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the hypoxic condition was considered as the primary component of a high - altitude environment in past decades, which is known to lead physical and pathological changes in mammals. it has been widely reported that hypoxic exposure can attenuate hypertension, heart disease, diabetes, and respiratory diseases, as well as central nervous system diseases. however, despite these potential beneficial effects of the hypoxic condition, no studies to date have investigated the effects of hypoxia on osteoporosis. osteoporosis is the most common metabolic skeletal disease worldwide and is characterized by a reduction in bone mass and bone strength, and an increase in fracture risk with great morbidity. although multiple mechanisms have been implicated to be involved in osteoporosis, menopause and age - related bone loss have been the major causes of primary osteoporosis. estrogen deficiency is considered to be the most important contributing factor in the pathological process of osteoporosis, and more than 90% of the osteoporosis - related morbidity worldwide is observed in postmenopausal women more than 60 years old. after menopause, bone resorption (carried out by resorptive cell - osteoclasts) outpaces bone formation (carried out by bone - forming cell - osteoblasts) in the bone - remodeling process. recent in vitro studies reported that the hypoxic condition reduced osteoblastogenesis via inhibition of phosphatidylinositol 3-kinase (pi3k)/akt signal pathway. however, no evidence has been presented so far indicating whether hypoxic exposure impairs the differentiation of osteoblasts and affects bone metabolism in vivo either with or without the postmenopausal state. the current study investigated whether hypoxic exposure (in comparison to normoxic conditions) affects bone metabolism in normal adult rats or in ovariectomized (ovx) rats and expression of some associated major regulatory molecules in osteoblastic cultures. thirty - seven female sprague dawley (sd) rats (12 weeks old) were purchased from the experimental animal center of sun yat - sen university (guangzhou, china) and were housed under specific pathogen - free (spf) conditions with day and night cycle of 12 h each. the temperature and humidity were maintained at 253c and 655%, respectively. thirteen rats were randomly separated into the hypoxic group (n=7) and the control normoxic group (n=6) without any operation. following the separation, meanwhile, the other 24 female rats underwent an ovariectomy after being anesthetized via intraperitoneal injection of pentobarbital sodium - phosphate - buffered saline (pbs) solution with the dose of 30 mg / kg body weight at 12 weeks of age according to the classic protocol. four weeks after the operation, the ovx rats were randomly separated into hypoxic (n=12) or normoxic (n=12) groups. exposure of rats to the hypoxic and normoxic conditions was performed according to the classic protocol for experimental high - altitude imitation. after hypoxic or normoxic treatment for 14 days, rats were anesthetized via intraperitoneal injection of pentobarbital sodium - pbs solution with the dose of 30 mg / kg body weight and were sacrificed for collecting blood samples and harvesting femur samples for various analyses described below. serum bone turnover markers were analyzed by using rat enzyme - linked immunosorbent assay (elisa) kits of c - telopeptide of collagen (ctx, a marker for bone resorption), alkaline phosphatase (alp, a marker for bone formation), and fatty acid binding protein-4 (fabp-4, a marker for fatty acid metabolism and metabolic syndrome) (all purchased from bohua biotech, shanghai, china) to examine treatment systemic effects on bone remodeling. assays were carried out as instructed with absorbance being measured at 450 nm using an el800 automated microplate reader (bio - tek instruments, winooski, vermont, usa). the resulting concentrations of ctx (ng / ml), fabp-4 (ng / ml), and alp (u / l) were measured using the respective standard curves of the assays. to examine treatment effects on bone structure and volume, proximal femoral metaphyses (a region known to be most active in bone remodeling in rats) were analyzed by micro - computed tomography (-ct), using the zkks - mct - sharp - iii scanner (caskaisheng, guangzhou, china). the femoral trabecular region was measured from 30 spongiosa slices (30 m thick), and the growth plate was treated as the label to identify a consistent location to start the region of interest for analysis. the 3d - med 3.0 software (institute of automation, chinese academy of science, xian, china) was used to measured the bone mineral density (bmd) and bone volume / tissue volume (bv / tv). for histological analyses, the right femurs were decalcified by glycerinum - edta (10% quality) for 12 weeks after fixation, dehydrated with concentrated ethanol, and embedded in paraffin wax after being washed with xylene ; they then were cut into 5 m sections. femurs were cut sagittally at the distal femoral region and cut transversally at the mid - diaphyseal level. for examining general bone morphology and measuring densities of osteoblasts and bone marrow adipocytes, sections were dewaxed and rehydrated, and then were subjected to routine hematoxylin and eosin (h&e) staining. according to the classic method, the trabecular bone surface - adhered cuboidal cells whose nuclei were co - stained with hematoxylin were counted as osteoblasts (cells / mm trabecular perimeter) in the secondary spongiosa. meanwhile, the bone marrow adipose stacks whose nuclei were co - stained with hematoxylin were counted as adipose cells in the lower secondary spongiosa (cells / mm bone marrow area). for identification of osteoclasts, one set of sections were stained for tartrate - resistant acid phosphatase (trap, a marker of osteoclasts) using the trap staining kit (sigma - aldrich, california, usa) and counter - stained with methyl green. in trap - stained sections, the irregular red cells whose nuclei were co - stained in green were counted in the secondary spongiosa as osteoclasts (cells / mm trabecular perimeter). numbers of the above types of cells were counted by three volunteers in four fields randomly selected (400) under blinding using imagepro 4.5 software (leeds precision instruments, minneapolis, minnesota, usa). to examine the effects of hypoxic exposure on osteoblasts in vitro, human osteosarcoma - derived osteoblastic mg-63 cells were cultured in 75 cm flasks for 3 days in dulbecco s modified eagle medium (dmem, high glucose) supplemented with 10% fetal calf serum, penicillin 100 u / ml, and streptomycin 100 mg / ml. confluent cultures were trypsinized, passaged, and sub - cultured prior to being subjected to hypoxic / normoxic treatment. hypoxic / normoxic conditions were applied after detached mg-63 cells were transplanted into six - well plates (210 cells / well) and cultured under normal conditions for 12 hours. for the hypoxic condition, mg-63 cells were gassed with 95% n2 and 5% co2 at 37c for 1 h. then cells were transferred into a normoxic incubator (95% air, 5% co2) for an additional 12 h for re - oxygenation. to examine potential hypoxic treatment effects on cell viability following exposure to hypoxic or normoxic condition in vitro, 110 mg-63 cells were plated in a 96-well plate (150 l of medium / well), then incubated at 37c for 12 hours. then, 20 l of 5 mg / ml mtt solution in pbs was added to each well and mixed for 5 minutes prior to being incubated (37c, 5% co2), allowing mtt incorporation for 3 to 4 hours. following incubation, the medium was discarded and the wells were set to dry prior to formazan (mtt metabolic product) being thoroughly mixed and resuspended in 200 l of dmso. the optical density was measured at 560 nm after subtracting the background measurement at 670 nm. to examine the treatment effects in osteoblasts on protein expression of major regulatory genes of bone formation and resorption, mg-63 osteoblastic cells were washed with cold pbs, then lysed on ice for 30 min with radio immunoprecipitation assay (ripa) buffer. then, protein samples (30 mg respectively) were separated on 8% sodium dodecyl sulfate (sds) gels and then transferred to polyvinylidene difluoride (pvdf) membranes. the membranes were incubated with rabbit - derived antibodies against receptor activator of nuclear factor kappa - b ligand (rankl) and osteoprotegerin (opg), runt - related transcription factor 2 (runx-2), osterix, osteocalcin (1:1000 ; all from santa cruz biotech, california, usa), and -actin (1:1000 ; bioworld technology, st. louis, missouri, usa) after being blocked with dry skim milk - infused pbs buffer (4%) for 1 h. following these steps, the membranes were incubated with peroxidase - conjugated secondary antibody (goat anti - rabbit or a goat anti - mouse ; 1:1000 ; cell signaling technology, shanghai, china) for 1 h and then were visualized by enhanced chemiluminescence (ecl ; santa cruz biotech) using kodak x - omat ls film (eastman kodak, rochester, new york, usa) post pbs washing three times. all western blot analyses in this study were repeated at least three times data were expressed as means sd, and one - way analysis of variance (anova) was performed for the analysis of statistical significance. thirty - seven female sprague dawley (sd) rats (12 weeks old) were purchased from the experimental animal center of sun yat - sen university (guangzhou, china) and were housed under specific pathogen - free (spf) conditions with day and night cycle of 12 h each. the temperature and humidity were maintained at 253c and 655%, respectively. thirteen rats were randomly separated into the hypoxic group (n=7) and the control normoxic group (n=6) without any operation. following the separation, meanwhile, the other 24 female rats underwent an ovariectomy after being anesthetized via intraperitoneal injection of pentobarbital sodium - phosphate - buffered saline (pbs) solution with the dose of 30 mg / kg body weight at 12 weeks of age according to the classic protocol. four weeks after the operation, the ovx rats were randomly separated into hypoxic (n=12) or normoxic (n=12) groups. exposure of rats to the hypoxic and normoxic conditions was performed according to the classic protocol for experimental high - altitude imitation. after hypoxic or normoxic treatment for 14 days, rats were anesthetized via intraperitoneal injection of pentobarbital sodium - pbs solution with the dose of 30 mg / kg body weight and were sacrificed for collecting blood samples and harvesting femur samples for various analyses described below. serum bone turnover markers were analyzed by using rat enzyme - linked immunosorbent assay (elisa) kits of c - telopeptide of collagen (ctx, a marker for bone resorption), alkaline phosphatase (alp, a marker for bone formation), and fatty acid binding protein-4 (fabp-4, a marker for fatty acid metabolism and metabolic syndrome) (all purchased from bohua biotech, shanghai, china) to examine treatment systemic effects on bone remodeling. assays were carried out as instructed with absorbance being measured at 450 nm using an el800 automated microplate reader (bio - tek instruments, winooski, vermont, usa). the resulting concentrations of ctx (ng / ml), fabp-4 (ng / ml), and alp (u / l) were measured using the respective standard curves of the assays. to examine treatment effects on bone structure and volume, proximal femoral metaphyses (a region known to be most active in bone remodeling in rats) were analyzed by micro - computed tomography (-ct), using the zkks - mct - sharp - iii scanner (caskaisheng, guangzhou, china). the femoral trabecular region was measured from 30 spongiosa slices (30 m thick), and the growth plate was treated as the label to identify a consistent location to start the region of interest for analysis. the 3d - med 3.0 software (institute of automation, chinese academy of science, xian, china) was used to measured the bone mineral density (bmd) and bone volume / tissue volume (bv / tv). for histological analyses, the right femurs were decalcified by glycerinum - edta (10% quality) for 12 weeks after fixation, dehydrated with concentrated ethanol, and embedded in paraffin wax after being washed with xylene ; they then were cut into 5 m sections. femurs were cut sagittally at the distal femoral region and cut transversally at the mid - diaphyseal level. for examining general bone morphology and measuring densities of osteoblasts and bone marrow adipocytes, sections were dewaxed and rehydrated, and then were subjected to routine hematoxylin and eosin (h&e) staining. according to the classic method, the trabecular bone surface - adhered cuboidal cells whose nuclei were co - stained with hematoxylin were counted as osteoblasts (cells / mm trabecular perimeter) in the secondary spongiosa. meanwhile, the bone marrow adipose stacks whose nuclei were co - stained with hematoxylin were counted as adipose cells in the lower secondary spongiosa (cells / mm bone marrow area). for identification of osteoclasts, one set of sections were stained for tartrate - resistant acid phosphatase (trap, a marker of osteoclasts) using the trap staining kit (sigma - aldrich, california, usa) and counter - stained with methyl green. in trap - stained sections, the irregular red cells whose nuclei were co - stained in green were counted in the secondary spongiosa as osteoclasts (cells / mm trabecular perimeter). numbers of the above types of cells were counted by three volunteers in four fields randomly selected (400) under blinding using imagepro 4.5 software (leeds precision instruments, minneapolis, minnesota, usa). to examine the effects of hypoxic exposure on osteoblasts in vitro, human osteosarcoma - derived osteoblastic mg-63 cells were cultured in 75 cm flasks for 3 days in dulbecco s modified eagle medium (dmem, high glucose) supplemented with 10% fetal calf serum, penicillin 100 u / ml, and streptomycin 100 mg / ml. confluent cultures were trypsinized, passaged, and sub - cultured prior to being subjected to hypoxic / normoxic treatment. hypoxic / normoxic conditions were applied after detached mg-63 cells were transplanted into six - well plates (210 cells / well) and cultured under normal conditions for 12 hours. for the hypoxic condition, mg-63 cells were gassed with 95% n2 and 5% co2 at 37c for 1 h. then cells were transferred into a normoxic incubator (95% air, 5% co2) for an additional 12 h for re - oxygenation. to examine potential hypoxic treatment effects on cell viability, following exposure to hypoxic or normoxic condition in vitro, 110 mg-63 cells were plated in a 96-well plate (150 l of medium / well), then incubated at 37c for 12 hours. then, 20 l of 5 mg / ml mtt solution in pbs was added to each well and mixed for 5 minutes prior to being incubated (37c, 5% co2), allowing mtt incorporation for 3 to 4 hours. following incubation, the medium was discarded and the wells were set to dry prior to formazan (mtt metabolic product) being thoroughly mixed and resuspended in 200 l of dmso. the optical density was measured at 560 nm after subtracting the background measurement at 670 nm. to examine the treatment effects in osteoblasts on protein expression of major regulatory genes of bone formation and resorption, mg-63 osteoblastic cells were washed with cold pbs, then lysed on ice for 30 min with radio immunoprecipitation assay (ripa) buffer. then, protein samples (30 mg respectively) were separated on 8% sodium dodecyl sulfate (sds) gels and then transferred to polyvinylidene difluoride (pvdf) membranes. the membranes were incubated with rabbit - derived antibodies against receptor activator of nuclear factor kappa - b ligand (rankl) and osteoprotegerin (opg), runt - related transcription factor 2 (runx-2), osterix, osteocalcin (1:1000 ; all from santa cruz biotech, california, usa), and -actin (1:1000 ; bioworld technology, st. louis, missouri, usa) after being blocked with dry skim milk - infused pbs buffer (4%) for 1 h. following these steps, the membranes were incubated with peroxidase - conjugated secondary antibody (goat anti - rabbit or a goat anti - mouse ; 1:1000 ; cell signaling technology, shanghai, china) for 1 h and then were visualized by enhanced chemiluminescence (ecl ; santa cruz biotech) using kodak x - omat ls film (eastman kodak, rochester, new york, usa) post pbs washing three times. data were expressed as means sd, and one - way analysis of variance (anova) was performed for the analysis of statistical significance. short - term hypoxic exposure did not appear to affect trabecular bone structure and volume in the normal rats. after 14 days of hypoxia exposure, as analyzed by -ct, no statistical differences were observed in bmd, bone mineral content (bmc), bv / tv%, and trabecular number (tb.n) between the hypoxic and normoxic groups (figure 1a, 1b, 1e1h). however, hypoxic exposure for 14 days at 4 weeks after the ovariectomy procedure caused significant bone loss, although it did not affect the rate of body weight gain in ovx rats over 3 months following the ovariectomy procedure. following the hypoxic exposure in ovx rats, as examined by -ct scanning, there was a significant enhanced bone loss based on measurements in bmd (p0.05) and the osteoclast numbers (figure 2e, 2f, 2j ; p>0.05). however, in the ovx rats, the osteoblast number in the hypoxic group was statistically significantly lower than that in the normoxic group (figure 2c, 2d, 2l ; p0.05) and the osteoclast numbers (figure 2e, 2f, 2j ; p>0.05). however, in the ovx rats, the osteoblast number in the hypoxic group was statistically significantly lower than that in the normoxic group (figure 2c, 2d, 2l ; p<0.05), and the osteoclast number was significantly higher in the hypoxic ovx rats when compared with the normoxic ovx rats (figure 2 g, 2h, 2j ; p<0.01). on the other hand, compared with the normal - hypoxic group, the significant enhancement of osteoclast number and reduction of osteoblast number in the ovx group demonstrated the success of ovx - induced bone loss. the numbers of adipose cells were counted on the h&e - stained sections at the femur bone marrow (at the lower secondary spongiosa region). while there were no significant differences in bone marrow adipocyte density in the normal rats between the hypoxic group and the normoxic group (figure 2a, 2b, 2k), there was a higher density of bone marrow adipocytes in hypoxic ovx rats compared with normoxic ovx rats (figure 2c, 2d, 2k ; p<0.001). therefore, the short - term hypoxic condition could influence the osteogenesis - related adipose metabolism in ovx rats, and this influence may be an estrogen - dependent process. as expected, the adipocyte density in the ovx normoxic rats was higher than that in the normal normoxic rats. to examine treatment systemic effects on bone turnover and fat metabolism, serum levels of bone resorption marker ctx, bone formation marker alp, and fatty acid metabolism and metabolic syndrome marker fabp-4 were analyzed. in the normal rats, no differences in serum levels of ctx, alp, and however, in ovx rats, hypoxic exposure significantly increased serum levels of ctx and fabp-4 and decreased serum alp level (figure 3a3c). simultaneously, compared with the normal hypoxic group, the significant enhancements of ctx and fabp-4 levels and reduction of alp level in the ovx group demonstrated the success of ovx - induced bone loss. these data indicated that a short - term hypoxic exposure did not alter the skeletal metabolism in normal rats but could accelerate bone loss in ovx rats. to investigate some potential mechanisms for hypoxia - induced changes in bone / fat metabolism, treatment effects of hypoxia on cultured mg-63 osteoblastic cells were examined. a mtt cell viability test showed that there were no significant differences in cell viability between the hypoxic and normoxic groups of cells (figure 4a). through western blotting analyses, treatment effects were examined on the expression levels of osteoblast differentiation markers (runx2, osx, and ocn) in mg-63 cells following hypoxic exposure (figure 4b). while ocn expression in mg-63 cells was non - significantly different between the hypoxic condition and the normal condition, the levels of runx2 and osx expression were lower in the cells exposed to the hypoxic condition compared with the normoxic condition (p<0.05 and 0.01, respectively). furthermore, the expression of rankl in mg-63 cells was significantly higher and the opg level significantly lower after hypoxia exposure when compared with normal culture (figure 4c). currently, potential effects of high - altitude living (e.g., highland residents) and hypoxia exposure on the morbidity of osteoporosis are not exactly understood. recently, bozzini. reported the effects of different simulated high altitudes (1850, 2900, 4100, and 5450 m) on femur biomechanical properties in female growing rats that were exposed to the simulated high altitudes for more than 22 hours daily for 42 days. it was found that, due to a smaller bone mass, the long bones of the hypoxia - exposed rats were weaker than those of normoxia controls. in the present study, we investigated the effects of short - term hypoxia, representative of a transient or non - sustained high - altitude environment, on bone metabolism in both normal and ovx rats and in vitro mg-63 osteoblastic cells. this study showed that the short - term hypoxic exposure did not change the bone - remodeling process in normal adult female rats ; however, it decreased bone formation and increased bone resorption, and thus increased bone loss and enhanced bone marrow adipose tissue accumulation, in estrogen - deficient ovx rats. furthermore, the transient hypoxic culture impaired the osteoblastogenesis and enhanced expression of the osteoclastogenic signal (increased rankl but opg levels) of mg-63 osteoblasts in vitro. the post - hypoxic condition osteoclastogenic enhancement in our study is consistent with the past study of human osteoclasts that underwent hypoxia exposure. therefore, these data indicate that short - term hypoxic exposure may accelerate bone loss in ovx rats or in postmenopausal women with osteoporosis. in this study, bone loss was shown to be enhanced in ovx rats by short - term hypoxic exposure, which was accompanied by an increase in the number of bone - resorptive osteoclasts and a decrease in the number of bone - forming osteoblasts on trabecular bone surfaces. although ovx models were performed for more than 8 weeks in most studies, the hypoxic exposure in our study was measured at 4 weeks post ovx to avoid severe and irreversible bone loss of rats. in addition, there was an increased serum level of the bone resorption marker ctx but a decreased serum level of the bone formation marker alp, as well as an increase in adipocyte density in the bone marrow, following hypoxic exposure in ovx rats. while these changes were not observed in normal rats after hypoxic exposure, this suggests that imbalanced bone remodeling (decreased bone formation and increased bone resorption) caused by hypoxia occurs in female rats when estrogen is deficient. this can imply that short - term hypoxic exposure in postmenopausal women may cause accelerated bone loss. as multiple signal pathways are involved in the bone remodeling system, the factors contributing to the changes in bone metabolism as a result of hypoxia still need to be investigated. the hypoxic microenvironment has been considered as a vital factor for the hypoxia - induced factor (hif-1)-dependent angiogenesis that is critical for bone formation during bone development and regeneration after trauma. consistently, bone marrow stromal cell differentiation and osteoblastogenesis can be inhibited by hypoxic culture in vitro. however, potential molecular mechanisms for hypoxic exposure - induced bone loss in ovx rats remain to be clarified in future studies. in addition, although it has been reported in the past decades that hypoxia can increase adipogenesis, no clear evidence has indicated that the adipogenesis in bone marrow can be modulated by hypoxic exposure in vivo. as a means to investigate potential mechanisms of the impact of hypoxia on bone remodeling, the current study also examined hypoxic - exposure effects on osteoblast viability and expression of genes regulating osteoblastogenesis and osteoclastogenesis in treated mg-63 osteoblastic culture. while recent studies have shown that the hypoxic condition could damage the cell cycle of osteoblasts and block their proliferation, the current study showed that the hypoxic exposure did not affect osteoblast viability as measured by mtt assays ; this contradiction may be because the vitro hypoxia imitation in our study was a mild and short - term exposure (5% o2 for 1 hour). although the terminal biomarker of osteogenesis, osteocalcin, showed low expression with both hypoxic exposure and normal culture in vitro due to the limited culture time, our study demonstrated that the hypoxic condition suppressed protein expression of