men and women, black and white, and urban and rural residence. exclusion criteria included : inability to understand the recruiter, residing in nursing homes, or inability to independently coordinate an appointment for the baseline, in - home interview. the institutional review board at the university of alabama at birmingham approved the study and written informed consent was obtained prior to beginning study procedures. at baseline and in 2004, a two - hour, in - home assessment was performed, which included a medical history, cognitive screening test, screening for depression, assessment of activities of daily living, and questions regarding multiple facets of functional status. eligibility for the in - home follow - up in 2004 was the same as the baseline evaluation with 624 of 733 (85.1%) of surviving participants agreeing to the in - home assessment in 2004(15). these 624 participants were offered the opportunity to have a blood test obtained, and 408 (65.4%) agreed to the lab test. blood samples were used to measure multiple laboratory assays, but not vitamin d levels. vitamin d analyses were completed in 2013 for 350 of the 408 (85.8%) participants who had a blood sample obtained and had sufficient serum available for the vitamin d assays. of these participants with vitamin d data available, 187 /350 (53.4%) did not have ui in 2004. follow - up incident ui data were available on 175/187 (94.6%) of these participants. thus, prevalence of vitamin d deficiency and insufficiency was assessed using 350 participants ; while the 175 participants without ui in 2004 and with follow - up incident ui data were the focus of the current analysis. every six to twelve months, participants were contacted by phone to complete a questionnaire including information about ui. participants provided information about the presence of ui using the validated two - item incontinence severity index, which correlates well with ui measured with 24-hour pad weights and ui frequency on bladder diaries(16). participants were asked, have you ever leaked even a small amount of urine ? and how often does that occur ? incident ui was classified as an affirmative response to the first question and a response to the second question of at least monthly ui. analysis of serum concentrations of 25-hydroxyvitamin d [25(oh)d ] was performed on stored sera using an ids - isys analyzer. to ensure quality control of the vitamin d assay, the laboratory participates in the vitamin d external quality assessment scheme every 3 months and in the nist / nih vitamin d metabolites quality assurance program semi - annually. for the purposes of this evaluation, vitamin d deficiency was defined as a serum 25-hydroxyvitamin d level < 20 ng / ml and vitamin d insufficiency as a 25-hydroxyvitamin d level from 20 ng / ml to < 30 ng / ml. descriptive statistics were used to analyze baseline characteristics of the subsample (n=350) of uab study of aging participants for which sera were available for vitamin d assays in 2004 and the 175 participants who were included in the incident ui analysis. multivariable logistic regression and cox proportional hazard models adjusted for age, gender, and race / ethnicity were utilized to evaluate the relationship between vitamin d status and incident ui among those participants reporting no ui at baseline. all analyses were conducted using sas 9.2 (sas institute, cary, nc). the university of alabama at birmingham study of aging (soa) is a longitudinal cohort study of community - dwelling older adults designed to assess factors associated with changes in mobility. between 1999 and 2001 the study recruited 1,000 individuals 65 years of age and older from five counties in west central alabama, including 3 rural counties and 2 urban counties. individuals were randomly selected from medicare beneficiary lists stratified by race and gender so that the cohort included equal numbers of men and women, black and white, and urban and rural residence. exclusion criteria included : inability to understand the recruiter, residing in nursing homes, or inability to independently coordinate an appointment for the baseline, in - home interview. the institutional review board at the university of alabama at birmingham approved the study and written informed consent was obtained prior to beginning study procedures. at baseline and in 2004, a two - hour, in - home assessment was performed, which included a medical history, cognitive screening test, screening for depression, assessment of activities of daily living, and questions regarding multiple facets of functional status. eligibility for the in - home follow - up in 2004 was the same as the baseline evaluation with 624 of 733 (85.1%) of surviving participants agreeing to the in - home assessment in 2004(15). these 624 participants were offered the opportunity to have a blood test obtained, and 408 (65.4%) agreed to the lab test. blood samples were used to measure multiple laboratory assays, but not vitamin d levels. vitamin d analyses were completed in 2013 for 350 of the 408 (85.8%) participants