osteogenic biomarkers including transcriptional factors runx2 and osx, suggesting that hypoxic treatment of osteoblastic culture can reduce osteoblast differentiation. this finding is consistent with the recent study where osteoblastic differentiation was shown to be inhibited by the hypoxic condition. in addition, as osteoclastogenesis is modulated by osteoblasts through the rank / rankl / opg signal pathway, this study measured the expression of the key osteoclastogenic cytokine rankl and the major osteoclastogenic inhibitor opg in osteoblasts exposed to hypoxic conditions in vitro. we showed that the treated osteoblastic culture had elevated protein expression of rankl but a reduced level of opg, suggesting that hypoxia - treated osteoblasts have a higher ability to induce osteoclastic differentiation and formation and osteoclastic activity. these results suggest that a high - altitude environment may impair osteoblastic differentiation and enhance osteoclastic formation. in addition, while it has been demonstrated that estrogen can induce the apoptosis of osteoclasts and estrogen deficiency could stimulate osteoclastogenesis in ovx rats, potential interaction of estrogen action and the rank / rankl / opg system in osteoclastic over - formation under the hypoxic condition remains to be investigated. further studies are required to verify whether the hypoxia - induced accelerated bone loss in ovx rats may be due to the reduced differentiation of osteoblasts and over - formation and/or activation of osteoclasts resulting from higher level rankl but lower level opg in osteoblasts. on the other hand, the imbalance of bmsc differentiation is considered to be a major factor in menopausal osteoporosis ; the increased fat mass in the bone marrow has been shown to be associated with a reduction in bone mass, and this bone loss and bone marrow adiposity inverse relationship has been demonstrated. in our results, consistent with recent investigations, the fat mass was elevated in ovx rats ; furthermore, the hypoxic exposure also enhanced the adipogenesis of bone marrow significantly in ovx rats. although these results suggest that the hypoxic condition may have altered the balance of adipose / osteoblast differentiation of bone marrow mscs and resulted in bone loss and adipogenesis, deeper elaboration of a mechanism is still needed. simultaneously, consistent with our results, a recent report suggested that bone marrow adipocytes influenced the coupling of osteoblast and osteoclast differentiation, and may be relevant to bone - loss disorders. this study observed an enhanced bone loss in ovx rats (accompanied by reduced bone formation, increased resorption, and bone marrow adiposity) following hypoxic exposure. these findings suggest that high - altitude environments may play a harmful role in bone metabolism and contribute to the development / progression of postmenopausal osteoporosis. although our in vitro studies suggest that the hypoxic condition can suppress osteogenic differentiation and enhance osteoclastic formation, further studies are required to investigate whether it is so under an estrogen - deficient culture condition, and future work is required to study potential mechanisms for the enhanced bone loss in rats with estrogen deficiency. | backgroundalthough it has been reported that hypoxic exposure can attenuate hypertension, heart disease, diabetes, and some other diseases, effects of hypoxia on osteoporosis are still unknown.material/methodsthe current study investigated whether short - term hypoxic exposure (in comparison with normoxic conditions) affects bone metabolism in normal or ovariectomized (ovx) adult female rats in an vivo study. micro - computed tomography bone volume / structural analyses, histological examination, and serum bone turnover biochemical assays were used. in addition, the expressions of some associated major regulatory molecules were measured in osteoblastic cultures.resultswhile the 14-day hypoxic exposure did not change the bone - remodeling process in normal adult female rats, it decreased bone volume, osteoclast density, and serum bone formation marker (alkaline phosphatase) level, but increased osteoclast density and serum bone resorption marker (c - telopeptide of collagen) level in ovx rats. the bone marrow adipocyte number and serum fatty acid binding protein-4 level were increased in ovx - hypoxic rats compared with ovx - normoxic rats. consistently, in human mg-63 osteoblastic cultures, the hypoxic condition suppressed protein expression of osteogenic transcriptional factors runx2 and osterix, elevated protein expression of osteoclastogenic cytokine receptor activator of nuclear factor kappa - b ligand, but reduced that of osteoclastogenic inhibitor osteoprotegerin.conclusionsour results suggest that, although no change occurred in the bone - remodeling process in normal adult female rats after hypoxic exposure, under the estrogen - deficient osteoporotic condition, the hypoxic condition can alter the bone microenvironment so that it may further impair osteoblastic differentiation and enhance osteoclastic formation, and thus reduce bone formation, enhance bone resorption, and accelerate bone loss. |
surgical skill and strategy for the correction of tetralogy of fallot (tof) have been improved and resulted in relatively satisfactory outcomes. however, prematurity and low birth weight continue to remain risk factors for poor outcomes.1) consistent and optimal results have not been achieved in neonates who waited to reach adequate body weight for corrective surgery.2)3) stenting of the right ventricular outflow track (rvot) has been reported as a palliative procedure in low birth weight premature neonates.4 - 6) we herein report a case of successful rvot stenting in a low birth weight neonate born with tof and prostaglandin e1 dependency. a male neonate with a prenatal diagnosis of tof associated with pulmonary atresia was born through vaginal delivery following 36 weeks and 5 days of gestation with 2,150 g body weight. diameters of branch pulmonary arteries measured 4.3 mm on the right and 4.2 mm on the left. there was a u - shaped patent ductus arteriosus that measured 2.4 mm in diameter. because systemic oxygen saturation frequently fell below 60%, prostaglandin e1 could not be discontinued. seven days after birth, cardiac catheterization for rvot stenting was performed under general anesthesia. cefamandole was administered prophylactically and a single dose of heparin was administered immediately following puncture of the right femoral vein. we introduced a 5-f multi - purpose catheter (cook inc, bloomington, usa) into the left pulmonary artery using a 0.032-inch terumo wire (terumo corporation, tokyo, japan), which was exchanged for a 0.014 inch guide wire (balance middleweight, abbott vascular, santa clara, usa). after removing the 5-f catheter, a 7-f long sheath (shuttle sl flexor tuohy borst, cook inc, bloomington, il, usa) was introduced into the main pulmonary artery through a guide wire. a 515 mm stent (palmaz genesis amiia, cordis corporation, roden, netherlands) was placed to extend from above the level of the pulmonary valve to the infundibulum. because the proximal infundibulum was not covered by the stent, a 415 mm stent (palmaz genesis amiia, cordis corporation, roden, netherlands) was additionally inserted to fully cover the infundibulum (fig. oxygen saturation of arterial blood was increased to 92% following stent insertion, at which point prostaglandin e1 was deemed safe for discontinuation. six days following stent insertion, the patient was discharged with aspirin at 5 mg / kg / d. echocardiography measured peak velocity at 4.3 m / s in the rvot and stenosis of the rvot below the stent was suspected. the diameter of the main pulmonary artery measured 5.0 mm and the diameters of the branch pulmonary arteries measured 5.3 mm on the right, and 6.4 mm on the left. at this time his body weight was 4.9 kg and total corrective surgery the ventricular septal defect was closed with a dacron patch and rvot widening was performed by infundibulectomy and transannular autologous pericardial patch insertion following stent removal. after the initial echocardiographic examination of this patient, we did not consider waiting for his body weight to be optimal for the procedure. also, we discontinued infusion of prostaglandin e1, because it has been associated with higher mortality and morbidity.2)3) early neonatal correction of tof has been supported by some clinicians.7) according to the ' early repair ' strategy, cardiopulmonary bypass can be performed in infants weighing as little as 2 kg.7) therefore, our patient might have been a candidate for ' early repair '. palliative methods for tof include systemic to pulmonary arterial shunt insertion, particularly the modified blalock - taussig shunt, stenting of ductus arteriosus, balloon pulmonary valvuloplasty and rvot stenting. pulmonary artery hypoplasia and distortion are common complications following initial palliation by modified blalock - taussig shunt,9) particularly when palliation was performed during the neonatal period.10) according to gladman.10) neonatal palliation by modified blalock - taussig shunt was associated with moderate to severe distortion of the right pulmonary artery in 35% of patients, and the need for pulmonary artery intervention in 60% of patients. in addition, because the ductus arteriosus of our patient was ' u ' shaped, we excluded its stenting as an option, and severe infundibular hypertrophy excluded isolated balloon pulmonary valvuloplasty11) as a selective palliative method. he was discharged following the procedure, and gained adequate body weight for corrective repair of tof. dohlen.4) reported that utilization of low profile, flexible pre - mounted coronary stent had greatly simplified their practice of rvot stenting. we decided not to use it, because medical insurance does not cover use of coronary stent for stenting non - coronary vessels in the republic of korea. the optimal diameter of rvot stent suggested by dohlen.4) is 1 - 2 mm larger than the infundibular diameter during diastole, and the minimal size of stents used in their study was 4 mm. the length of the stent should be enough to cover the entire stenotic rvot, and careful attention is needed in the positioning. the 3 months gained appeared to be sufficient to overcome complications associated with repair during early infancy,12) but was a relatively short period to perform ' right ventricular infundibulum sparing ' strategy.8) in conclusion, the potential of growth of proximal infundibulum should be taken into account when considering stent positioning. | surgical skill and strategy for the correction of tetralogy of fallot (tof) have improved and resulted in satisfactory outcomes. however, prematurity and low birth weight continue to remain risk factors for poor outcomes. we present a case of a 2,150 g neonate born with tof, in whom palliation was achieved with right ventricular outflow tract (rvot) stenting. seventy - seven days after the procedure, stenosis of rvot below the stent was identified. at that time his body weight was 4.9 kg and total corrective surgery was deemed feasible. eight months following surgical repair, the patient remained well without medical intervention. rvot stenting may be a viable interim procedure while waiting for a low birth weight neonate born with tof and prostaglandin e1 dependency to reach optimal weight to undergo corrective surgery. |
meningiomas constitute approximately 20% of all brain tumors8). in 1922, harvey cushing first used the term meningioma16). according to the world health organization (who) histologic grading system, three grades of meningiomas with increasing risks of recurrence are distinguished8). benign or common - type tumors with a low rate of recurrence (7 - 20%) are assigned to grade i. grade ii meningiomas include atypical meningiomas as well as the rare chordoid and clear cell (intracranial) variants, and exhibit a higher risk of recurrence (29 - 40%). grade iii meningiomas are anaplastic meningiomas with high mitotic activity (20/10 high - power fields) and/or obviously malignant cytology, as well as the rare variants, papillary and rhabdoid meningiomas. the recurrence rate of anaplastic meningiomas is suggested to be 50 - 78%. whereas a retrospective review showed that resections of who grade ii meningiomas are less often classified by surgeons as gross total when compared with benign meningiomas, a majority of who grade ii meningiomas are still able to undergo gross total resection (gtr) as the initial treatment17). the subsequent prognosis and optimal management after gtr of who grade ii meningiomas remain unclear. we sought to define the long - term recurrence rate and predictive factors for recurrence of who grade ii meningiomas. between 1993 and 2005, 448 patients with an intracranial meningioma were treated surgically at our institution by one surgeon. the mean duration of follow - up was 45 months (range, 3 - 175 months). twenty - eight tumors had undergone simpson grade i (complete) resection, as confirmed by both the surgeon 's impression at the time of surgery and the first post - operative imaging scans. this study was approved by the institutional review board at our institution. the patient and tumor characteristics of 55 who grade ii meningiomas are summarized in table 1. age at the time of diagnosis was defined as the patient 's age at the time of his or her first surgery. tumor size was defined by the largest tumor diameter rounded to the nearest centimeter on imaging scans before the initial surgery. the tumors were sub - grouped according to location, as follows : convexity ; parasagittal ; falx ; sphenoid ridge ; cerebellopontine angle ; tuberculum sellae ; olfactory groove ; and tentorium (table 2). " complete resection " is defined as simpson grade i, and " incomplete resection " is defined as simpson grade ii, iii, or iv. the meningiomas were also divided into groups, as follows : convexity and non - convexity groups. the anatomic distribution of the 55 meningiomas that were totally resected during this period differed in having a higher proportion of tumors in the " convexity " group (19 of 25 patients) and a lower proportion in the " non - convexity " group (9 of 30 patients). patients with completely resected who grade ii meningiomas who do not receive radiation therapy are observed closely in our institution. we recommend post - operative radiation therapy in patients with grade ii meningiomas which are not completely resected. in recurrent cases gamma knife radiosurgery is an alternative treatment modality in small or surgically inaccessible meningiomas or meningiomas, and patients of advanced age and poor operative risk. logistic regression, mantel - cox, and breslow - gehan - wilcoxon univariate and multivariate models were used to compare the recurrence - free survival distributions across subgroups. the following variable data were analyzed by multiple regression analysis with recurrence as the dependent variable : age ; gender ; tumor size ; peritumoral edema ; extent of tumor removal ; and tumor location. between 1993 and 2005, 448 patients with an intracranial meningioma were treated surgically at our institution by one surgeon. the mean duration of follow - up was 45 months (range, 3 - 175 months). twenty - eight tumors had undergone simpson grade i (complete) resection, as confirmed by both the surgeon 's impression at the time of surgery and the first post - operative imaging scans. this study was approved by the institutional review board at our institution. the patient and tumor characteristics of 55 who grade ii meningiomas are summarized in table 1. age at the time of diagnosis was defined as the patient 's age at the time of his or her first surgery. tumor size was defined by the largest tumor diameter rounded to the nearest centimeter on imaging scans before the initial surgery. the tumors were sub - grouped according to location, as follows : convexity ; parasagittal ; falx ; sphenoid ridge ; cerebellopontine angle ; tuberculum sellae ; olfactory groove ; and tentorium (table 2). " complete resection " is defined as simpson grade i, and " incomplete resection " is defined as simpson grade ii, iii, or iv. the meningiomas were also divided into groups, as follows : convexity and non - convexity groups. the anatomic distribution of the 55 meningiomas that were totally resected during this period differed in having a higher proportion of tumors in the " convexity " group (19 of 25 patients) and a lower proportion in the " non - convexity " group (9 of 30 patients). patients with completely resected who grade ii meningiomas who do not receive radiation therapy are observed closely in our institution. we recommend post - operative radiation therapy in patients with grade ii meningiomas which are not completely resected. in recurrent cases gamma knife radiosurgery is an alternative treatment modality in small or surgically inaccessible meningiomas or meningiomas, and patients of advanced age and poor operative risk. logistic regression, mantel - cox, and breslow - gehan - wilcoxon univariate and multivariate models were used to compare the recurrence - free survival distributions across subgroups. the following variable data were analyzed by multiple regression analysis with recurrence as the dependent variable : age ; gender ; tumor size ; peritumoral edema ; extent of tumor removal ; and tumor location. of the 448 patients, 20 had who grade iii meningiomas, 55 had who grade ii meningiomas, and 373 had benign meningiomas. a multivariate analysis was then performed to assess the following eight variables as possible prognostic factors for tumor recurrence : 1) age ; 2) gender ; 3) tumor location ; 4) tumor size ; 5) peritumoral edema ; 6) post - operative radiation therapy ; 7) histologic type ; and 8) extent of resection. of these eight factors, three were predictive of recurrence based on multivariate analysis, as follows : pathologic subtype (p=0.001) ; degree of resection (p=0.001) ; and peritumoral edema (p=0.016). the extent of resection was a significant independent predictive factor for who grade ii meningiomas (p=0.05) (table 3). of the 55 patients diagnosed with who grade ii meningiomas at our institution between 1993 and 2005, 26 were men and 29 were women. the mean age at the time of diagnosis was 55 years (range, 19 - 82 years). clinical follow - up included serial imaging for an average of 45 months after tumor resection (range, 3 - 175 months) with 40% of the patients followed either to recurrence or for at least 5 years after surgery. in who grade ii patients, 14 had recurrences 7 - 96 months post - operatively ; 11 patients had recurrences 5 years post - operatively (mean time to recurrence, 42.9 months), and 3 had recurrences 10 years post - operatively. complete resection was achieved in 28 patients and incomplete resection was achieved in 27 patients. the recurrence rate between the two groups of ' who grade ii meningioma patients (complete resection) ' and ' who grade i meningioma patients (complete or incomplete resection) ' were 14.2% and 11.5%, respectively (table 4, fig. 1, 2c). although it was difficult to show statistical significance, the recurrence rates were similar between the benign meningioma treatment group and the who grade ii meningioma without radiotherapy treatment group (p=0.844). however, the relative risk (rr) of recurrence was significantly higher for patients with who grade ii meningiomas with incomplete resections (10/27, rr=37%) than patients with complete resections (4/28, rr=14%), independent of post - operative radiotherapy. 2b represents the recurrence - free survival between the two treatment groups (p=0.05). in the incomplete resection group five of 7 patients had recurrences after post - operative radiotherapy (table 4). we analyzed the recurrence - free survival between these two groups, but it was difficult to interpret the statistical significance of the influence of radiotherapy on recurrence (p=0.314). when subdividing this group based on simpson grade, the simpson grade iii or iv group had a high risk of recurrence regardless of post - operative radiotherapy (n=3, rr=100%). however, if the degree of resection was simpson grade ii, the recurrence rate was similar to the complete resection group, although post - operative radiotherapy was not administered (3/18, rr=15.5%). table 5 shows the information regarding patients who did not receive radiotherapy after incomplete resection ; five patients had recurrences. this group had distinguishing features from other groups. with the exception of one patient, all patients were in the non - convexity area, and in whom it was more difficult to perform complete resections than in patients in the convexity area. most of the patients had moderate - to - severe degrees of peritumoral edema ; three of the patients had several recurrences. we delivered post - operative radiotherapy to this patients who had more risky conditions for recurrence than other patients. anatomically, there were higher proportions of convexity meningiomas in the completely resected group [67% (19/28) ] than in the incompletely resected group [22% (6/27) ]. considering the importance of the degree of resection and post - operative radiation therapy, we compared the simpson grade i resection group without radiotherapy in convexity and simpson grade ii resection followed by post - operative radiotherapy group in non - convexity. the recurrence rate was significantly higher in the latter (60%) than the former (15%). in this study, we sought to define the long - term recurrence rate of who grade ii meningiomas after complete or incomplete resection, as well as factors influencing recurrence, including post - operative radiation. thus, we attempted to reveal what clinicopathologic features may be useful for predicting recurrence of who grade ii meningiomas. it is well - known that the histologic type is a significant prognostic factor in overall meningiomas. in our study, according to the kaplan - meier survival analysis, the recurrence rate between the pathologic types was shown to be lowest in who grade i meningiomas and highest in who grade iii meningiomas (fig. the recurrence rate increased with the histologic grade rising, consistent with the findings of ayerbe.1) mantle.9) reported that meningiomas invade brain tissue along cells, forming bridges between the cortex and surface of the tumor. mantle.9) suggested that the invasive cells were the main reason for tumor recurrence. some authors state that tumor size may be helpful in predicting the recurrence of who grade ii meningioma14). also, several studies reported that males have higher recurrence rate4,15), and development of tumors in patients at a young age (< 40 years) was associated with higher likelihood of recurrence15), although these were not clear in this or other studies7,12). in the present study, the degree of peritumoral edema is a significant prognostic factor in overall meningiomas, but has no predictability for who grade ii meningiomas (fig. 2d, table 3). some authors have reported statistical significance as predictive factors for recurrence of meningiomas20) because edema represent a variety of pathologic changes, such as a cerebral - pial vascular supply and cerebral white matter hypoperfusion13,18,20). the degree of resection as a significant independent predictive factor of recurrence in who grade ii meningiomas as well as in overall meningiomas, many authors had reported similar results. mirimanoff.11) reported recurrence - free survival rates after total resection of 93% at 5 years, 80% at 10 years, and 68% at 15 years ; compared with partial resection, the recurrence - free survival rates dropped to 63%, 45%, and 9%, respectively12). jaaskelainen5) reported that after complete resection the recurrence rates were 19% at the 20-year follow - up. the same group reported that in patients with atypical or malignant meningiomas after complete resection the risk of recurrence was 38% and 78% at 5 years, respectively6). goyal.3) reported that 8 of 22 patients had local recurrences, including 2 of 15 with gtr, 3 of 4 with str, and all 3 patients had resection of unknown extent. at 10 years, patients with gtr had a higher local control rate than those who had a str or a resection of unknown extent (87% vs. 17% ; p=0.02). the most important factors that determine the likelihood of meningioma recurrences is the extent of tumor resection. although location was not a significant predictive factor for recurrence of meningiomas in our study, location is an important factor influencing the extent of resection of meningiomas. convexity meningiomas are an indication for complete resection, simpson grade i can also identify complete resection most significantly than other locations. we reasoned that comparing the two groups, simpson grade i resection without rt in the convexity group and simpson grade ii resection followed by post - operative radiotherapy in the non - convexity group was meaningful to minimize bias. for amplifying the clarity of the relationship between the extent of resection and recurrence considering the potential difficulty of radical resection in non - convexity, we compared two groups to the exclusion of the simpson grade iii or iv group ; the recurrence rate of the former was 15.7% and the recurrence rate of the latter was 60%. it was reconfirmed that the extent of resection was a powerful factor for decreasing recurrence of who grade ii meningiomas. post - operative adjuvant radiotherapy after surgical resection of who grade ii meningiomas continues to be controversial. in our study furthermore, the incompletely resected group with radiotherapy had a higher recurrence rate than the group without radiotherapy. in contrast to our expectations, some patients did well for many years after subtotal resections alone. we presumed the cause of a higher recurrence rate was due to the fact that the patients had been expected to have a worse prognosis because of factors, such as severe edema or complex site or previous recurrence underwent radiotherapy (table 5). most of the authors tend not to recommend adjuvant radiotherapy in grade i meningiomas. if gross total resection was achieved, however, there are significant variations in opinion about the effect of post - operative radiotherapy in cases of completely resected grade ii meningiomas10) therefore, we compared the recurrence rate between two groups (the overall treatment group of benign meningiomas and the complete resection group of who grade ii meningiomas without radiotherapy). although it was difficult to show statistical significance (p=0.844), the recurrence rate between the two groups were similar to each other (table 4, fig. 2c). this suggests that complete resection of who grade ii meningiomas reduced the recurrence rate to that of who grade i meningiomas, regardless of post - operative radiotherapy. however, in the completely resected group, the effect of post - operative radiation therapy was uncertain in this study. we subdivided the incomplete resection group through simpson grade for researching the effect of post - operative radiotherapy. the group performed by simpson grade iii or iv had a significantly high risk of recurrence without reference to post - operative radiotherapy. if the degree of resection is simpson grade ii, the recurrence rate is similar to the complete resection group, although post - operative radiotherapy was not performed. it is questionable whether or not post - operative radiotherapy must be performed in who grade ii meningiomas after simpson grade ii resection. some authors have found statistical significance between radiotherapy and the recurrence rate, and they recommended irradiation, irrespective of the extend of resection2,19). however, in another study3) in which 8 of 22 patients with atypical meningiomas received post - operative radiotherapy, local control was 87% at 5 and 10 years following gtr, and radiotherapy had no significant impact on local control or overall survival. mccarthy.11) reported studies which included patients with grade ii meningiomas who underwent gross total resection, and failed to demonstrate a statistically significant change in local control or survival in patients receiving adjuvant radiotherapy. a number of authors do not recommend radiotherapy for atypical meningiomas regarding the extent of surgical resection because the majority of patients with atypical meningiomas would ultimately experience local recurrence despite a course of conventional radiation treatment5,7) there are no randomized trials to support any opinion regarding the role of post - operative radiotherapy, and it is still debatable whether or not these patients should be carefully observed or treated pre - emptively. because of the rarity of who grade ii meningiomas and differences in the way who grade ii meningiomas are classified, the literature on the role of radiation in treating such patients are difficult to interpret. this study had a limitation with respect to the small sample size. also, the data for our study were collected retrospectively, and thus a potential for bias may exist. the gold standard for evaluating our treatment policy is a prospective randomized trial, but this approach is thought to be difficult due to the rarity of atypical meningiomas. nevertheless, the fact that the surgical procedure was performed by one surgeon adds credibility to our study. 1) in who grade ii meningiomas, complete resection (simpson grade i) is the most important factor for preventing recurrence. however, in the completely resected group, the effect of post - operative radiation therapy is uncertain in this study. 2) if the degree of resection is simpson grade ii, the recurrence rate is similar to the complete resection group, although post - operative radiotherapy is not performed. it is questionable whether or not post - operative radiotherapy must be performed in who grade ii meningiomas after simpson grade ii resection. 3) regardless of post - operative radiotherapy, if the degree of resection is simpson grade iii or iv, the risk of recurrence is significantly high. 1) in who grade ii meningiomas, complete resection (simpson grade i) is the most important factor for preventing recurrence. however, in the completely resected group, the effect of post - operative radiation therapy is uncertain in this study. 2) if the degree of resection is simpson grade ii, the recurrence rate is similar to the complete resection group, although post - operative radiotherapy is not performed. it is questionable whether or not post - operative radiotherapy must be performed in who grade ii meningiomas after simpson grade ii resection. 3) regardless of post - operative radiotherapy, if the degree of resection is simpson grade iii or iv, the risk of recurrence is significantly high. the extent of resection is the most powerful independent predictive factor for recurrence of who grade ii meningiomas. if complete resection was performed, the recurrence rate of who grade ii meningiomas was reduced to that of benign meningiomas, regardless of adjuvant treatment. post - operative adjuvant radiotherapy was not a significant factor in this study, also it continued to be controversial according to previous reports. however, because of several limitations of this study, re - estimation of the role of post - operative radiotherapy is required. | objectiveintracranial meningiomas are primarily benign tumors with a good prognosis. although who grade ii meningiomas are rare (2 - 10%), who grade ii meningiomas have higher recurrence and mortality rates than benign. we evaluated the patient recurrence rate and investigated the prognostic factors of who grade ii meningiomas.methodsbetween 1993 and 2005, 55 patients were diagnosed with who grade ii meningiomas in our hospital. who grade ii meningiomas (n=55) were compared with other who grades meningiomas (i, n=373 ; and iii, n=20). the patients had a median age of 48.4 years (range, 14 - 17 years), a male - to - female ratio of 26 : 29, and a mean follow - up time of 45 months (range, 3 - 175 months).resultsin who grade ii meningiomas, only the extent of resection was a significant prognostic factor. post - operative radiotherapy had no significant influence on tumor recurrence (p=0.053). the relative risk of recurrence was significantly higher in who grade ii meningiomas with incomplete resection (10/27, rr=37%) than in who grade ii meningiomas with complete resection (4/28, rr=14%) regardless of post - operative radiotherapy. in the incomplete resection group, simpson grade iii or iv had a significantly high risk of recurrence regardless of post - operative rt (n=3, rr=100%) however, if the degree of resection was simpson grade ii, the recurrence rate was similar to the complete resection group even though post - operative rt was not performed.conclusioncomplete resection was the most powerful independent predictive factor of the recurrence rate in who grade ii meningiomas. post - operative adjuvant rt was not a significant factor in this study. |
the heart and the kidneys share responsibility for maintaining hemodynamic stability and end - organ perfusion through a tight - knit relationship that controls cardiac output, volume status, and vascular tone. connections between these organs ensure that subtle physiologic changes in one system are tempered by compensation in the other. as such in particular, some of the key mediators include the sympathetic nervous system, the renin - angiotensin - aldosterone axis, and atrial natriuretic peptide. these agents have receptors in the heart, the kidneys, and the vasculature that affect volume status, vascular tone, cardiac output, and inotropy. a change in the performance of one of these organs elicits a cascade of mediators that affects the other. in the setting of underlying heart disease or chronic kidney disease, the capacity of each organ to respond to perturbation caused by the other acute or chronic heart failure may push the kidneys beyond their ability to maintain glomerular filtration, regulate fluid and electrolytes, and clear metabolic waste. similarly, acute kidney injury or chronic kidney disease affects cardiac performance through electrolyte dysequilibration, volume overload, and negative inotropy. clinical, cardiac, and renal parameters associated with dysfunction in the other organ are identified in table 1. this special relationship and the interdependence of the kidneys and the heart is well recognized. the manner in which dysfunction of one organ affects the other has recently led to the characterization of the cardiorenal syndrome (crs). at a consensus conference of the acute dialysis quality initiative (adqi), the cardiorenal syndrome was subclassified into 5 types based upon the organ that initiated the insult as well as the acuity or chronicity of the precipitating event. this review will primarily focus on crs type 1, where acute cardiac decompensation results in activation of hemodynamic and neurohormonal factors that lead to an acute drop in gfr and the development of aki. we will examine the scope and impact of this problem, the pathophysiology associated with this relationship, diagnostic clues for earlier detection, and therapeutic interventions to prevent and treat this complication. heart failure is a common chronic condition affecting 2% of the adult population and resulting in over 1 million annual admissions, making it the leading cause of hospitalization in the united states among adults over the age of 65. acute kidney injury may complicate one - third of these admissions, resulting in a three fold increase in length of stay, a greater likelihood for hospital readmission, and a 22% higher mortality rate [69 ]. this reduction in outcomes occurs with increases in serum creatinine of as little as 0.33 mg / dl, regardless of its presence at admission or its development during the course of heart failure treatment [10, 11 ]. in addition, approximately 25% of patients with chronic heart failure have been found to have reduced gfr, independent of their level of left ventricular function. a prospective cohort of 754 patients with chronic heart failure found in the large acute decompensated heart failure national registry (adhere), reduced gfr affected 30% of the 107,362 individuals. furthermore, 21% of patients had serum creatinine concentrations > 2 mg / dl, and 9% had serum creatinine concentrations > 3 this reduction in kidney function has significant impact on both morbidity and mortality [17, 18 ]. in a meta - analysis of 80,098 hospitalized and nonhospitalized patients, an egfr 90 ml / min was associated with a 24% 1-year mortality. the heart, by way of regulating the systemic circulation, and the kidneys, through their effect on extracellular fluid volume, share responsibility for the hemodynamic balance in the body. the kidneys produce a glomerular filtrate that is dependent upon perfusion pressure and afferent and efferent arteriolar tone. the arteriolar resistance is under intrinsic myogenic control, and responsive to several neurohormonal systems. the renin - angiotensin - aldosterone system (raas), the sympathetic nervous system (sns), and local vasodilators such as nitric oxide (no), adenosine, and prostaglandins contribute to maintaining the glomerular filtration rate (gfr) through conditions of increased or decreased perfusion pressure. when renal perfusion pressure decreases, angiotensin ii (aii) preferentially increases the efferent arteriolar resistance to preserve intraglomerular hydrostatic pressure and maintain gfr. simultaneously, the afferent arteriole, under control of tubuloglomerular feedback and prostaglandins, dilates to increase the transmission of perfusion pressure into the glomerulus. an elegant system senses decreased glomerular perfusion from hypovolemia or decreased cardiac output at the macula densa and the juxtaglomerular apparatus, then activates the raas, nitric oxide, adenosine, and prostaglandin production to prevent dramatic changes in kidney function. we will now explore some of the mechanisms that effect kidney function during decompensated heart failure. acute decompensated heart failure (adhf) results in reduced effective arterial filling volume (eafv). this decreased eafv diminishes renal blood flow and subsequently renal perfusion pressure. decreased tubular sodium and chloride delivery is sensed by the macula densa and the juxtaglomerular apparatus, activating the raas. raas enhances sodium and water retention to increase eafv and stroke volume, but comes at the detrimental cost of volume overload. whereas angiotensin causes efferent arteriole constriction, norepinephrine induces both afferent and efferent arteriole constriction and increases renal vascular resistance. in a setting of low cardiac output, both angiotensin and norepinephrine cause decreased renal blood flow (rbf), diverting blood to the coronary and cerebral circulations. when the normal compensatory mechanisms such as no, bradykinin, adenosine, and prostaglandins are unable to maintain gfr in the setting of decreased rbf, the groundwork for renal ischemia is laid. it appears, therefore, that the cardiovascular effects on hemodynamics and the renal effects on extracellular fluid volume are in constant flux. an imbalance in this relationship results in the crs. in the setting of heart failure where low cardiac output and an overactive neurohormonal system push the compensatory limits, a simple insult such as nsaids or aggressive diuresis can precipitate acute kidney injury. nsaids inhibit the protective effect of prostaglandins to dilate the afferent arteriole, while over diuresis might lead to further decreased eafv. diuretics are effective when properly dosed to allow reequilibration of fluid from the interstitial compartment into the intravascular compartment. other observations have suggested that rbf is the most important determinant of gfr in patients with chf. while it is true that decreased forward flow as a result of decreased cardiac output in adhf can cause acute deterioration in kidney function, there are several reasons why this mechanism fails to completely explain the development of the crs. first, altered hemodynamics alone are inadequate to explain the mechanism of kidney injury in adhf as redundant feedback mechanisms exist to prevent it. second, the crs has been observed in patients with diastolic dysfunction who have normal left ventricular systolic function. in the adhere registry, acute kidney injury occurred at similar rates in patients with both systolic and diastolic dysfunction. and finally, subgroup analysis of the escape trial showed evidence that poor forward flow alone was insufficient to explain worsening kidney function. in this trial, an improved cardiac index was not associated with improved renal outcomes, but increased cvp and atrial pressures were associated with decreased kidney function. observations dating back to the 1930s have suggested that renal venous congestion could also contribute to decreased glomerular filtration. experiments conducted on canine models revealed that increased venous pressure in the kidneys caused changes in urinary sodium, chloride, and urea excretion similar to decreased arterial pressure. this also led to a drop in glomerular perfusion pressure, and a reduction in gfr. this leads to compression of the tubule, increased tubular fluid pressure, and backleak of filtrate into the interstitium. furthermore, as hydrostatic pressure within the bowman 's capsule increases, gfr fails and the raas is activated and the sns is triggered. the sequence of events is shown in figure 1. studies in human subjects have also demonstrated that increased central venous and right atrial pressure are associated with worsening kidney function as well as increased mortality [29, 30 ]. damman and colleagues have demonstrated that increased venous pressure is an independent determinant of glomerular filtration in patients with heart failure. in this study the lowest glomerular filtration rate was observed in patients with lowest renal blood flow and highest right atrial pressures. elevated intraabdominal pressure from ascites and abdominal wall edema is also prevalent in patients with adhf, and associated with worsening kidney function. several studies have suggested that the deterioration in the kidney function is not due to direct parenchymal pressure on the kidneys, but rather due to elevated central venous pressure, arterial underfilling, and renal venous congestion [32, 33 ]. the decline in kidney function from increased intraabdominal pressure is mechanistically related to the venous congestion described above. there are a variety of neurohormonal mediators associated with the deterioration of kidney function in adhf. understanding these - mediators and effectors yields insight into the diagnosis and therapy of crs. the crs occurs with both hypoperfusion associated with decreased cardiac output as well as venous congestion. the actions of the raas, beyond its role to maintain hemodynamics, may explain this cardiorenal connection. activation of raas by hypoperfusion activates the sympathetic nervous system (sns), and mediates the release of reactive oxygen species (ros) and mediators of vascular inflammation. angiotensin ii activates both nadh - oxidase and nadph - oxidase, which then generates reactive oxygen species. studies have demonstrated this activity in vascular smooth muscle cells, cardiac myocytes, and both renal tubular cells and glomeruli in the kidneys. the ensuing oxidative stress results in a proinflammatory state activating chemokines such as il-1, il-6, and tnf alpha, and attracting leucocytes. furthermore, studies have shown that the effect of nadph - oxidase mediated ros release can be attenuated by angiotensin converting enzyme (ace) inhibition. angiotensin ii increases monocyte chemoattractant protein-1 (mcp-1) in mesangial and mononuclear cells by a mechanism dependent on nuclear factor-b (nf-b) activation. angiotensin ii also activates the sympathetic nervous system through its effect on the vasomotor center in the brain. this was established by showing increased muscle sympathetic nerve activity (msna) in patients with kidney failure. studies using ace inhibitors and angiotensin receptor blockers (arb) have shown decreased msna and decreased sympathetic activity [41, 42 ]. thus aii seems to play a direct role in renal injury and direct damage to the glomerular filtration barrier [44, 45 ]. nitric oxide, an endothelium - derived relaxing factor, is a vasodilator that acts to regulate vascular tone, blood pressure, and smooth muscle hypertrophy through downregulation of ace and the aii type 1 receptor. no therefore represents a physiologic antagonist of aii at both the glomerular and tubular levels [46, 47 ]. it also plays a role in tubuloglomerular feedback through dilation of the afferent arteriole. in decompensated heart failure, raas activation causes angiotensin mediated hypertension through increased systemic vascular resistance, greater renal perfusion pressure through preferential efferent arteriolar vasoconstriction, and renal oxidative stress through enhanced nadph - oxidase activity in rats. reduced activity of superoxide dismutase (sod) is thought to be involved in increased ros generation. subsequently, there is a shift in the no / ros system to the ros side. even mild heart failure is associated with decreased renal perfusion by way of no inhibition. also, endothelin i (et 1) is implicated in vasoconstriction, causing mesangial cell contraction and mesangial cell mitogenesis. whereas aii stimulates the release of et 1, no inhibits et 1 release from endothelial cells. an imbalance in favor of more et 1 production causes endothelial dysfunction as well as glomerular and interstitial damage. atrial natriuretic peptide (anp) and brain natriuretic peptide (bnp) are released in response to stretch of the cardiac chambers, and play a role in regulation of ecfv by inducing sodium and water loss. although they are an ideal therapeutic target, their role in the pathophysiology of crs is not known. erythropoietin is purported to decrease apoptosis in renal cells and cardiac myocytes by decreasing oxidative stress. small trials have revealed that heart failure patients who received erythropoietin had improved kidney function, but their place in the treatment of crs can not be confirmed without long - term studies. antidiuretic hormone (adh) levels are elevated in hf due to nonosmotic stimuli from baroreceptor stimulation. antagonism of adh would seem to have a role in the crs, but studies of vasopressin receptor 2 antagonists did not result in improvement in kidney function. a recent study by damman and colleagues showed that congestive heart failure is associated with increased markers of tubulointerstitial damage such as n - acetyl - beta - d - glucosaminidase (nag), kidney injury molecule 1 (kim-1), and neutrophil gelatinase associated lipocalin (ngal). other studies have also demonstrated renal tubular and interstitial damage as well. in summary, it appears that regardless of whether decreased perfusion occurs as a result of hypoperfusion or venous congestion, the consequent processes resulting in kidney injury are the same. the resulting nadph / nadh suppresses super - oxide dismutase, and increases reactive oxygen species. this results in the well known cascade of hypoxic ischemic injury, inflammation, apoptosis and cell death as shown in figure 2. one of the cornerstones of crs therapy is the early identification of worsening kidney function. this can be accomplished with the use of biomarkers that become detectable before the traditional tests for kidney function, including glomerular filtration rate or serum creatinine (figure 3). biomarkers such as ngal, nag, and kim-1 have been implicated in tubulointerstitial damage and have been used to identify acute kidney injury [6163 ]. furthermore, while cystatin c in the serum is a marker of reduced glomerular filtration, urinary cystatin c is a marker of tubular dysfunction. other biomarkers that have proven useful include bnp, il-18, and fatty acid binding protein (fabp). thus detection of these biomarkers might be used to diagnose crs at an earlier time point, facilitate targeted therapy for crs by modifying pharmacologic therapy, and monitor progression of disease. nevertheless, a higher index of suspicion for identifying patients at crs is needed as testing for biomarkers at this time is expensive. tests for volume status and end - organ perfusion are also useful in the diagnosis of crs. bioimpedence vector analysis is effective at assessing hydration status and bnp measurement provides an assessment of cardiac filling, although it is often elevated in patients with aki without overt fluid overload. urine sediment examination should be performed in differentiating crs from other causes of aki by excluding pathologic cells, casts, or crystals. hyponatremia, when present, may indicate excess adh and portend an overall poor prognosis. although patients with adhf have a poor prognosis to begin with, ensuing aki that accompanies the crs confers an even more dire condition. in patients with adhf who present with worsening kidney function, management is challenging and effective therapies are lacking. this is in large part due to the exclusion of patients with kidney dysfunction in many of the trials analyzing treatment for heart failure. a rational approach would be multi - modal, focusing on the underlying pathophysiology of crs with the goal of disrupting the cardiorenal connections. this requires use of biomarkers in appropriate settings to detect early changes in kidney function, and represents an opportunity for initiation of immediate treatment. although diuretics have a major role in the symptomatic treatment of heart failure, their effectiveness is limited due to diuretic resistance in crs. although renal hypoperfusion may require