who had a blood sample obtained and had sufficient serum available for the vitamin d assays. of these participants with vitamin d data available, 187 /350 (53.4%) did not have ui in 2004. follow - up incident ui data were available on 175/187 (94.6%) of these participants. thus, prevalence of vitamin d deficiency and insufficiency was assessed using 350 participants ; while the 175 participants without ui in 2004 and with follow - up incident ui data were the focus of the current analysis. every six to twelve months, participants were contacted by phone to complete a questionnaire including information about ui. participants provided information about the presence of ui using the validated two - item incontinence severity index, which correlates well with ui measured with 24-hour pad weights and ui frequency on bladder diaries(16). participants were asked, have you ever leaked even a small amount of urine ? and how often does that occur ? incident ui was classified as an affirmative response to the first question and a response to the second question of at least monthly ui. analysis of serum concentrations of 25-hydroxyvitamin d [25(oh)d ] was performed on stored sera using an ids - isys analyzer. to ensure quality control of the vitamin d assay, the laboratory participates in the vitamin d external quality assessment scheme every 3 months and in the nist / nih vitamin d metabolites quality assurance program semi - annually. for the purposes of this evaluation, vitamin d deficiency was defined as a serum 25-hydroxyvitamin d level < 20 ng / ml and vitamin d insufficiency as a 25-hydroxyvitamin d level from 20 ng / ml to < 30 ng / ml. descriptive statistics were used to analyze baseline characteristics of the subsample (n=350) of uab study of aging participants for which sera were available for vitamin d assays in 2004 and the 175 participants who were included in the incident ui analysis. multivariable logistic regression and cox proportional hazard models adjusted for age, gender, and race / ethnicity were utilized to evaluate the relationship between vitamin d status and incident ui among those participants reporting no ui at baseline. all analyses were conducted using sas 9.2 (sas institute, cary, nc). of the 350 participants who had serum samples available for vitamin d assays in 2004 (the baseline year for this analysis) (table 1), 54% were vitamin d deficient [25(oh)d < 20 ng / ml ] and 25% were vitamin d insufficient [25(oh)d 20 ng / ml to < 30 ng / ml ] (table 1). among the 187 subjects with no ui at baseline, 57% (107/187) were vitamin d deficient and 24% (45/187) were vitamin d insufficient. for these 187 subjects, the mean baseline 25(oh)d level was 20.5 ng / ml 9.7. mean length of follow - up was 34.5 months (range 6 42). for the 175 of the 187 subjects who had follow - up evaluation for incident ui over 42 months, incident ui occurred in 37% (65/175, 37% of women and 38% of men). these 175 participants generally reflected the demographic distribution of the original soa cohort and those included in a previous analysis of incident ui(17). among those with vitamin d deficiency, 37/97 (38%) developed ui and among those with vitamin d insufficiency, 20/44 (45%) developed ui. this compared to 8/34 (23%) of those with sufficient levels of vitamin d. however, there was no statistically significant difference in the proportion of individuals developing incident ui during the study period by vitamin d status (p=0.13). after adjustment for age, gender and race / ethnicity, cumulative incident ui at 42 months was associated with vitamin d insufficiency status at baseline [aor 3.18 (95%ci 1.148.87), p = 0.03 ]. there was a trend toward association between baseline vitamin d deficiency and incident ui [2.47 (95%ci 0.936.53), p=0.07 ]. however, there was no association between vitamin d insufficiency or deficiency and time to incident ui in unadjusted (log - rank p=0.19) or adjusted analyses (cox model overall test of significance, p=0.14). these results suggest a potential association between vitamin d and the development of ui among a racially diverse cohort of older men and women. an evaluation of the longitudinal association between vitamin d status and incident ui among older adults is novel. previous cross - sectional data have suggested an association between low vitamin d status and lower urinary tract symptoms in men and women ; however, these studies are limited because they can not evaluate for causality(11, 12). the university of alabama at birmingham soa is unique among longitudinal studies of aging populations because the study population is racially diverse, validated questionnaires were used to assess urinary symptoms, and stored serum samples were available. there are several mechanisms by which vitamin d could exert influence on lower urinary tract function. vitamin d receptors are present in the bladder and striated muscle of the pelvic floor musculature(8, 9). skeletal muscle control and strength are vital for the voluntary control of the urethral sphincter and pelvic floor muscles and likely a