a reduction in the dose of diuretics, venous congestion may necessitate additional diuresis. thus, delicate fluid management may involve monitoring urine flow, central venous pressures, and possible cardiac output to optimize renal physiology. nevertheless, cvp monitoring is cumbersome and costly. a forthcoming trial, determining optimal dose and duration of diuretic treatment in people with acute heart failure (dose - ahf) study, is designed to answer these questions with regard to the role of diuretics in crs. several studies have explored the pharmacologic properties of natriuretic peptides in the treatment of heart failure. nesiritide, a recombinant natriuretic peptide, decreases preload, after load, and pulmonary vascular resistance, while inducing diuresis. because of its natriuretic and aquaretic properties, these agents seem to be an ideal candidate to relieve the venous congestion in crs. nevertheless, no studies have shown benefit on kidney function. in fact, a meta - analysis demonstrated poorer renal outcomes with nesiritide. in one study, nesiritide when compared to placebo had no effect on glomerular filtration rate, renal plasma flow, urine output, and sodium excretion in patients with crs. to address these controversies, the acute study of clinical effectiveness of nesiritide in decompensated heart failure trial (ascend) is underway. by making use of their aquaretic properties, vasopressin (v2 receptor) antagonists have been used in severe heart failure. however, clinical trials such as the efficacy of vasopressin antagonism in heart failure outcome study with tolvaptan (everest) trial showed no benefit of tolvaptan, a vasopressin antagonist, on all - cause mortality or the combined end point of cardiovascular mortality or hospitalization for adhf. kidney function remained stable throughout this trial, and the use of vasopressin antagonists in the crs conundrum may be limited to those patients complicated by hyponatremia. although other studies showed there was some renal benefit, the cost of these medications would prohibit them from being used routinely. adenosine is generated locally in the macula densa in response to diuretics that block sodium and chloride absorption, resulting in afferent arteriolar constriction and decreased gfr. antagonizing adenosine might have a role in preserving kidney function in crs. to this extent, kw-3902, an adenosine a1-receptor antagonist, was found to improve kidney function and decrease diuretic resistance in patients with adhf and crs. however, in crs it might have an early role by rapidly reducing venous pressure. in two trials of ultrafiltration in patients with adhf, the relief for acutely fluid - overloaded patients with decompensated congestive heart failure (rapidchf) and ultrafiltration versus intravenous diuretics for patients hospitalized for acute decompensated congestive heart failure (unload), there was marked weight loss and relief of heart failure symptoms [73, 74 ], but no improvement of kidney function. cardiorenal rescue study in acute decompensated heart failure (carress - hf) study which will assess the effectiveness of ultrafiltration in adhf and crs. although the use of inotropes in systolic heart failure may improve the eafv and cardiac output, the inherent adverse effects of these agents, including arrhythmias and myocardial ischemia, have limited their utility. in fact, the outcomes of a prospective trial of intravenous milrinone for exacerbations of chronic heart failure (optime - chf) trial revealed increased mortality and poorer outcomes in the milrinone arm. levosimendan, a phosphodiesterase inhibitor, has been studied in crs. in one study, levosimendan resulted in improved gfr when compared to dobutamine. however, another study of levosimendan and dobutamine did not show any benefit. at this time the role of raas blockade with ace inhibitors, arb, direct renin inhibitors, or aldosterone antagonists in crs is also unclear. while most of these medications cause an acute drop in gfr through the dilatory effect on the efferent arteriole, they have long - term reno- and cardioprotective effects. therefore, patients who are prone to develop crs yet able to tolerate a small reduction in gfr, up to 30% from the baseline, may benefit from these agents. as raas has been implicated in oxidative damage, its interruption though ace inhibition or angiotensin blockade may prevent the development of crs. similar to raas blockade, beta blockers through their effect on the sns may have a role in the long - term prevention of adverse cardiac events and in remodeling. however in crs, their role is limited by the altered hemodynamics. unless the underlying etiology of adhf is myocardial infarction, beta blockers are often held until the patients are hemodynamically stable. cardiorenal syndrome represents a disruption of the robust relationship between the kidneys and the heart to preserve hemodynamics and maintain organ function. despite the ability to adjust filling pressures, afterload, inotropy, cardiac output, and volume status in order to compensate for a wide range of perturbations, dysfunction in either of these organs creates a susceptibility to dysfunction in the other. the mechanisms for worsening kidney function in adhf are likely due to underperfusion from reduced cardiac output, venous congestion impairing tubular function and glomerular filtration, and activation of neurohormonal mediators that effect renal blood flow and glomerular autoregulation. the raas, sns, and no pathways are instrumental in preserving kidney function in compensated hf, but play an aggravating role once hf acutely worsens. measures to reverse kidney dysfunction in adhf require the early recognition and immediate treatment of crs. agents that target the physiologic mechanisms of crs may be effective in restoring kidney function. these include diuretics, natriuretic peptides, or ultrafiltration to reduce venous congestion, inotropes to augment cardiac output, and raas and sns blockade. despite these interventions, crs identifies patients at the limits of hemodynamic compensation and most susceptible to increased morbidity and mortality. | the heart and the kidneys share responsibility for maintaining hemodynamic stability and end - organ perfusion. connections between these organs ensure that subtle physiologic changes in one system are tempered by compensation in the other through a variety of pathways and mediators. in the setting of underlying heart disease or chronic kidney disease, the capacity of each organ to respond to perturbation caused by the other may become compromised. this has recently led to the characterization of the cardiorenal syndrome (crs). this review will primarily focus on crs type 1 where acute decompensated heart failure (adhf) results in activation of hemodynamic and neurohormonal factors leading to an acute drop in the glomerular filtration rate and the development of acute kidney injury. we will examine the scope and impact of this problem, the pathophysiology associated with this relationship, including underperfuson and venous congestion, diagnostic tools for earlier detection, and therapeutic interventions to prevent and treat this complication. |
in recent years, nanostructures of transition metal oxides have gained a great attention from material scientists and engineers due to their different properties compared with the corresponding bulk counterparts, which in turn provides promising applications in various fields of technology. preparation of high quality nanostructures of defined, controllable size and morphology is a critical requirement in order to develop nanodevices or other different applications for catalyst, sensing, pharmacy [17 ], and so on. cuo, categorized into transition metal oxide group, is a p type, narrow bandgap semiconductor. it has monoclinic structure and many interesting characteristics : super thermal conductivity, photovoltaic properties, high stability, and antimicrobial activity. due to such exclusive properties, cuo can be used in many technological fields, for example, active catalyst, gas sensor [4, 6 ], high efficiency thermal conducting material, magnetic recording media, with very good selectivity, or solar cell applications. in addition to some shared properties of metal oxide nanostructures, such as tio2, zno, wo3, and sno2, cuo nanostructures have other unique magnetic and super - hydrophobic properties. furthermore, these nanostructures show very promising applications in heterogeneous catalysis in the complete conversion of hydrocarbons into carbon dioxide, enhancement of thermal conductivity of nanofluid, nanoenergetic materials, and super - hydrophobic surfaces or anode materials for lithium ion batteries (libs). however, this material has not got attention of scientists at right level until recent years. compared with other oxides of transition metal such as fe2o3, tio2, and zno only few reports have described the synthesis strategies adopted for cuo nanostructures along with the introduction of their related applications. in this paper we would like to systematically discuss the effect of some factors in solution based process on the nanoproducts rather than focus on each individual synthesis process. for each factor, critical comments will be provided based on our knowledge and related research experience. some properties and potential applications of cuo nanostructures reported in the literature to date the potentials of cuo nanostructures as functional components for fabrication of micro / nanodevices are also evaluated and highlighted. in particular, we focus on the fundamental properties and various nanostructured forms of cuo that have been reported in the literature to date and summarize the various synthetic strategies. precisely controlling the synthetic strategies is very critical because it helps to obtain cuo nanostructures with manageable dimensions and morphology which is crucial for obtaining corresponding unique properties. this in turn enables a variety of promising applications that would not be possible for bulk material. the aim of this review is to provide critical discussion about important factors that can affect morphologies and size of cuo nanoproducts prepared by solution methods. the review hopefully can contribute some useful information that helps to better understand the relation between synthetic process, final morphology, and the properties of corresponding cuo nanostructures. a better understanding of the fundamental properties of cuo nanostructures is essential for their application as building blocks for functional devices. the development of synthetic methods has been widely accepted to contribute an important part to fundamental study for understanding properties and realizing applications of nanoscale materials. it allows material scientists to control different parameters of the products such as shape, particle size, size distribution, and composition. numerous methods such as thermal evaporation, sonochemical, sol - gel, hydrothermal, and electrochemical methods [2, 1115 ] and microwave irradiation approach [1618 ] have been developed to synthesize cuo nanostructures with diverse morphologies, sizes, and dimensions using various chemical, physical, or chemistry physics combined strategies. this paper focuses only on direct solution methods to successfully prepare copper (ii)-oxide (cu = o) nanostructures with different sizes, shapes, some of their properties and different applications in daily life and technology. the main reason why we would like to limit our considerations only to the wet chemical methods is that these approaches offer many advantages compared with physics synthesis processes such as the possibility to use low cost and high - throughput equipment, low wastage of raw materials, high uniformity of size and shape of the nanoproduct, and, finally, potential deployment of large scale production with low capital investment. in this review, we would like not to present the synthetic strategies in detail but rather to discuss the effect of major factors of the synthetic process such as solvents, starting materials, and additive materials on cuo nanoproducts. a typical direct solution method to prepare cuo nanostructures usually involves the following steps : preparing the precursor solution, modification of nanoproducts with additives or surfactants, heat treatment, and washing and drying process. cuo nanoproducts are prepared according to the following equations : (1)copper salt+alkaline hydroxide cuoh2+salt of alkaline metal(2)cuoh2cuo+h2o the entire process was summarized by the diagram in figure 1. in the next part, we will discuss in detail possible effects of each factor on the morphology and properties of final products. solvent is one of the most important components of wet chemical methods as solvent has a crucial effect on the product. due to the critical role of solvent, it is sometimes used to name a particular wet chemical approach, for example, alcohol - thermal synthesis or dmso (dimethyl sulfoxide) route. two primary criteria for the solvents used to synthesize cuo nanostructures are as follows : (i) they dissolve copper and alkali hydroxide compounds and (ii) they can be washed away easily or decomposed during the washing and drying process without leaving any detrimental impurities or residues in the final nanoproduct. there are many secondary factors that great attention should be paid for the synthesis process such as viscosity, surface tension, volatility, reactivity, toxicity, and cost. in order to dissolve well copper salts and alkali hydroxide, which are ionic compounds, polar solvents are normally used for synthesizing cuo nanostructures. in fact, in most of the reports, cuo nanostructures were prepared by utilizing water as solvent ; however, there are only few reports of using water solely without any additives or surfactants (see table 1). preparation of nanostructures in water without any additives normally leads to large (several hundreds of nanometers) and nonuniform size and shape particles or complex structures like 3d flower - like structures. these results imply that water itself has some disadvantages that make scientists prefer to use other organic solvents such as alcohols of different carbon chain length or number of functional groups (oh). the main disadvantage of using water as solvent is that water tends to promote the coarsening process during the growth of nanostructures prepared from liquid phase. coarsening involves the growth of larger crystals at the expense of smaller crystals and is governed by capillary effects. since the chemical potential of a particles increases with decreasing particle size, the equilibrium solute concentration for small particles is much higher than that for large particles or, in other words, larger particles are more energetically favored than smaller particles. the resulting concentration gradients lead to transport of solute from the small particles to the larger particles. the rate law for this process, derived by lifshitz, slyozov, and wagner (lsw), is given by (3)r3ro3=kt, where r- is the average particle radius, r - o is the average initial radius, k is the rate constant, and t is time. the rate constant k is given by (4)k=8vm2cr=54ana, where is the surface energy, vm is the molar volume, cr= is the equilibrium concentration at a flat surface (i.e., the bulk solubility), is the viscosity of the solvent (at room temperature, water = 8.94 10 pas, absolut ethanol = 10.74 10 pas and iso propanol = 19.45 10 pas), and a is the solvated ion radius. from (4) it is apparent that the rate constant k is inversely proportional to if cr= and are independent of the solvent. the viscosity of water is several times higher than the viscosity of alcohols such as ethanol or isopropanol, and hence the coarsening process of nanostructures in water takes place much faster and results in big agglomerated clusters. in addition, the polarity of water is also more favorable for the formation of large agglomerated clusters. the idea that polarity of solvent can alter the morphology of nanoparticles was reported theoretically and experimentally by the group of leekumjorn. in their report, the authors showed that solvent polarity has a better correlation with the simulation and experimental results than other parameters such as dielectric constant or dipole moment. computational results showed that the nonpolar solvents of hexane, toluene, and benzene (polarity index en < 0.120) kept oleate - capped nanoparticles in suspension and solvated the oleate chains so that the oleate layer swelled to full extension. in contrast, as the most polar solvent tested (en = 1.000), water caused nanoparticles to aggregate and precipitate. for solvents of intermediate polarity like ethanol, acetone, and chloroform, quantum dots were colloidally stable in solvents below a critical polarity index value (en = 0.307). some other organic solvents rather than water have been also tested for the synthesis of cuo nanostructures. different alcohols will then be the next choice to synthesize copper oxide nanoparticles due to their excellent characteristics as solvents. alcohols have a very polar hydroxyl (oh) group, with the high electronegativity of oxygen allowing hydrogen bonding to take place with other molecules. also, alcohols have nonpolar carbon chains ; therefore, they can dissolve both polar and nonpolar substances, while water can dissolve only polar ones. alcohols are low toxic, and ethanol is the least toxic of the alcohols, which makes it more suitable for use in industry and consumer products. the most common used alcohols are ethanol (boiling point 78.4c), glycerol (290.0c), propanol (97c), propylene glycol (188.2c), and ethylene glycol (197.3c). another advantage of alcohols is that they could be removed easily during the washing process without leaving unwanted impurities on the products. because in liquids phase process, cuo nanoparticles were formed through the decomposition of cu(oh)2, organic solvents of boiling points higher than the decomposed temperature of cu(oh)2 should be chosen. however, copper hydroxide is decomposed into copper oxide at a quite low temperature, which is only 80c ; then most of alcohols fulfill those criteria and can be utilized to prepare cuo nanostructures. even ethanol, of which boiling point (78.4c) is slightly lower than the decomposed temperature of cu(oh)2, could be used. however, following the above discussion, as the carbon chain gets longer, alcohols become less polar and have higher viscosity ; it is more favored for synthesis of small, uniform, and nonaggregated nanoparticles. some other organic solvents such as, dimethylformamide (dmf) and dimethyl sulfoxide (dmso) were also used but only in a few publications and the influence of solvent on the size and shape of the product was not well discussed. in principle, any kind of soluble copper salts could be used as precursor to prepare cuo nanostructures without much difference or at least there seems to be no report on the influence of copper salt precursor. various copper salts such as chloride, nitrate, sulfate, acetate were used to prepared cuo nanomaterials, however the effect of different copper salts were not discussed in details. however, particle size and uniformity of copper nanoparticles prepared from copper acetate seem better than those from inorganic copper salt. a reasonable explanation is that carboxylate groups are still adsorbed on the surface of the copper oxide nanoparticles and play the role of a surfactant and suppress nanoparticles from growth and aggregating process. the other main necessary precursor for synthesizing cuo nanoparticles is the base agent which provides hydroxyl ion to react with copper salt and gives the cu(oh)2 precipitation. naoh seems to be more preferred just because it is much less expensive than koh, while both compounds give almost the same effect due to their similar properties. nh4oh could also be utilized ; however, the high volatility of nh4oh brings some limitation during the synthesis process and, hence, nh4oh appears only in few reports of preparing cuo nanoparticles. sun. reported that using ammonia may enhance the agglomeration of the products due to the high polar nature of ammonia. oh group should be 1 : 2 according to (1), (2) ; however, many salts of cu readily hydrolyze in water and thus induce high solution acidity (ph < 2), while ph can play an important role in the dynamic process during the reaction. hence, the cu / oh ratio in the precursor solution could be adjusted from report to report in order to obtain the nanoproduct of desired morphology and size. a slower reaction rate, which leads to small size and narrow size distribution of the products, can be achieved by using low concentration of reactants. however, if the concentration of cu salts was too low, the amount of the cuo product would become negligible. concentrations that were too high, on the other hand, make the product agglomerate so the concentration of precursor solution should be chosen carefully to balance between the quantity and quality which refers to small size and good separation of the nanoproduct. limitation of using low concentration solution could be an obstacle to the mass production of nanoproduct due to the wastage of solvent, but recycle of the used solvent could be a solution for such problem. after the nucleation and growth process are completed, the average size of cuo nanoparticles could continue to increase due to agglomeration. this process reduces the quality of cuo nanoparticles in particular and nanoproducts in general, so restraining the particles from self - aggregating is a very important task. in nanostructure fabrication and processing, it is difficult to prepare small nanoparticles due to the challenge in huge surface energy ; then preventing the as produced nanoproducts from aggregating together is also a real problem that is needed to be solved. electrostatic stabilization keeps the system at kinetic equilibrium while steric stabilization keeps the system at thermodynamically stable case. electrostatic stabilization is based on the repulsion between equally charged particles. by considering the combination of van der waals attractions and electrostatic repulsion derjaguin, landau, verwey and overbeek even though some of the assumptions in the dlvo theory, which is named after the scientist, such as infinitely flat surface or constant charge density of the particles, were far from reality, it explained well interaction between two approaching particles and hence is widely accepted by the science comunity. however, there is no report of using electrostatic solely to stabilize the cuo nanoparticles prepared in liquid phase. instead, most groups used various surfactants to provide the steric barrier or to combine both mechanisms to achieve the best result in preventing the cuo nanoproduct from aggregation. normally, surfactants have a hydrophilic head and a hydrophobic tail. the polar heads of surfactants are absorbed on to the surface of nanoparticles, while the hydrophobic tail provides the steric repulsion to stop agglomeration. to provide sufficient repulsion between nanoparticles, the length of the stabilizer needs to be significantly longer than the characteristic size of the nanoparticles and also the polar head must have a tight bonding with nanoparticles. depending on the nature of each different polymer, another beneficial effect that surfactant may bring is to increase viscosity of the liquid media and thus minimize the rate of coarsening, as presented in the previous part. it is extremely useful if both stabilization mechanisms above were achieved at the same time. many groups had taken advantages of such combination by using certain polymers, of high ion density. the ionic nature of the polymer results in the electrostatic repulsion between nanoparticles while the long polymer chain simultaneously keeps the nanoparticles away from each other by steric effect. among the polymers of this kind, polyvinylpyrrolidone (pvp) is one of the most commonly used as it can be dissolved in both polar and nonpolar solvents ; the polar amide group in polymeric chain of pvp is readily attached to the surface of nanoparticles to protect them from aggregation by the two stabilization mechanisms. when dissolved into a solvent, these polymers will form a network, which can also play as templates to guide the growth of nanoproducts. it is then the network formed by polymer that could alter the morphology of the nanostructures. although polymer stabilizers are introduced primarily to form a layer on the surface of nanoparticles, occupy the growth sites, and reduce the growth rate of nanoparticles so as to prevent agglomeration of nanoparticles, the presence of such polymer stabilizers during the formation of nanoparticles can have various influences on the growth process of nanoparticles. interaction between the surface of a solid particle and polymer stabilizer or stabilizer with each other may vary significantly depending on the surface chemistry of solid, the polymer, solvent, and temperature. the complicated dynamic process of the interaction between polymer and nanoparticles leads to a strong dependence of morphology and size of nanoparticles on type of surfactants or even the amount of surfactant that was used. further increasing the amount of polymer in the reaction mixture it can be easily understood by considering the fact that increased amount of polymer produces steric resistance for the diffusion and consequently results in a diffusion controlled growth, which favors the formation of spherical particles. cuo nanoproducts could be prepared by various methods such as sol - gel, spray pyrolysis, precipitation, solvothermal or sonochemical methods. each method more or less has some limitations ; for example, solvothermal could be used to prepare material in extreme conditions such as high temperature, high pressure ; the bad side is that it takes quite long time, sol - gel process is quite complicated, and there are so many parameters need to be controlled. among the methods that could be applied to synthesize nanomaterials, microwave irradiation method which is a chemistry physics combined method currently shows its many advantages and cut down the number of limitations, which are usually brought by other methods. chemical reactions that took long time to complete can now be accomplished in minutes with the aid of microwave. microwave assisted synthesis not only helps in implementing green chemistry but also led to the revolution in organic synthesis. microwave irradiation is well known to promote the synthesis of a variety of compounds, where chemical reactions are accelerated because of selective absorption of microwave by polar molecules. it was found that the main advantages of microwave irradiation method compared with conventional method are fast, mild, energy - efficient, and friendly with the environment. the effectiveness of microwave irradiation in the preparation of nanoparticles can be explained by the preeminent advantages of microwave as a means of heating. the mechanism of traditional heating process is conductive heat or heating by convection currents and hence this is a slow and energy inefficient process due to the energy lost at the wall of the vessel. normally the wall of the vessel absorbs heat first ; then heat is transferred to the liquid inside so the temperature of the outside surface needs to be in excess of the boiling point of liquid for the temperature inside the liquid volume to reach boiling point. these disadvantages can be overcome easily by applying microwave technique because when heating with microwave, vessel wall is transparent to microwave and solvent / reagent absorbs microwave energy directly. the direct in core heating and instant on - off pulse of heat lead to the formation of a homogenous temperature gradient and reduce the time reaction. these advances in turn produce smaller particles of uniform size and shape. by using microwave irradiation, different nanostructures of cuo of uniform size and shape could be prepared in a few minutes [1618 ]. the coordination number of copper atom is 4, which means that it is linked to four oxygen neighbor atoms in an approximately square planar configuration in the (110) plane. in all crystallized solids, divalent copper surroundings are always very distorted by a strong jahn - teller effect which often leads to more stable square planar groups. the cu - o bond lengths in this plane are 1.88 and 1.96, respectively, which are larger than those in the cuprous oxide. the next two cu - o bond lengths perpendicular to the plane are much greater, so an octahedral type of coordination can be ruled out. the o atom is coordinated to four cu atoms in the form of a distorted tetrahedron. it is often accepted that the cuo has a mixture bonding of ionic and covalent bonding, even though the oxidation state of cu in cuo is unquestionably cu. the lattice parameters of cupric oxide are a = 4.6837, b = 3.4226, c = 5.1288, = 99.54, and = = 90. some other basic physics constants of cuo were also summarized in table 2. x - ray diffraction is obviously the most common tool to study the crystal structure of materials and confirm the purity of the product. from x - ray diffraction pattern, the lattice constants, lattice strain, or particles size can be extracted following the debye sherrer formula. according to aparna, elastic strain calculated from xrd results shows that cuo nanoparticles smaller than 20 nm have high strain and greater particles have less strain. this phenomenon relates to the pressure different between the inside and outside of a curved surface, resulting from the surface tension. raman spectroscopy, which is a sensitive probe to the local atomic arrangements and vibrations of the materials, has been also widely used to investigate the microstructural nature of the nanosized materials in general and cuo nanomaterial in particular. raman scattering could help to detect the existence of unintended phases such as cu2o or cu(oh)2 or show the crystallinity of the product. the space group of cuo is c2h with two molecules per primitive cell so the zone center raman active normal modes of cuo are ra = 4au + 5bu + ag + 2bg. among these vibration modes, there are three acoustic modes (au + 2bu), six infrared active modes (3au + 3bg), and three raman active modes (ag + 2bg). three well known bands of cuo are ag (296 cm), bg (346 cm), and bg (631 cm). figure 3 shows raman spectra of cuo nanostructures prepared by microwave irradiation method with three typical modes. xu. studied raman spectra of cuo nanocrystals with different grain sizes at room temperature and high temperatures up to 873 k. samples of smaller grain size show stronger and sharper raman peaks which also shift to smaller wavenumbers. the red shifts could be explained by the phonon confinement effect in nanometer size materials [25, 26 ]. it should be noted that crystal defects, of which number increases rapidly as the grain size decreases due to the large surface / volume ratio, could contribute significantly to raman spectra as all of the three raman modes in cuo relate only to the vibration of oxygen atoms as was pointed out by irwin and wei. apart from the three main vibration modes above, wang. reported multiphonon band of cuo nanostructures, which appears at wavenumber of 1130 cm and relates to the inharmonic coupling between phonons in polar solid. in particular, the multiphonon band 2bg in cuo was suggested to be the stretching vibration in the x - y plane, induced by the electronic density variation in this layer. the intensity of multiphonon raman peak is much weaker than that of the one phonon band and varies with morphology and the size of the as prepared cuo nanostructures. the authors reported that the multiphonon band of the as prepared cuo nanostructures with belt - like morphology possesses higher intensity than that of the cuo nanostructures with shuttle - like morphology, while raman intensity of multiphonon band of the shuttle - like morphology is higher than that of the cuo nanostructures with bamboo leaf - like morphology. the difference in the raman intensity of different morphology was explained by anisotropy of different nanostructures. the electronic movement along the x - y plane becomes significant in x - y plane and promotes the intensity of 2bg mode in belt-, shuttle-, and bamboo leaf - like nanostructures. another explanation for the variation in the raman intensity of this mode is the phonon - plasmon coupling due to high local density of anisotropic carriers in cuo nanostructures. the variation in the multiphonon intensity shows a finite size and crystallinity effect of cuo nanostructures. compared with other properties such as electrical conductivity and field emission, optical property of cuo nanostructures has been much less investigated and discussed so far. as a p type semiconductor, a narrow bandgap of around 1.2 ev was reported for bulk cuo. in fact, reported values of bandgap for cuo were not in good agreement ; for example, bandgap in the range of 1.56 and 1.85 ev was reported for cuo thin films [29, 30 ]. in addition, the variation of bandgap could also relate to quantum size effect in different cuo nanostructures [31, 32 ]. for nanomaterials, several methods could be applied to characterize the optical properties or to estimate the bandgap in particular. among these methods, uv - vis absorption spectroscopy, as a nondestructing and quick technique, is one of the most convenient methods to reveal the energy structures and optical properties of semiconducting materials. the optical bandgap of semiconductor material can be calculated from the absorption spectra by using tauc 's relation (5)h=hegn, where h is the energy of incident photon and n is the exponent factor that determines the type of electronic transition causing the absorption and can take the values 1/2 and 2 depending whether transition is direct or indirect, respectively. bandgap is determined from the intercept of the straight line with the horizontal axis. by uv - vis studies, some groups have reported significant blue shift (up to 1.7 ev) in the absorption edge compared with the bandgap of bulk material, which was explained by the quantum confinement effect in these nanostructures. in literature, optical behavior of cuo nanomaterials has been mainly assessed by absorption techniques, while luminescence techniques, which are important tools to investigate electronic transitions in semiconductors including band edge or near band edge transitions have been seldom used. the low emission efficiency of cuo is the main reason for the lack of luminescence data for cuo nanomaterials ; also the results on the origin of luminescence of cuo remain contradictory. there are several photoluminescence bands generally reported for cuo nanostructures which expand from uv to near ir region ; however the most frequent peaks fall in the region from 400 to 600 nm. generally, the deep level emission in cuo consists of a green emission at around 605 nm and a near - yellow emission at around 680 nm. though the origin of deep level emission in cuo is under debate, and little information is available on the cuo defect structure, the deep emissions are generally supposed to relate to defects in cuo nanomaterial. however, recent theoretical calculations indicate that although cu vacancies are the most stable defects in cuo, they do not make any changes in the electronic structures of cuo. otherwise, oxygen vacancies or ocu antisite defects are likely responsible for these emissions while their formation energy is not much different from formation energy of cu vacancies. the green emission is commonly assigned to the singly ionized oxygen vacancies ; the yellow or red emission has been supposed to relate to the interstitial metal ion in the oxide. therefore, the evolution of green and yellow bands in cuo is competitive with each other. the blue shift behavior of the near band edge transition in comparison with that of the bulk cuo in combination with the findings from uv - vis analysis was normally attributed to the enhancement of the quantum confinement effect resulting from the decrease in the dimensional structure and the size of the nanoparticles. one can also evaluate the concentration of structural defects by comparing the photoluminescence intensity ratio of near band edge emission to green deep level emission. vila. observed luminescence bands centered at 1.33, 1.23, and 1.11 ev in cuo nanomaterial and suggested that the emission with highest energy corresponds to near band edge transition in cuo while the two other emissions are most probably introduced by oxygen vacancies and oxygen on copper antisite defects [35, 37, 38 ]. besides emissions in visible or ir region, other authors announced emissions in the uv region. mageshwari and sathvamoorthy reported several photoluminescence peaks at 325, 339, and 356 nm and explained the difference photoluminescence emission peaks of cuo as agreed with earlier reports by various sizes and shapes of cuo nanostructures. this fact indicates that luminescence properties of cuo are strongly dependent on the morphology of the nanocrystals. gizhevski. studied the influence of temperatures and annealing time during sample preparation on the photoluminescence properties of cuo nanocrystals and reported three main broad emission bands centered at about 305 nm (4.07 ev), 505 nm (2.46 ev), and 606 nm (2.05 ev) [4, 14, 39, 42, 43 ]. the 2nd band was attributed to band edge emission while the band at 600 nm region was also assigned to defect related states. the emission at the band edge contains several subpeaks due to the band edge emission from 1 to the new sublevels which might arise by the interaction of two excitons or the d - d splitting in cu (3d 4s) at 300 k. pl intensities of the 305 nm (4.07 ev) band were seen to increase with the increasing temperature treatment and were explained by the enhancement of the crystallinity of the sample. as mentioned in the section of optical properties, photoluminescence and magnetic properties are aspects of least study for cuo nanomaterials ; however cuo nanostructures showed interesting and unique magnetic properties so it is worth making a summary on this topic. cuo, which is different from other antiferromagnetic transition metal monoxides such as nio, mno, and coo, shows magnetic order even above its neel temperature. kimura. reported that the ferromagnetic or antiferromagnetic ordering in cuo single crystal could be controlled by fine tuning the bond angle between in plane cu - o - cu. large bond angle cu - o - cu was believed to result in large super exchange interaction, favored for antiferromagnetism while spiral ordering in certain crystallographic directions favors a ferroelectric phase in the same material. due to the complex dependence on temperature of spin structure, cuo normally shows two antiferromagnetic transitions at 213 k and 230 k, respectively. the first neel temperature related to commensurate to incommensurate transition, whereas the second one is attributed to incommensurate to paramagnetic transition. a hysteresis loop or a bifurcation in fc - zfc curves was reported by a few groups as evidences for ferromagnetism in cuo nanostructures. for cuo nanomaterials, some reports showed that magnetic properties could vary, depending on size of the nanostructures. for example, punnoose. showed that susceptibility of cuo nanoparticles is inverse proportional to the particle size for particle smaller than 10 nm. the magnetic behaviors of cuo nano particles larger than 10 nm are similar to bulk material. however, morphology of cuo nanostructures should play an important role as well, because other authors showed that ferromagnetism could also arise in nanosheets, nanoneedles, and so on of larger size [47, 48 ]. hysteresis loop of weak ferromagnetism was observed at 5 k in cuo nanosheets prepared by hydrothermal synthesis by the group of zhao.. temperature dependence of magnetization showed neel temperature of cuo nanosheets of 219 k while the fc and zfc data show obviously maxima at about 40 k, which probably corresponded to blocking temperatures. below 40 k, the magnetization increases rapidly in fc data which reconfirms the existence of weak ferromagnetism in cuo nanosheets. the influence of impurities such as fe, ni at low content (0.5%) was excluded as the most probable ferromagnetic material is cufe2o4 but the magnetization of this phase is only half of as - measured magnetization of cuo nanosheets. the ferromagnetism resulted from the uncompensated spins on the surface of nanomaterials, which will be orientated under magnetic field and results in weak ferromagnetism as observed in cuo nanostructures. as the surface area of nanomaterials is much larger than that of bulk cuo, the surface effect, which is in this case shown by the ferromagnetic property, becomes dominant and hence cuo nanomaterials will exhibit ferromagnetic property more clearly. this argument could explain the size and morphology dependence of the ferromagnetism of cuo nanostructures. according to bhalerao - panajkar., the core shell nature of their cuo nanoparticles with a ferromagnetic shell and antiferromagnetic core may be responsible for bifurcation of fc - zfc curves because in pure antiferromagnetic or dominantly antiferromagnetic particles no bifurcation in fc - zfc curves occurs. the bifurcation in fc - zfc could also arise due to the ferromagnetic nature of the shell supporting the core shell nature of the particles. ferromagnetic / antiferromagnetic core shell structure was also supported by the asymmetry of the coercivity plot, which indicates the presence of an exchange field in core / shell system as suggested by other groups. cuo first attracted attention of chemists as a good catalyst in organic reactions but recently discovered applications of cuo such as high - tc superconductors, gas sensors, solar cells, emitters, electronic cathode materials also make this material a hot topic for physicists and materials science engineers. some of the most interesting applications of cuo nanomaterials are sensing, photocatalyst, and super capacitor that will be highlighted in this section. it is surface conductivity that makes cuo an ideal material for semiconductor resistive gas sensor applications and in fact cuo nanomaterials were used for detection of many different compounds such as co, hydrogen cyanide, and glucose. as sensing properties closely relate to the chemical reaction on the surface of sensor, the specific area is a key factor to achieve high sensitivity sensor. due to the high surface area / volume ratio, the shape of cuo nanostructures was also believed to affect significantly the sensing properties of cuo nanomaterial ; for example, spherical crystals often show higher sensitivity than columnar one. aslani and oroojpour studied co - sensing properties of different cuo nanoparticles prepared by solvothermal route as a function of morphology and size of nanoparticles. the results show that cloud like structures with high surface area / volume have higher response and detection limit than other morphologies. yang. also showed that the specific surface area of these cuo nanostructures plays an important role in the sensitivity for detecting hcn. both sides (5 mm in diameter) of a silver - coated quartz crystal microbalance (qcm) resonator were covered with cuo nanostructures ; the resonator was used as sensing probe in a quartz crystal resonator. the absorbance of hcn gas on sensor is indicated by the shift of resonant frequency. as specific area of cuo nanostructure used for coating the probe changes from 9.3 m / g to 1.5 m / g, the sensitivity reduces from 2.26 to 0.31 hz/g. in both reports, the authors showed that the sensitivity of sensors depends not only on the surface area but also on the morphology of the nanostructure. the change in sensitivity of different nanostructures could be explained by the variation in the chemical reactivity of different crystal planes. glucose detection is another important application of cuo in sensing field. in conventional methods, glucose detection is based on the use of glucose oxidase which is an enzyme used in the sensor. however, the main disadvantages of conventional methods are high cost and lack of enzyme stability, complicated immobilization procedures of enzyme, and the coexisting interferences in the biological fluids together with critical operating conditions. most of those limitations could be solved by using cuo nanostructures as an alternative oxidase, where cuo nanomaterials act as catalyst to convert glucose into gluconolactone and finally to glucose acid. the better efficiency of the oxidized reaction in cuo based sensor resulted from high surface area, surface energy which enhanced electron transfer ability of cuo nanomaterials. pseudocapacitors also known as one type of supercapacitors have attracted significant attention of researcher as efficient energy storage devices with superior properties such as high power density, excellent reversibility, and long cycle life time - dependent power, which are necessary properties of electronics portable devices. as the demand for high capacity energy storage in modern life was raised continuously, pseudocapacitors have become a hot topic recently. among transition metal oxides which are considered as ideal electrode materials for pseudocapacitors, cuo is a really promising candidate for its abundant resources, environmental compatibility, cost effectiveness, and favorable pseudocapacitive characteristics. it was found that the morphology and particle size of cuo remarkably affected its specific capacity. cauliflower like, nanobelt - shaped, and feather - like cuo nanocrystals were synthesized by the chemical deposition method by group of h. zhang and m. zhang. according to the authors, morphologies of the cuo nanostructures can influence the electrochemical properties significantly. the electrochemical properties of cuo as electrode material were enhanced by the improving of morphology. cauliflower - like cuo exhibited a higher specific capacitance (116.9 f g) than nanobelt - shaped and feather - like cuo and also showed good reversibility. specific capacitance of cauliflower - like cuo (115.3 f g) was 343.5% higher than cuo bought (26 f g) at 5 ma cm. the cuo cauliflower - like exhibited a higher utilization efficiency and better property for electrolyte diffusion than the feather - like and nanobelts structures. the increasing order of the specific capacitance was consistent with increasing sequence of cuo specific surface area, indicating that the highly mesoporous structure and high specific surface area of the electrode facilitate the ions to transfer into the porous structure more easily which would lead to more redox faradic reactions and surface adsorption of electrolyte cations. cuo nanomaterials could substitute for graphite anode in libs due to its superiorities such as high theoretical capacity (670 mahg), improved safety, low cost and environmental benignity. however, it also suffers very rapid capacity decay caused by huge and uneven volume variations (around 174%) during the lithium uptake / releasing process. one possible approach to improve the electrochemical performance of cuo materials is to use well - configured nanostructures ranging from zero - dimensional nanoparticles to multidimensional assemblies. in these nanostructures, not only lithium diffuses much easier, but also the strain associated with lithium uptake could be well accommodated, leading to better electrochemical performance. the unique nanostructural features endower them excellent electrochemical performance with high capacities of 450650 mah g at 0.52 c and almost 100% capacity retention over 100 cycles after the second cycle. recently composite material of cuo nanomaterial was developed to further increase the capacities of libs. rai. successfully used cuo / reduced graphene oxide nanocomposite as anode materials for lithium ion batteries. the initial discharge capacity of the pure cuo nanoparticles and their nanocomposite is 785.2 mah g and 1043.3 mah g with reversible capacity retention of 392.1 mah g and 516.4 mah g after 45 cycles, respectively. water pollution due to organic wastage from industry production has become a serious problem in the world today. most of organic compounds in waste water are toxic and can not be decomposed naturally so they need to be treated with care before disposal. water treatment using semiconductor catalysts under solar uv or visible light seems to be the most effective way as it has shown that this method could be employed to totally decompose many different organic compounds into biodegradable without complex technologies. cuo is a promising candidate due to low cost and abundance. as a p type semiconductor of narrow bandgap in visible region, cuo is expected to be a good material for application in photocatalyst and solar energy conversion. however, some groups reported that cuo shows almost no or very little photocatalyst properties under visible light. adding some amount of h2o2 could help to greatly improve the photocatalyst efficiency under visible light. study the degradation of brominated flame retardants by copper oxide nanoparticles and saw that adding an amount of h2o2 enhances the photocatalyst properties of cuo nanoparticles. they also showed that the interaction of cuo nanoparticles with h2o2 results in an electron spin resonance spectrum similar to spectrum of cu ion. this fact might indicate a release of cu ion to the solution or changes in the electron configuration of cuo nanoparticles in the solid phase. based on these effects, the authors suggested that h2o2 may have a role in the activation of cuo catalyst besides being an oxidative agent. it is noteworthy that photocatalyst properties also show dependence on size and shape of cuo nanostructures which again can be explained by the enhancement