significant factor in achieving continence. a vitamin d analog was evaluated as a potential adjuvant treatment with tolterodine and demonstrated improved bladder capacity in an animal model of bladder outlet obstruction(18). additionally, prostatic cells express 1-hydroxylase and can synthesize 1,25 di - hydroxyvitamin d (the active form of vitamin d)(10). a recent study found a specific polymorphism of the vitamin d receptor uniquely linked to a protective effect for the development of lower urinary tract symptoms in men(19). current treatment guidelines for ui care recommend lifestyle changes and behavioral therapy as a first step followed by bladder relaxant drug therapy(5). older adults are more susceptible to the anticholinergic adverse events from bladder drug therapy (constipation, reflux, cognitive changes, xerostomia), which occur frequently with the most commonly prescribed bladder relaxants. additionally, these drugs can be costly and many patients discontinue drug therapy after a few months(6, 7). vitamin d is a low cost, well - tolerated supplement and could provide an adjuvant treatment to currently recommended exercise - based behavioral therapy. the results from the study of aging showing more than half of participants were deficient in vitamin d and more than 80% were insufficient or deficient are consistent with population - based estimates(20). vitamin d has been implicated in multiple health conditions and the institute of medicine has called for additional studies to provide information about the role of vitamin d(21). longitudinal studies evaluating vitamin d replacement rarely include outcomes related to bladder health(22, 23). the current results add to the growing body of literature focused on the potential for vitamin d status to impact the health of older adults. the assessment of the association of vitamin d deficiency with ui may have been limited by the small number of persons with a baseline serum 25-hydroxyvitamin d level of 30 ng / ml or greater (n=34). the cox proportional hazard analyses are limited because ui was reported every 612 months, which could lead to left censoring. we were not able to ascertain if participants received interval non - pharmacologic treatments for their bladder symptoms during the 612 month assessments. the uab study of aging was designed to assess factors impacting social mobility among community - dwelling older adults and did not query a comprehensive set of factors that might impact the development of incontinence in this population. lastly, vitamin d could be a marker of better health and physical activity, which was not controlled for in this analysis. urinary incontinence is a burdensome condition with significant impact on quality of life for many adults as they age. treatment strategies for geriatric conditions such as ui are often multicomponent ; however, current drug therapy is limited because of the potential for adverse events and cost. these results add to a growing body of literature suggesting physiologic mechanisms, cross - sectional association studies, and now preliminary longitudinal research demonstrating a potential association between vitamin d and optimal pelvic floor function. vitamin d offers a low cost, well - tolerated option that warrants further evaluation in an interventional trial, particularly focused on a population of older adults with vitamin d insufficiency. | objectiveto determine if vitamin d status is associated with incident urinary incontinence (ui) among community - dwelling older adults.methodsthe university of alabama at birmingham study of aging is a prospective cohort study of community - dwelling medicare enrollees. standardized assessment of ui using the validated incontinence severity index. analysis of 25-hydroxyvitamin d (25(oh)d levels was performed on stored baseline sera. ui was assessed every 612 months for up to 42 months. analyses included multivariable logistic regression and cox proportional hazard models.resultsof 350 participants (175 male, 147 black, mean age 73.6 5.8), 54% (189/350) were vitamin d deficient [25(oh)d < 20 ng / ml ] and 25% (87/350) were vitamin d insufficient [25(oh)d 20 ng / ml to < 30 ng / ml ]. among the 187 subjects with no ui at baseline, 57% (107/187) were vitamin d deficient and 24% (45/187) were vitamin d insufficient. 175 of the 187 subjects had follow - up evaluation for incident ui over 42 months and incident ui occurred in 37% (65/175). after adjustment cumulative incident ui at 42 months was associated with baseline vitamin d insufficiency (p=0.03) and demonstrated a trend association with deficiency (p=0.07). there was no association between baseline vitamin d status and time to incident ui.conclusionsthese preliminary results support an association between vitamin d and incident ui in community - dwelling older adults. future studies may target specific at risk groups, such as men with bph or women with pelvic floor disorders for evaluation of the impact of vitamin d supplementation on urinary symptoms. |
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