due to large surface area as well as the anisotropic of single crystals nanostructures of cuo, meaning that the photocatalyst of different crystal plane in cuo could be different. cuo could also be good candidate in solar energy conversion due to many properties : high absorption coefficient, narrow bandgap in visible region which is expected to give high conversion efficiency, being nontoxic, stability, good electrical conductance, simple manufacture process, and so on. a more direct way to convert solar energy to electricity is to use cuo as absorber in solar cell. efficiency of solar cell based on cuo is far lower than efficiency of leading chalcogenide system such as cis or cigs, but due to its low cost, abundant resource, and simple preparation process it was shown that efficiency of only several percents in cell based on cuo is good enough to make commercial solar cells. different from its counterpart cu2o, cuo is used less for solar cell as the achieved efficiency for cu2o is higher. number of reports on cuo solar cell is rare but recent results show very promising achievement, which shows that further development of cuo nanomaterials based solar cell has a bright future. prepared solar cells based on cuo nanoparticles / c60 junction which provided efficiency of 1.8 10%, fill factor of 0.25, jsc of 0.18 10 ma cm and voc of 0.04 v. a crystallite size of cuo was determined to be 3.4 nm, and higher crystallinity of cuo would increase the efficiency of the cuo / c60 solar cells. or more recently, using solvothermal method, chandrasekaran prepared cuo nanoparticles and used the product to make a solar cell with efficiency of 0.863%, which is compared with other reported values [15, 54, 55 ]. up to now, the record efficiency of solar cell based on copper oxide is about 2%, while the theoretical value is about 20%, so efficiency of several percents is obviously achievable. cuo nanomaterials could also be used as good substitution for expensive noble metal cathode in dye solar cell. this topic was first introduced by anandan. in 2005 and the optimal power conversion efficiency when using cuo nanorods as electrode was 0.29% compared with 1.23% when using pt as electrode in the same condition. by using cuo nanoneedles of higher surface active area, liu. obtained an efficiency of 1.12%, for tio2 based dye solar cell. this result shows that nanomaterials of cuo could replace well pt electrodes and can even give better efficiency under optimization process. field emission displays are now in a more dominant position in the market compared with crt displays because of their advanced properties such as high brightness, good color rendition, short response time, and low power consumption. among the various nanomaterials studied for field emission applications, 1d nanostructures of cuo emerged as very promising field emitters because of some advantages : low turn - on field, high current density, and low fabrication cost [5862 ]. liu. investigated the field emission properties of an individual cuo nanoneedle by in situ microscopy. the authors showed that individual nanoneedle possesses good field emission properties, such as low turn - on field of 5.3 v/m, high maximum current of 1.08 a at 9.7 v/m. the field emission properties of the single cuo nanoneedle and cuo nanoneedle 's film arrays are also compared and the results showed that the screening effect played a key role in the field emission properties. hu. used a simple method of direct heating of bulk copper plates in air to obtain cuo nanowire films on a large scale. the length and density of nanowires could be controlled by growth temperature and growth time. the as produced cuo nanowires have high density, good preferred orientation, and sharp tip, which is very beneficial to field emission. field emission measurements showed that cuo nanowires have a low turn - on field of 3.54.5 v/m and a large current density of 0.45 ma cm under an applied field of about 7 vm. the authors also showed that cuo nanowires having large length / radius ratio can effectively improve the local field, which enhance field emission. by varying the oxidant concentration, the authors can modulate the morphology of the nanoproducts from nanorod to nanotubes. the tip morphologies of cuo nanostructures were found to be crucial for the field electron emission, and the nanorods with needle - like tips showed superior emission properties with a turn - on field of 3.5 v/m and a field enhancement factor of 2107, compared to other structures. apart from improving the field emission efficiency by optimizing the aspect ratio (length / diameter) of 1d nanostructures, some other methods were also utilized to enhance the field emission current. wang and li found that laser irradiation could effectively enhance the field emission current of cuo nanowire arrays. the effects of laser intensity, wavelength, emission current, and working vacuum on the enhancement have been investigated in detail. among these factors, the contribution from extra excited electrons, which increases the number of electrons in conduction band of cuo for subsequent tunneling, is dominant. the observed laser induced enhancement in field emission current is attributed to the interplay of two factors, namely, laser induced electron transition to excited states and surface oxygen desorption. based on the idea of light induced field emission of their work, new vacuum nanodevices of cuo nanowires such as photodetectors or switches another example is the work of maji., where the authors also prepared cuo nanowire arrays by thermal oxidation. in order to improve the field emission properties of cuo nanowires they coated a zno layer on a cu substrate before the thermal oxidation process. the zno layer was deposited by immersing a cu foil into an aqueous solution of zinc nitrate and hexamethylenetetramine at 95c for several hours. the turn - on field of the zno - coated cuo nanowire array was 0.85 v/m compared with turn - on field 6.5 v/m of cuo nanowires without zno coating layer at the same current density of 10 a / cm. the authors suggested that in addition to the enlarged nanowire density and aspect ratio, crack elimination may be the reason for the enhancement of field emission properties. in conclusion, cuo nanostructures have been widely studied and are receiving more and more attention from material scientists and engineers recently because of their interesting properties and potential applications in various fields. in this study, we make a summary on the influences of different factors of synthesis process, some unique properties, and some promising applications of cuo nanostructures. we focus on the some chemical synthetic strategies along with associated influence of basic factors of synthesizing process for cuo nanostructures, as well as their interesting fundamental properties, and interesting applications. understanding the synthesizing process as well as the characteristics of cuo nanostructures is fundamental for further purposes to realize application of cuo nanostructures in daily life and technology. some unique properties of cuo nanostructures which make cuo different from other transition metal oxides were also summarized and highlighted. although encouraging developments and fascinating achievements in cuo nanostructures have been obtained as overviewed in this paper, better understanding for controlling morphology, structures, and properties of cupric oxide nanostructures and finding ways to take advantages of these interesting properties of such nanostructures still require much effort from scientists but also bring in opportunities for further development. | cupric oxide (cuo), having a narrow bandgap of 1.2 ev and a variety of chemophysical properties, is recently attractive in many fields such as energy conversion, optoelectronic devices, and catalyst. compared with bulk material, the advanced properties of cuo nanostructures have been demonstrated ; however, the fact that these materials can not yet be produced in large scale is an obstacle to realize the potential applications of this material. in this respect, chemical methods seem to be efficient synthesis processes which yield not only large quantities but also high quality and advanced material properties. in this paper, the effect of some general factors on the morphology and properties of cuo nanomaterials prepared by solution methods will be overviewed. in terms of advanced nanostructure synthesis, microwave method in which copper hydroxide nanostructures are produced in the precursor solution and sequentially transformed by microwave into cuo may be considered as a promising method to explore in the near future. this method produces not only large quantities of nanoproducts in a short reaction time of several minutes, but also high quality materials with advanced properties. a brief review on some unique properties and applications of cuo nanostructures will be also presented. |
the existence of a fountain of youth, a legendary spring that can restore the youth of anyone who drinks its water was believed by a large population in the 16 century influenced by the spanish explorer juan ponce de león y figueroa. such a fountain was mentioned also in the writing of herodotus some thousands of years ago. in the modern era, even though most may not believe in the existence of such a fountain, it is certain that everyone dreams of living longer. life expectancy has increased tremendously in recent years in many developed countries including the usa it is close to 80 years, whereas in underdeveloped countries such as zambia and angola life expectancy is below 40 years. the primary parameter responsible for increased life expectancy is certainly the lifestyle [2, 3 ]. stress is likely to be the single most important factor that can reduce life expectancy significantly. although ageing is certainly a genetically and epigenetically regulated process, modification of the lifestyle is likely to be the best way to increase life expectancy. an existing proof of the concept are the mormons, a specific group of the world population who live longer than average. among the many reasons why mormons live longer is possibly their lifestyle forbidding smoking and allowing regular fasting [2, 3 ] on the first sunday of each month. in the later study, 59% of the mormon fasters were diagnosed with heart disease compared to 67% of the non - fasters (after age, weight and other health conditions were taken into account) ; the non - mormon fasters showed same benefits as the mormon fasters. during day - to - day activities, accumulation of toxins or damaged particles appearing as misfolded or cross - linked aggregated macromolecules retard the biological and physiological cell functions, acting as sinks for important signalling components of the body. fasting and calorie restriction (cr) have been proven to stimulate anti - ageing processes and prolonging lifespan by activating detoxifying reactions. ageing, on the other hand, promotes progressive accumulation of damaged macromolecules and organelles [6, 7 ]. the question arising is how can fasting perform such a critical task of promoting longevity ? probably serves as a boost mechanism for cellular clean - up, preventing accumulation of toxic components and promoting longevity. in fact, suppressing autophagy accelerates accumulation of protein aggregates leading to triggering cell death signals. interestingly, the ageing process involves a co - ordinated program involving a large number of signalling cascades include but not limited to insulin / insulin - like growth factor (igf)-1, mammalian target of rapamycin (mtor), jun nh2-terminal kinase (jnk) and transforming growth factor (tgf) signalling pathways [1013 ]. at the same time, the ageing process deactivates several crucial survival protein cascades such as phosphatidylinositol 3-kinase (pi3k)- acutely transforming retrovirus akt8 in rodent t cell lymphoma (akt), b cell cll / lymphoma-2 (bcl-2) and/or sirtuin - forkhead box (sirt - foxo) signalling pathways. [this would tend to suggest that fasting induces autophagy, which in turn would promote anti - ageing signalling. this review intends to show that anti - ageing effects through lifestyle modification by fasting or chemicals / drugs potentiation of autophagy can clean up the toxic components and induce expression of pro - survival / anti - ageing proteins. the large plethora of signalling pathways intersecting at the three major degradative pathways (lysosomal, autophagosomal and proteasomal) is ultimately what decides the balance between ageing and longevity (fig. although the death due to ageing for human beings is difficult to correlate because many old people die from other factors such as cardiovascular disease, cancer or diabetes independent of ageing, lifespan and ageing are easy to correlate for lower eukaryotic species. for example, the lifespan of caenorhabditis elegans can be extended simply by blocking pro - ageing genes. compared to wild - type c. elegans, mutant species lacking insulin / igf-1 receptor (daf-2) live twice their age. insulin receptor in turn activates class i pi3k - akt signalling pathway, leading to the inhibition of foxo-putative player in the human ageing process (figs. 1 and 2) this would tend to suggest that inactivation of pi3k / akt and activation of foxos would extend the lifespan of c. elegans[22, 23 ]. a mitogen - activated kinase member jnk also modulates foxo and plays a role in the ageing process. the cytokines also appear to be involved as tgf- signalling seems to be instrumental for autophagy. in addition, mtor signalling and ampk (amp - activated protein kinase) signalling also appear to be involved in the ageing process [26, 27 ]. signalling cascades regulating the ageing process : pathways intersecting along the way from ageing to longevity. akt, acutely transforming retrovirus akt8 in rodent t cell lymphoma ; ampk, amp - activated protein kinase ; bcl-2, b cell cll / lymphoma-2 ; igf, insulin - like growth factor ; mtor, mammalian target of rapamycin ; pi3k, phosphatidylinositol 3-kinase ; ros, reactive oxygen species ; tgf, transforming growth factor ; ub, ubiquitin. sirt1, sirtuin 1 ; foxo, forkhead box ; sch9, saccharomyces cerevisiae kinase 9 ; pka, protein kinase a. ageing is a process in which the organisms gradually loose their ability to adapt to the changing environments, become more vulnerable to stress and accumulate damaged products due to overall decrease in the protein degradation time. autophagy also known as autophagocytosis, is a catabolic process involving the degradation of a cell s own components through the lysosomal machinery. in addition, it is responsible for elimination of damaged organelles and intracellular pathogens as well as aberrant or aggregated proteins that can not be removed via the proteasomal pathway. low levels of autophagy in general is considered to be pro - survival up to a certain threshold at which higher level of autophagy cause cellular stress and therefore death described as type ii programmed cell death [28, 29 ]. based on the selective uptake of cargo destined for degradation macroautophagy in which portions of the cytosol and complete organelles are engulfed by double - membrane structures known as autophagosomes (early autophagic vacuoles), which readily fuse with the lysosome to form single membrane structures autophagolysosomes (late autophagic vacuoles). by this time, luminal content is degraded and resulting elements are returned into the cytosol to undergo metabolic processes. according to the ability of the autophagosomes to degrade different organelle waste material, the intracellular engulfment has been referred as pexophagy (in case of peroxisomes), xenophagy (for intracellular bacteria and viruses) or mitophagy (for mitochondria). very likely, the future will describe other subtypes of autophagy, possibly erophagy (for endoplasmic reticulum), ribophagy (for ribosomes) or any other cell organellephagy. during ageing, age - associated increase in reactive oxygen species (ros) release and consequent mitochondrial dna (mtdna) damage occurs (fig. the mitochondria quality control system has been tightly associated with autophagy to segregate damaged, potentially harmful ross inside the cell. hypoxia has been associated with modulation of various cellular processes including autophagy and as a cause of mitochondrial dysfunction and tissue damage the brain, heart [37, 38 ] and kidney are all known to be affected in this process. in the brain, for example oxidative stress in the irreplaceable post - mitotic neurons can cause epigenetic or specific gene promoters silencing in response to unrepaired dna damage, therefore evading apoptosis. from worms and flies to mice and human beings, induction of autophagy has been associated with prolonged lifespan and decreased neurodegeneration due to decreased accumulation of ubiquitylated proteins [42, 43 ]. attenuation of the mtor signalling pathway has been show to extend lifespan in yeast, worms and flies. rapamycin, an inhibitor of the mtor pathway extends the lifespan of treated mice by reducing the toxic - aggregate formation in huntington s disease model mice. autophagy in the heart has dual role : under normal conditions, autophagy has a housekeeping role in the turnover of cytoplasmic constituents ; failure to do so, may result in defective autophagic degradation (deficiency of lysosomal - associated membrane protein-2 (lamp-2) causes cardiomyopathy) ; cardiomyocytes expressing polyglutamine exhibit increased autophagosomal content eventually causing heart failure ; missense mutation in the ab - crystallin (cryab) gene increases insoluble cryab - associated aggregate formation by reducing autophagy causing severe form of desmin - related cardiomyopathy. autophagy also removes damaged organelles in the heart after cellular distress (hypoxia/ reoxygenation), working against the mitochondrial protein bnip3 (bcl-2/adenovirus e1b 19 kd interacting protein) death signalling or apoptosis. regardless of the organ system involved, cr in general attenuates mitochondrial dysfunction and affects cell adaptation to hypoxia. cr prolongs the lifespan through a silent information regulator 1 (sirt1) by enhancing autophagy. in the kidney, hypoxia - induced expression of bnip3 is positively regulated by the hypoxia - inducible factor 1 (hif1) and forkhead box o3 (foxo3) being essential for enhancement of autophagy (fig., the efficiency of autophagic degradation reduces resulting in an accumulation of intracellular waste products. 2. microautophagy is a process where engulfment occurs directly by the lysosomal membrane. although microautphagy similar to macroautophagy is responsible for the disposal of long - lived proteins and organelles, it does not exhibit adaptation to nutritional deprivation. its distinction from macroautophagy comes from the fact that here small portions of the cytosol are internalized through small lysosomal invaginations for continuous protein degradation even under normal conditions. studies of this type of autophagy are still limited to yeast, but implications for importance of this process in antigen presentation and the major histocompatibility complex (mhc) do exist [55, 56 ]. chaperone - mediated autophagy (cma) is the third type of autophagic processes in which cytosolic proteins containing specific lysosome - targeting motif are recognized by a complex of chaperone protein such as heat shock cognate protein (hsc) 70 and delivered to the lysosomal membranes. after binding to lamp2a, besides the great interplay among the three types of autophagy, cma in contrast, specifically targets single soluble cytosolic proteins for translocation across the lysosomal membrane and degradation [58, 59 ]. this specificity is based upon recognition of an amino acid motif on the protein destined for degradation by a cytosolic chaperone complex. nevertheless, the role of cma in the turnover of long - lived proteins and intracellular quality control, as well as it role in different diseases has been similar to other types of autophagy. almost all proteins targeted for cma degradation contain a fingerprint pentapeptide kferq recognized by the cma cytosolic chaperone complex, but the same proteins can also contain targeting signals for other proteolytic systems, the ultimate decision for degradation being made upon current cellular conditions or reasons for degradation. a definite ticket for degradation of a substrate via cma is recognition by the hsc70 chaperone / co - chaperone complex. lamp2a, a single - span lysosomal membrane protein serves to bind and translocate the protein destined for lysosomal degradation. upon binding, substrate unfolding takes place and lysosomal lumen associated hsc70 protein (lys - hsc70) together with a lysosomal hsp90 protein somehow translocate the cytosolic protein for degradation, which takes only min.. under normal conditions, cma has housekeeping and stress - response function, but it can also be activated by accumulation of misfolded or oxidized proteins or starvation itself. interestingly, under nutritional deprivation lamp2a receptor levels increase not due to increased expression of this protein, but due to decreased rate of degradation ; supply from the pooled lamp2a on the luminal side of the lysosome regulate the activity of the cma during starvation. several pathologic conditions associate with cma : lysosomal storage diseases (galactosialidosis, mucolypidosis, danon s disease), nephropathies (diabetic, acidotic and chronic kidney disease), neurodegenerative disorders (alzheimer s, parkinson s and huntington s disease), oncogenic and immunological diseases. during ageing, the cma activity also declines mainly as a consequence of decreased levels of lamp2a and altered turnover of this protein on the lysosomal membrane. a proof of concept was recently found in an inducible exogenous copy of lamp2a (tet - off - lamp2a mouse) where activation of the transgene in the liver of old mice restored the cma levels close to those of young mice. overall, comparing macroautophagy to cma, one would say that the first one takes place early during nutritionally deprived conditions with the second one being actually activated later, when the macroautophagic activity starts to decrease. accumulated evidence suggests that during the ageing process, the ability of autophagy to digest oxidatively damaged macromolecles and organelles is significantly diminished resulting in an accumulation of long - lived post - mitotic cells such as cardiomyocytes and neurons as well as lipofuscin (a non - degradable intralysosomal polymer) and aberrant proteins forming aggresomes (fig. 1). mitochondria become defective and interaction between the defective mitochondria with lipofuscin - loaded lysosomes promotes cellular ageing process. lipofuscin, an ageing pigment, is considered a creditable marker for the ageing of cells. lipofuscin tends to accumulate even at an early age, but rapidly progresses with the advancement of ageing process suggesting the inability of autophagy to handle the garbage disposal capacity. the mitochondrial - lysosomal axis theory of ageing predicts that lysosomal dysfunction associated with ageing process is the hallmark for lipofuscin accumulation. thus, decline of autophagy during ageing appears to be the cause for lipofuscin accumulation. in fact, both the in vivo and in vitro function of autophagy is reduced with the advancement of age. interestingly, such reduction in age - dependent autophagy is reversed with fasting and/or cr [53, 66 ]. in the next section, we will discuss a number of pharmacological manipulations that can induce autophagy when it is needed. accumulated evidence suggests that during the ageing process, the ability of autophagy to digest oxidatively damaged macromolecles and organelles is significantly diminished resulting in an accumulation of long - lived post - mitotic cells such as cardiomyocytes and neurons as well as lipofuscin (a non - degradable intralysosomal polymer) and aberrant proteins forming aggresomes (fig. 1). mitochondria become defective and interaction between the defective mitochondria with lipofuscin - loaded lysosomes promotes cellular ageing process. lipofuscin, an ageing pigment, is considered a creditable marker for the ageing of cells. lipofuscin tends to accumulate even at an early age, but rapidly progresses with the advancement of ageing process suggesting the inability of autophagy to handle the garbage disposal capacity. the mitochondrial - lysosomal axis theory of ageing predicts that lysosomal dysfunction associated with ageing process is the hallmark for lipofuscin accumulation. thus, decline of autophagy during ageing appears to be the cause for lipofuscin accumulation. in fact, both the in vivo and in vitro function of autophagy is reduced with the advancement of age. interestingly, such reduction in age - dependent autophagy is reversed with fasting and/or cr [53, 66 ]. in the next section, we will discuss a number of pharmacological manipulations that can induce autophagy when it is needed. before discussing pharmacological manipulation to enhance autophagy, it is necessary to understand the signalling processes for autophagy regulation (fig. interestingly, there is striking similarities in the signalling pathways between autophagy and ageing (fig. aat, area at risk ; ampk, amp - activated protein kinase ; foxo, forkhead box ; igf, insulin - like growth factor ; lkbi, tumour suppressor kinase ; pi3k, phosphatidylinositol 3-kinase ; sirt, sirtuin ; ee2f, eukaryotic elongation factor 2f ; eif4f, eukaryotic translation initiation factor 4f. ampk, amp - activated protein kinase ; igf, insulin - like growth factor ; mtor, mammalian target of rapamycin ; pka, protein kinase a ; sch9, saccharomyces cerevisiae kinase 9. 1. mtor signalling. in yeast, there are two functionally distinct mtor complexes, one (torc1) is rapamycin sensitive and responds to nutrition and the other (torc2) is rapamycin insensitive and does not respond to nutrition. if mtor signalling is inhibited, physiological characteristics of starvation appear including induction of autophagy. in yeast cells, autophagy is controlled by torc1 through autophagy - related genes (atg) 1, atg13 and atg17, all being involved in the formation of the autophagosomes and inactivation of torc1 by rapamycin alters the phosphorylation of atg13 thereby inducing autophagy. reduction of mtor signalling by rnai or knocking down of mtor, let-363/cetor or the regulatory associated protein of mtor (raptor) can extend the lifespan in c. elegans. similar to ageing, insulin / igf-1 pathway is critically involved in the autophagy process. it has been known that low insulin levels can induce autophagy (such as during fasting) whereas high insulin concentration suppresses autophagy. although the precise relationship of insulin / igf-1 between ageing and longevity is not clear, autophagy appears to be involved in the regulation of ageing through this signalling pathway. this hypothesis is supported by the observation that knockdown of atg7 and atg12 partially inhibits the lifespan extension of the insulin - like receptor daf-2 mutants and causes significant shortening of the lifespan of wild - type worms. increased longevity of daf-2 mutants is partially inhibited by the mutants of atg6 (also known as beclin-1), atg-18 or atg 8 (ubiquitin - like protein also known as lc3). these reports suggest that insulin / igf-1 signalling pathway is involved in the autophagy regulation of ageing. this concept receives further support from the observations that mutations of daf-2, age-1 (encoding the catalytic subunits of pi3k) and pdk-1 [encoding pdk-1 (pyruvate dehydrogenase kinase) ] extend lifespan. this pathway plays a crucial role in the stress response and proliferation as well as longevity (fig. three autophagy - related proteins have been identified, atg1, atg13 and atg 18, as protein kinase a (pka) substrates in saccharomyces cerevisiae, which act at the early stages of autophagy indicating that ras / pka inhibits early in the process. in response to nutrient - rich conditions, two ras gtpases, ras1 and ras2 activate adenylate cyclase to produce camp, which after binding results in the dissociation of pka regulatory subunit bcy1 leading to the activation of pka catalytic subunits tpk1, tpk2 and tpk3. a recent study has indicated that ras / pka regulates the lifespan based on the observation that type 5 adenylyl cyclase knockout mice live 30% longer compared to corresponding wild - type littermates. sch9, the yeast pkb homologue, is a negative regulator of autophagy as its hyperactivation reduces autophagy when mtor is inactivated by rapamycin whereas simultaneous inactivation of sch9 and pka without suppression of torc1 induces autophagy, suggesting that they control autophagy in parallel (fig. it appears that longevity phenotype of pka and sch9 signalling are linked to each other with autophagy activation as msn2/4 and rim15 are involved in pka- and sch9-mediated regulation of autophagy. pka, sch9 and mtor are likely to regulate the biological processes in concert because they integrate nutrient signalling and cellular growth. ampk activates cellular processes to restore intracellular energy, and thus functions like an energy sensor. in a recent study, ampk was found to activate the lifespan of c. elegans and promote autophagy in human cells. another related study showed that myocardial ischaemia stimulated autophagy through an ampk - dependent mechanism. ischaemic areas at risk (aat) are capable of inducing the tumour suppressor kinase lkbi, which acting through the ampk pathway and eukaryotic elongation factor 2f (ee2f) can activate autophagy (fig. the pi3k pathway working through akt / pkb as well as the sirtuins have been found to activate foxo (fig. up - regulation of foxo induces autophagy as shown in drosophila, c. elegans and mouse muscle fibres. 1. fasting / cr. in most species, fasting and cr have been associated with an enhancement of lifespan by simultaneously inducing autophagy. fasting or cr reduces plasma insulin levels and indeed exposure of daf-2 mutant worms to cr extends lifespan, suggesting cr and insulin / igf-1 signalling function in parallel in regulating ageing. a recent study indicates that sch9 and mtor pathways mediate the lifespan extension of cr. have shown that transgenic expression of sirt1 induces autophagy in human cells in vitro and in c. elegans in vivo. caloric restriction and resveratrol the knockdown or knockout of sirt1 prevented the induction of autophagy by resveratrol and by nutrient deprivation in human cells as well as by dietary restriction in c. elegans. conversely, sirt1 was not required for the induction of autophagy by rapamycin or p53 inhibition, neither in human cells nor in c. elegans. the knockdown of pharmacological inhibition of sirt1 enhanced the vulnerability of human cells to metabolic stress, unless they were stimulated to undergo autophagy by treatment with rapamycin or p53 inhibition. along the similar lines, resveratrol and dietary restriction only prolonged the lifespan of autophagy - proficient nematodes, whereas these beneficial effects of longevity were abolished by the knockdown of the essential autophagic modulator beclin-1. this study indicates that autophagy is universally required for the lifespan - prolonging effects of caloric restriction and pharmacological sirt1 activators. recently, the resveratrol s ability to directly activate sirt1 and its anti - ageing effect have been questioned [9698 ]. spermidine, a precursor of spermine, is a ubiquitous polycation, which is synthetized from putrescine. these polyamines including putrescine, spermidine and spermine participate in diverse biological processes. exogenous supply of spermidine prolongs the lifespan of several organisms including yeast, nematodes and flies simultaneously reducing age - related oxidative protein damage in mice, indicating this compound may fulfill the definition of anti - ageing drug. spermidine induces autophagy in cultured yeast and mammalian cells, as well as in nematodes and flies and genetic inactivation of essential autophagy genes abolishes the lifespan prolonging effect of spermidine in yeast, nematodes and flies [99, 100 ]. although not proven beyond any doubt, several nutriceuticals including curcumin, piperine, trehalose and lithium have been found to induce autophagy. whether such autophagy is linked with lifespan extension is not yet known. there is overwhelming evidence that cellular mechanisms and signalling pathways regulating ageing are related to autophagy. key factors that regulate longevity and enhance autophagy such as p53, foxos and sirtuins, as well as protein translation factors such as eif4e and ee2f are among the putative targets in the fight against ageing. nutriceuticals including resveratrol, spermidine, curcumin and piperine all seem to work in a complex way towards enhancement of autophagy. future targeting of players involved in the autophagic pathway holds promise in anti - ageing or lifespan extension research. | abstractthis review focuses on the interrelationship between ageing and autophagy. there is a striking similarity between the signalling aspects of these two processes. both ageing and autophagy involve several of the signalling components such as insulin / igf-1, ampk, ras - camp - pka, sch9 and mtor. ageing and ageing - mediated defective autophagy involve accumulation of lipofuscin. components of anti - ageing and autophagy include sirts and foxos. nutritional deprivation or calorie restriction as well as several nutriceuticals including resveratrol, spermidine, curcumin and piperine can enhance autophagy and increase lifespan. such striking similarities indicate that lifespan is strongly dependent on autophagy. |
relaxin - like peptides are members of the insulin superfamily and, like insulin and insulin - like growth factors (igf), are small peptides (~60 amino acids) that share a common two - domain structure (a and b domains) in their mature form. functionally, however, relaxin family peptides are different from insulin and igf : they bind to unrelated receptors and play diverse roles in reproduction and neuroendocrine regulation as opposed to carbohydrate / fat metabolism and growth. four relaxin family peptide - encoding genes (rln, rln3, insl3, and insl5) originated early in vertebrate history and are shared by most vertebrates. the receptors for the rln / insl peptides belong to two distinct groups of g protein - coupled receptors (gpcr), collectively named the relaxin family peptide receptors (rxfp). in mammals, there are four known receptors, rxfp14, associated with the four relaxin family ligands. rxfp1 and rxfp2 are evolutionarily related to glycoprotein hormone receptors (e.g., luteinizing and follicle - stimulating hormone receptors), containing a large extracellular domain made up of ten leucine - rich repeats (lrr) and a low - density lipoprotein receptor type a (ldla) module ; they are the cognate receptors for the ligands rln and insl3 in humans, both of which primarily have reproductive actions. on the other hand, rxfp3 and rxfp4 are classic type i peptide gpcrs with short n - terminal domains ; they are evolutionarily related to somatostatin and angiotensin receptors and, in humans, are the cognate receptors for rln3 and insl5, both of which are associated with neuroendocrine signaling. the two hormones with reproductive functions in mammals, rln and insl3, are the best understood. the hormone rln is well known for its role in parturition, where it softens connective tissues of the reproductive tract via tissue remodeling and prepares the mammary glands for lactation, but it has numerous other physiological actions as well ; its receptor (rxfp1) also exhibits a wide distribution suggesting endocrine action in mammals (table s1, see supplementary materials available online at doi : 10.1155/2012/310278). in teleosts, the peptide sequence of rln is highly similar to that of rln3 ; although its function remains unknown, the rln gene exhibits substantial overlap in expression with rln3, both being highly expressed in brain, although teleost rln is also significantly expressed in gonads. while mammalian and teleost rlns differ somewhat in their expression patterns, insl3 has a more similar expression pattern in the two lineages ; it is highly expressed in leydig cells in both mammals and teleosts, and at lower levels in other tissues (see table s1). in mammals, the receptor for insl3, rxfp2, is also highly expressed in testes suggesting paracrine action, but lower levels of rxfp2 expression are observed in a wide array of tissues. the peptides rln3 and insl5 exert their influence primarily through the hypothalamic - pituitary - gonadal (hpg) axis [11, 12 ]. rln3 is the most conserved member of the family ; it is predominantly expressed in the nucleus incertus (ni) in mammalian brain and its homologous region in teleosts. ascending rln3-producing projections from the ni innervate a broad range of rxfp3-expressing regions of the forebrain in mammals, including the hypothalamus and it is implicated in the acute stress response and regulation of food intake [12, 15 ]. collectively, these lines of evidence suggest that rln3 acts through the hpg axis and may play a dual role linking nutritional status to reproductive function. lastly, insl5 is the least well understood member of the family, but in humans its primary sites of expression are rectum, colon, and uterus [16, 17 ] (see table s1). the receptor for insl5 in mammals, rxfp4, has a wide distribution being found in colon, placenta, testis, thymus, prostate, kidney, and brain in human, strongly suggesting endocrine action. despite the evolutionary distance separating rxfp1/2 and rxfp3/4-type receptors, experimental studies have shown that some rln / insl peptides can bind additional (secondary) receptors at lower affinity. for example, in addition to rxfp3, rln3 can bind to and activate rxfp1 and rxfp4, rln can bind to rxfp2 in addition to rxfp1, and insl5 can bind to (but activate only weakly) rxfp3 in addition to its primary receptor rxfp4. such primary and secondary ligand - receptor interactions have been demonstrated for human rln / insl - rxfp pairs, but analogous pairings in other vertebrates, such as teleosts, in which relaxin family peptide - receptor signaling and diversification have taken an evolutionary pathway distinct from that in mammals, remain to be established. recent evolutionary analyses revealed that vertebrate rln / insl genes and their receptors primarily diversified through the two rounds (2r) of whole genome duplication (wgd) that occurred in early vertebrate evolution and, in teleosts, during the teleost fish - specific wgd (3r) (figure 1). to summarize, mammals retained 4 ligand and 4 receptor genes following 2r, while teleosts have 10 (most teleosts) or 11 (zebrafish) receptor and 6 ligand genes following 3r (figure 1) and after-3r local duplications (figure 2, table s2). many of the genes retained in duplicate in fish (rln3-, insl5-, and rxfp3-type genes) are hypothetically involved in neuroendocrine regulation (figure 3). but due to a lack of understanding of the evolutionary history of rln / insl and rxfp genes in teleosts, the ligand - receptor pairings in teleosts are virtually unknown. one of the interesting aspects of the evolution of rln / insl peptides is how a set of relatively closely related ligands signals via two unrelated types of receptors. yegorov and good hypothesized that this dual - functioning arose in the ancestral pre-2r rln / insl peptide that had roles in both reproductive (via rxfp1/2-receptor) and neuroendocrine (via rxfp3/4) regulations in primitive vertebrates (figure 3). as a result of the wgds, the ancestral tripartite system gave rise to two distinct parties of rln / insl - rxfp ligand - receptor pairs (figure 3). curiously, it can be observed that, with the exception of the rxfp1 receptor and its ligand rln, each of the duplication events resulted in a single ligand that potentially could function with two related receptors (figure 3). in most mammals, this tripartite model became reduced to a 1 : 1 relationship for ligands and receptors after the divergence of tetrapods from the gnathostome ancestor (as described above), but in teleosts, there are multiple receptors for some ligands, which may have occurred through receptor subfunctionalization (figure 3). based on the evolutionary history of duplication, and the ligand - receptor pairings in mammals, we developed hypotheses concerning which ligand - receptor pairings we expect in teleosts (figure 4). the primary goal of this paper is to test our hypotheses about the rln / insl - rxfp ligand - receptor pairs in teleosts using selection analyses and experimental qpcr data from zebrafish. we performed two kinds of molecular evolutionary analyses to (1) hypothesize which ligand - receptor pairings may occur in teleosts and (2) examine differences in selection among mammalian and teleost genes. (1) previous studies have used the correlation of evolutionary distances between putative ligand - receptor pairs as evidence of cofunctioning [20, 21 ]. here, we employed a similar correlation approach, but rather than comparing the mean evolutionary distances among gene pairs, we compared the proportion of sites under different forms of selection (purifying, neutral, or positive) in pairs of teleost genes to the primary ligand - receptor pairs known to exist in mammals, rln - rxfp1, insl3-rxfp2, and rln3-rxfp3 - 1. if the genes coding for the ligands and receptors coevolve, we expect a correlation in the rates and types of selection on ligand - receptor pairs. this would correspond to values falling along the (0, 0 : 1, 1) plane of the xy - plot. on the other hand, a similar [x, y]-value for the same ligand - receptor pair in mammals and teleosts would suggest that the pair plays a similar role in the two lineages. (2) we tested for evidence of (a) codon - specific positive selection in mammalian and teleost ligand and receptor genes and (b) codon - specific positive selection in mammalian versus teleost genes using the branch - site model of positive selection. while the first analysis (a) tests whether specific codons have been positively selected within lineages, the second (b) looks for evidence that codons have been differentially selected in mammalian versus teleost lineages. (1) evidence for ligand - receptor coevolution for mammalian and teleost orthologsbetween 70 and 93% of the sites across all genes, and in both mammals and teleosts, have been subject to purifying selection (figure 5(a)). additionally, the extent of purifying selection was symmetric for the ligand - receptor pairs rln3-rxfp3 and insl3-rxfp2 suggesting close coevolution, while for the remaining two pairs, rln - rxfp1 and insl5-rxfp4, the proportion of sites under purifying selection was higher for the receptor genes (between 0.7 and 0.92) than for the ligands (ranging from 0.40.95), suggesting a more diffuse coevolution (or no coevolution), and more relaxed evolution on the ligand.on the other hand, there are significantly fewer sites which are evolving neutrally (figure 5(b)) or are subject to positive selection (figure 5(c)). for the receptor genes, from 3 to 20% of the sites were found to be evolving neutrally (figure 5(b)), and from 2 to 13% were subject to positive selection ; rxfp3 exhibits the fewest neutral or positively selected sites, rxfp4 has the highest proportion of sites under neutral evolution and rxfp2 exhibits the highest proportion of sites under positive selection. largely due to the anomalous nature of asymmetric selection on the rln - rxfp1 ligand - receptor system in mammals, the extent of neutral and positive selection among ligand genes varied widely between mammals and teleosts, primarily because teleost rln was found to have a large number of sites evolving neutrally, whereas mammalian rln has a large proportion of sites subject to positive selection (figures 5(b) and 5(c), resp.). between 70 and 93% of the sites across all genes, and in both mammals and teleosts, have been subject to purifying selection (figure 5(a)). additionally, the extent of purifying selection was symmetric for the ligand - receptor pairs rln3-rxfp3 and insl3-rxfp2 suggesting close coevolution, while for the remaining two pairs, rln - rxfp1 and insl5-rxfp4, the proportion of sites under purifying selection was higher for the receptor genes (between 0.7 and 0.92) than for the ligands (ranging from 0.40.95), suggesting a more diffuse coevolution (or no coevolution), and more relaxed evolution on the ligand. on the other hand, there are significantly fewer sites which are evolving neutrally (figure 5(b)) or are subject to positive selection (figure 5(c)). for the receptor genes, from 3 to 20% of the sites were found to be evolving neutrally (figure 5(b)), and from 2 to 13% were subject to positive selection ; rxfp3 exhibits the fewest neutral or positively selected sites, rxfp4 has the highest proportion of sites under neutral evolution and rxfp2 exhibits the highest proportion of sites under positive selection. largely due to the anomalous nature of asymmetric selection on the rln - rxfp1 ligand - receptor system in mammals, the extent of neutral and positive selection among ligand genes varied widely between mammals and teleosts, primarily because teleost rln was found to have a large number of sites evolving neutrally, whereas mammalian rln has a large proportion of sites subject to positive selection (figures 5(b) and 5(c), resp.). the selection analysis supports our hypothesis for many ligand - receptor pairs in teleosts, but the receptors for the two insl5 paralogs remain uncleargiven the presence of additional ligand and receptor genes in teleosts for which no ortholog was present in mammals, the correlation approach could not be used for the additional ligand - receptor genes in teleosts because there was no reference comparison in mammals and too many possible pairs to consider. thus, to examine the possible pairings of these additional genes, we simply plotted the proportion of sites subject to each form of selection in teleosts for visual comparison (figure 6). this revealed that the gene coding for rln has a higher number of neutrally evolving sites than the gene of its proposed receptor, rxfp1, although this may be an artifact of the comparison to mammalian rln. on the other hand, the numbers of selected sites in the genes of the proposed ligand - receptor pairs insl3-rxfp2 (as demonstrated above), rln3a - rxfp3 - 2a / rxfp3 - 2b, and rln3b - rxfp3 - 1 were similar, supporting possible cofunctioning, although rxfp3 - 2a shows a higher fraction of positively selected sites than either of the rln3 ligand genes. lastly, however, there was also a poor correlation in the expected selection profile of insl5 compared with its proposed receptor genes : both teleost insl5a and insl5b evolve relatively neutrally but none of their proposed receptors do, with the exception of rxfp4, which has a slightly higher rate of neutral and positive selection. the remaining three rxfp3 - 3 receptor genes are very conserved (figure 6). thus, although teleost insl5 and rxfp4 genes had similar selection profiles to those of mammals (see above), suggesting a conserved function between the two lineages, the other three proposed receptors for the insl5 paralogs (i.e., rxfp3 - 3a1, rxfp3 - 3a2 and rxfp3 - 3b) exhibited strong purifying selection and did not closely parallel the selection profile of either candidate ligands. given the presence of additional ligand and receptor genes in teleosts for which no ortholog was present in mammals, the correlation approach could not be used for the additional ligand - receptor genes in teleosts because there was no reference comparison in mammals and too many possible pairs to consider. thus, to examine the possible pairings of these additional genes, we simply plotted the proportion of sites subject to each form of selection in teleosts for visual comparison (figure 6). this revealed that the gene coding for rln has a higher number of neutrally evolving sites than the gene of its proposed receptor, rxfp1, although this may be an artifact of the comparison to mammalian rln. on the other hand, the numbers of selected sites in the genes of the proposed ligand - receptor pairs insl3-rxfp2 (as demonstrated above), rln3a - rxfp3 - 2a / rxfp3 - 2b, and rln3b - rxfp3 - 1 were similar, supporting possible cofunctioning, although rxfp3 - 2a shows a higher fraction of positively selected sites than either of the rln3 ligand genes. lastly, however, there was also a poor correlation in the expected selection profile of insl5 compared with its proposed receptor genes : both teleost insl5a and insl5b evolve relatively neutrally but none of their proposed receptors do, with the exception of rxfp4, which has a slightly higher rate of neutral and positive selection. the remaining three rxfp3 - 3 receptor genes are very conserved (figure 6). thus, although teleost insl5 and rxfp4 genes had similar selection profiles to those of mammals (see above), suggesting a conserved function between the two lineages, the other three proposed receptors for the insl5 paralogs (i.e., rxfp3 - 3a1, rxfp3 - 3a2 and rxfp3 - 3b) exhibited strong purifying selection and did not closely parallel the selection profile of either candidate ligands. (2a) evidence for codon - specific positive selection in mammalian and teleost ligand and receptor genesto look for evidence of codon - specific positive selection in mammalian and teleost lineages, we compared models 7 (purifying selection), 8 (positive selection), and 8a (relaxation of purifying selection) using maximum likelihood - based comparisons in mammals and teleosts. genes are considered to be under positive selection if the support for model 8 is greater than model 7, but also model 8a. for genes that exhibited evidence of positive selection, determination of the amino acid sites estimated to be under selection was tested using bayesian empirical bayes (beb). we found evidence of positive selection for mammalian insl5 and mammalian rln ; however, the hypothesis that the positive selection found in mammalian insl5 is actually caused by a relaxation of purifying selection (i.e., tested by comparing model 8a versus model 8) could not be rejected. the extent of positive selection on mammalian rln is extensive ; however, in total, 12 amino acid positions were identified as having a beb probability > 0.9 that > 1.0 (i.e., to be under positive selection) and another five had a probability > 0.8 that > 1.0 (table s3). this suggests the presence of strong diversifying selection on mammalian rln. in teleosts, only insl3 showed evidence of having codons subject to positive selection at two sites (table s3). there was some, but limited, evidence of positive selection on the receptor genes within mammalian or teleost lineages. only one codon was found to exhibit strong evidence of positive selection in mammalian rxfp1, and two for rxfp2, while three codons showed evidence of positive selection in fish rxfp2, but the latter hypothesis was more likely attributed to a relaxation of purifying selection. additionally, a few codons were found to have evidence of positive selection in mammalian rxfp3 and teleost rxfp4 (stronger evidence). although mammalian rxfp4 also showed evidence of positive selection (model 8 was preferred over models 7 and 8a) ; no specific codons had a beb probability of being under strong positive selection. overall, this suggests similar patterns of selection on ligand - receptor pairs, with the notable exception of rln - rxfp1 in mammals for which strong evidence of positive selection exists for the ligand, but no strong evidence of positive selection on the mammalian receptor gene, rxfp1. to look for evidence of codon - specific positive selection in mammalian and teleost lineages, we compared models 7 (purifying selection), 8 (positive selection), and 8a (relaxation of purifying selection) using maximum likelihood - based comparisons in mammals and teleosts. genes are considered to be under positive selection if the support for model 8 is greater than model 7, but also model 8a. for genes that exhibited evidence of positive selection, determination of the amino acid sites estimated to be under selection was tested using bayesian empirical bayes (beb). we found evidence of positive selection for mammalian insl5 and mammalian rln ; however, the hypothesis that the positive selection found in mammalian insl5 is actually caused by a relaxation of purifying selection (i.e., tested by comparing model 8a versus model 8) could not be rejected. the extent of positive selection on mammalian rln is extensive ; however, in total, 12 amino acid positions were identified as having a beb probability > 0.9 that > 1.0 (i.e., to be under positive selection) and another five had a probability > 0.8 that > 1.0 (table s3)., only insl3 showed evidence of having codons subject to positive selection at two sites (table s3). there was some, but limited, evidence of positive selection on the receptor genes within mammalian or teleost lineages. only one codon was found to exhibit strong evidence of positive selection in mammalian rxfp1, and two for rxfp2, while three codons showed evidence of positive selection in fish rxfp2, but the latter hypothesis was more likely attributed to a relaxation of purifying selection. additionally, a few codons were found to have evidence of positive selection in mammalian rxfp3 and teleost rxfp4 (stronger evidence). although mammalian rxfp4 also showed evidence of positive selection (model 8 was preferred over models 7 and 8a) ; no specific codons had a beb probability of being under strong positive selection. overall, this suggests similar patterns of selection on ligand - receptor pairs, with the notable exception of rln - rxfp1 in mammals for which strong evidence of positive selection exists for the ligand, but no strong evidence of positive selection on the mammalian receptor gene, rxfp1. (2b) evidence for differential selection on teleost versus mammalian lineages for orthologous receptorsalthough the above analyses suggested that only mammalian rln has experienced high levels of codon - specific positive selection, using the branch - site model of codon - specific positive selection, we tested whether mammalian and teleost lineages have been subject to lineage - specific positive selection, that is whether they have been selected to be fixed for different amino acids (table s5). this analysis revealed considerable evidence of lineage - specific selection indicating that mammalian and teleost lineages have evolved in different ways, and it also highlighted some important differences in the regions of the receptors that have been subject to positive selection. by mapping, codons were found to have evidence of positive selection to their position in the mature proteins ; we find that (1) the low - density lipoprotein / leucine rich repeat (ldl / lrr) region of rxfp1/2-type genes is an important region of diversification among lineages ; (2) for the 7 transmembrane (7tm) region shared between the two receptor types, all regions have more selected sites in rxfp3/4- than in rxfp1/2-type genes, except extracellular loop 2 (ecl2), and (3) intracellular loops 1 (icl1) and 3 (icl3) have many positively selected sites for rxfp3/4 genes while icl3 also has many amino acids selected for rxfp1/2 type genes (figure 7).closer examination of the sites that were selected in mammalian versus teleost lineages revealed somewhat different regions of selection in teleosts versus mammals. for rxfp1, mammals had more selection on the first few domains of the ldla / lrr region, while teleosts exhibit greater selection on the terminal lrr domains. additionally, in general mammalian, rxfp1 genes were found to have more selected sites in the icls (icl1 and icl3), while teleosts exhibit more selection in the ecls (ecl1 and ecl3) (figure s1). this suggests that while the overall patterns of selection are similar among mammalian and teleost putative ligand - receptor orthologs, divergent selection has operated in both lineages for all genes, and some of this selection could be associated with intra- versus extracellular signaling (figure s1). although the above analyses suggested that only mammalian rln has experienced high levels of codon - specific positive selection, using the branch - site model of codon - specific positive selection, we tested whether mammalian and teleost lineages have been subject to lineage - specific positive selection, that is whether they have been selected to be fixed for different amino acids (table s5). this analysis revealed considerable evidence of lineage - specific selection indicating that mammalian and teleost lineages have evolved in different ways, and it also highlighted some important differences in the regions of the receptors that have been subject to positive selection. by mapping, codons were found to have evidence of positive selection to their position in the mature proteins ; we find that (1) the low - density lipoprotein / leucine rich repeat (ldl / lrr) region of rxfp1/2-type genes is an important region of diversification among lineages ; (2) for the 7 transmembrane (7tm) region shared between the two receptor types, all regions have more selected sites in rxfp3/4- than in rxfp1/2-type genes, except extracellular loop 2 (ecl2), and (3) intracellular loops 1 (icl1) and 3 (icl3) have many positively selected sites for rxfp3/4 genes while icl3 also has many amino acids selected for rxfp1/2 type genes (figure 7). closer examination of the sites that were selected in mammalian versus teleost lineages revealed somewhat different regions of selection in teleosts versus mammals. for rxfp1, mammals had more selection on the first few domains of the ldla / lrr region, while teleosts exhibit greater selection on the terminal lrr domains. additionally, in general mammalian, rxfp1 genes were found to have more selected sites in the icls (icl1 and icl3), while teleosts exhibit more selection in the ecls (ecl1 and ecl3) (figure s1). this suggests that while the overall patterns of selection are similar among mammalian and teleost putative ligand - receptor orthologs, divergent selection has operated in both lineages for all genes, and some of this selection could be associated with intra- versus extracellular signaling (figure s1). quantitative expression of all ligand and receptor genes in zebrafish across multiple tissuesto infer functional ligand - receptor relationships, we assessed the expression of both ligand and receptor genes in male and female zebrafish heart, intestine, gonads, muscle, gills, brain, and eyes using real - time, quantitative pcr. overall, the fold increase of the target to housekeeping genes, especially the receptors, was similar for both sexes in all tissues (except gonad) confirming the reliability of the data (figures s2 and s3). to allow comparison of the relative amounts of mrnas produced per tissue, the relative mrna expression levels were normalized to the total amount of rna isolated per tissue (figure 8). this revealed that for all tissues studied, the expression levels of all rxfp genes appeared to be higher than the expression levels of all rln / insl genes, except for the very high expression levels of insl3 in testis tissue (figure 8).the ligand rln was most abundantly expressed in gonads and male intestine (figures s2 and 8) ; its primary hypothesized receptor, rxfp1, was also highly expressed in gonads, as was a potential secondary candidate receptor, rxfp2a (figure 8). the rxfp1 transcript was also detected in male heart and brain, while rxfp2b expression was found in brain and eyes. expression of the zebrafish - specific rxfp2-like transcript, a candidate receptor for rln and insl3, was only found in brain at high levels. very high expression of insl3 mrna was found in testes and somewhat lower levels in ovaries and eyes. the primary candidate receptors for insl3 are rxfp2a and rxfp2b, and high expression of both rxfp2a and rxfp2b was observed in gonads, while rxfp2-like was not detected in testes or ovaries. as expected, rln3a and rln3b expression was found predominantly in brain and gonad, but we also identified rln3a expression in heart (figures s2 and 8). on the other hand, all of the rxfp3 - 1, rxfp3 - 2, and rxfp3 - 3 genes showed a similar expression pattern : high expression in brain with lower levels in testes and eye, only rxfp3 - 3a3 exhibited relatively low expression in brain. relatively high levels of insl5a and insl5b mrna were found in intestine, but additionally insl5a expression was found in gonads and brain. our hypothesized candidate receptors for insl5a are rxfp3 - 3a1, rxfp3 - 3a2, and rxfp3 - 3b and for insl5b is rxfp3a3 (figure 4) : of the genes coding for these receptors, only rxfp3 - 3b showed high expression in the intestine (figure 8). to infer functional ligand - receptor relationships, we assessed the expression of both ligand and receptor genes in male and female zebrafish heart, intestine, gonads, muscle, gills, brain, and eyes using real - time, quantitative pcr. overall, the fold increase of the target to housekeeping genes, especially the receptors, was similar for both sexes in all tissues (except gonad) confirming the reliability of the data (figures s2 and s3). to allow comparison of the relative amounts of mrnas produced per tissue, the relative mrna expression levels were normalized to the total amount of rna isolated per tissue (figure 8). this revealed that for all tissues studied, the expression levels of all rxfp genes appeared to be higher than the expression levels of all rln / insl genes, except for the very high expression levels of insl3 in testis tissue (figure 8). the ligand rln was most abundantly expressed in gonads and male intestine (figures s2 and 8) ; its primary hypothesized receptor, rxfp1, was also highly expressed in gonads, as was a potential secondary candidate receptor, rxfp2a (figure 8). the rxfp1 transcript was also detected in male heart and brain, while rxfp2b expression was found in brain and eyes. expression of the zebrafish - specific rxfp2-like transcript, a candidate receptor for rln and insl3, was only found in brain at high levels. very high expression of insl3 mrna was found in testes and somewhat lower levels in ovaries and eyes. the primary candidate receptors for insl3 are rxfp2a and rxfp2b, and high expression of both rxfp2a and rxfp2b was observed in gonads, while rxfp2-like was not detected in testes or ovaries. as expected, rln3a and rln3b expression was found predominantly in brain and gonad, but we also identified rln3a expression in heart (figures s2 and 8). on the other hand, all of the rxfp3 - 1, rxfp3 - 2, and rxfp3 - 3 genes showed a similar expression pattern : high expression in brain with lower levels in testes and eye, only rxfp3 - 3a3 exhibited relatively low expression in brain. relatively high levels of insl5a and insl5b mrna were found in intestine, but additionally insl5a expression was found in gonads and brain. our hypothesized candidate receptors for insl5a are rxfp3 - 3a1, rxfp3 - 3a2, and rxfp3 - 3b and for insl5b is rxfp3a3 (figure 4) : of the genes coding for these receptors, only rxfp3 - 3b showed high expression in the intestine (figure 8). the main goal of this paper was to explore possible ligand - receptor pairings for the rln / insl - rxfp genes in teleosts. based on previous bioinformatic analyses, we describe how teleosts preferentially retained 2r- and 3r - derived paralogs of genes putatively involved in neuroendocrine functions (rln3/insl5-rxfp3/4), ultimately leading to a greater number (10 - 11) of receptor genes than ligands (6). given that the ligand - receptor pairings in teleosts are largely unknown, we employed selection and expression analyses to explore the possible ligand - receptor pairings. overall, the selection analyses showed that (1) the extent of purifying, neutral, and positive selection acting on the four rln - rxfp orthologs was highly similar between mammalian and teleost genes suggesting that, with the exception of mammalian rln, ligands and receptors have the same binding relationships in both lineages and (2) the ligand - receptor pairs rln3-rxfp3 and insl3-rxfp2 exhibited highly similar selection profiles suggesting close coevolution, while the pair insl5-rxfp4 exhibited a more diffuse coevolution, and rln - rxfp1 exhibited much faster evolution of the ligand in mammals than in teleosts. the overall similarity between the genes in teleosts and mammals is supported by the observation that all of the teleost ligand genes exhibit predominant expression in the same tissues as their orthologs in mammals : rln and insl3gonad, rln3brain and insl5intestine. however, even if the binding relationships are the same, it does not mean that the gene pairs have the same function in mammals and teleosts ; indeed, the branch - site test of positive selection suggests that differentiation in function has occurred between the two groups. secondly, although the binding relationships of the genes with orthologs in mammals and teleosts may be the same, it was difficult to resolve the ligand - receptor pairing relationships for the additional genes found in teleosts, but not in mammals. the rln3-rxfp3 system shows strong evidence of ligand - receptor coevolution with almost all amino acids being subject to purifying selection for both genes, and exhibiting a nearly perfect correlation in both mammals and teleosts. these findings are in accordance with previous studies and further support hypotheses about the highly conserved nature of the rln3-rxfp3 genes, and their probable parallel function across most vertebrates. however, teleosts possess two 3r - derived rln3 paralogs (rln3a and rln3b) and multiple rxfp3-type genes, not all of which are orthologous to mammalian rxfp3. based on the duplication history of the genes, we proposed that the rln3 peptide together with rxfp3 - 1 and rxfp3 - 2 receptors formed a tripartite ancestral teleost ligand - receptor signaling system (figure 3), and hypothesized that the after-3r subfunctionalization of the rln3 paralogs would be associated with subfunctionalization of the rxfp3 - 1 and rxfp3 - 2 receptor genes (figures 3 and 4). taking into account that in tetraodon nigroviridis the loss of rln3b coincides with the pseudogenization of rxfp3 - 1 (figure 3), we further propose that rln3b is a cognate ligand of rxfp3 - 1, while rln3a has specialized to function with two receptors, namely, rxfp3 - 2a and rxfp3 - 2b (figure 9). for example, experimental studies performed in zebrafish and eel indicate that the expression of the rln3 paralogs in fish shows strong homology to mammalian rln3, where they are predominantly expressed in the periaqueductal grey, a region homologous to ni in mammals. additionally, it is known that rln3a is expressed in a broader range of tissues (including gonad) than rln3b, indicating that rln3a and rln3b exhibit spatial (and perhaps temporal) subfunctionalization [8, 9, 14 ]. our expression analyses indicate both coexpression of the rln3 paralogs with rxfp3 - 1 and rxfp3 - 2 genes, and also possible subfunctionalization of the receptor since all of the rxfp3 - 1 and rxfp3 - 2 (and even rxfp3 - 3) genes are highly expressed in brain, while rxfp3 - 2a and rxfp3 - 2b are additionally expressed in the ovary, but at lower levels, mimicking the expression pattern of its candidate ligand, rln3a. resolving the ligand - receptor pairings for the insl5-rxfp4 system in teleosts we hypothesized that the rxfp3 - 3 and rxfp3 - 4 descendents (figure 1) are the potential receptors for insl5a and insl5b (figure 2, supplementary figure s3). specifically, we hypothesized that, in teleosts, rxfp3 - 3a1, rxfp3 - 3a2, and rxfp3 - 3b are candidate receptors for insl5a while rxfp3 - 4 (aka rxfp4) is the receptor for insl5b ; in zebrafish, the loss of rxfp3 - 4 was compensated by the gain of rxfp3 - 3a3 (figure 2), and the latter could serve as the receptor for insl5b (figures 3 and 4). despite this prediction, the selection and expression data provided little evidence for which receptors may bind to the two teleost insl5 paralogs (figure 9). the selection profile of teleost rxfp4 is the best match for that of both insl5a and insl5b, but all three rxfp3 - 3-type receptors are dominated by purifying selection and have selection profiles similar to those of rln3. on the other hand, the experimental data in zebrafish (which lacks rxfp4) indicate that insl5a is expressed in intestine and gonads and insl5b is expressed predominantly in intestine, and both paralogs exhibit low but significant expression in brain. this is consistent with the pattern in mammals, but the only receptor expressed at high levels in intestine was rxfp3 - 3b. the failure to find stronger evidence of coexpression of additional receptors for the insl5 paralogs may be caused, in part, by the endocrine action of insl5 and its expression in peripheral tissues [18, 24 ], many of which were not examined here, or possibly by developmental regulation of one or both of the insl5 paralogs. three of the other rxfp3 - 3 receptor genes, rxfp3 - 3a1, rxfp3 - 3a2, and rxfp3 - 3a3, were all additionally expressed in brain and male gonads, therefore if insl5a is a ligand for these receptors, teleosts may have expanded and subfunctionalized the role of the insl5 peptides involved in the hpg axis. further experimental work, including in situ hybridization, should be performed on insl5 and rxfp3 - 3 receptors in teleosts to thoroughly assess this hypothesis. furthermore, the coexpression of insl5- and rxfp3/4-type genes in a teleost species other than zebrafish should be performed since zebrafish possesses a slightly unique suite of genes (table s2), which did not allow for qpcr analyses of rxfp4. while teleosts exhibit a clear expansion of the rln / insl and rxfp genes involved in neuroendocrine pathways, the 3r duplicates of rln and insl3 and their corresponding rxfp1/2-type receptors expanded minimally. we find good support for the hypothesis that insl3-rxfp2 are ligand - receptor pairs in teleosts : their selection profiles are highly similar and, in zebrafish, which contain two rxfp2 paralogs (rxfp2a and rxfp2b), both receptor genes are highly expressed in gonads, although rxfp2b is additionally quite highly expressed in brain. previously, it was shown that insl3 expression in zebrafish shows strong parallels to that in mammals : in situ and qpcr analyses on male gonads reveal that it is expressed predominantly in leydig cells, and the more thorough qpcr analyses presented here further demonstrate that it is very abundantly expressed in male gonads, but also in female ovaries. current in situ analysis (underway in our laboratory) has also revealed the specificity of rxfp2a and rxfp2b expression in leydig cells (unpublished data). on the other hand, although rxfp2-like (which among teleosts is only present in zebrafish) has a similar selection profile to insl3, we found it to be predominantly expressed in brain, rendering interpretation difficulty, and we favor the hypothesis that rxfp2-like is an alternate receptor for rln (see figures 4 and 9). the only ligand - receptor pair for which there was a poor correlation in the nature of selection was rln - rxfp1 in mammals. while rxfp1 genes in mammals and teleosts have evolved in similar ways, the gene coding for the hormone relaxin, rln, has been subject to purifying and neutral evolution in teleosts, but has been the target of strong positive selection in mammals (see figure 5(c), table s3). in accordance with two recent studies showing the strong role of selection on the relaxin locus [25, 26 ], we find that approximately 50% of the codons in mammalian rln show evidence of positive selection, whereas no sites in teleost rln do. additionally, the qpcr expression pattern of rxfp1 in zebrafish shows broad but low levels of expression across multiple tissues, including gonad and brain. using rt - pcr and in situ analyses in zebrafish, donizetti. showed that expression of rxfp1 in zebrafish brain begins early in development and shows strong overlap with that of rxfp1 in humans. based on the similar amino acid sequence of rln and rln3 in teleosts, they propose that rxfp1 could be an additional receptor for rln3a and/or rln3b in teleosts. a study comparing the expression of rln3a, rln3b, and rln in eel using in situ and qpcr analyses found that the expression of teleost rln is similar to that of rln3, but with lower expression in brain and higher in gonads, similar to that observed in which expression this pattern is supported by our hypothesis for the evolution of the system in which the ancestral ligand molecule is hypothesized to have functioned in both reproductive and neuroendocrine pathways (figure 3). although we have focused on the similarities in the evolution of mammalian and teleost rln / insl - rxfp genes, the analysis of codon - specific positive selection revealed that mammalian and teleost genes have been subject to differential selection and that some receptor domains are the targets of more selection than others. for this analysis, sites were deemed to be subject to codon - specific selection if, when comparing a particular branch of the phylogenetic tree, there was evidence that certain amino acids were selected to be different from those in the background, we found that for the rxfp1/2-type genes, the ldla - lrr region generally showed high levels of selection, not surprisingly, since they are involved in receptor - ligand signaling. functional studies have shown that the lrr region is important for the binding of the cognate ligand ; the ldla module is essential for camp accumulation which takes place after the ligand is recognized and bound. apart from these regions, the only other two regions which were identified as having more than 20% of the sites subject to selection for rxfp1/2 genes were icl3 and ecl2. in general, lineage - specific selection was higher for the rxfp3/4-type genes : all domains were found to have more than 20% of the amino acids subject to positive selection except for four regions of the transmembrane domain (tm1, tm2, tm3, andtm7) and ecl1. of particular interest is the fact that for the rxfp3/4-type genes, icl1 is equally important as icl3 in terms of selection. the finding that icl3 (both receptor types) and icl1 (rxfp3/4-type receptors) are targets of selection suggests that a major component of selection for the rxfp receptors concerns downstream receptor signaling rather than selection for ligand binding per se. although the majority of the relaxin family genes originated prior to the divergence of osteichthyans, the fate of the family in teleosts and mammals is markedly different owing to the differential retention and diversification of genes in each lineage. earlier studies suggested that teleosts only possessed relaxin 3- and rxfp3-like genes and proposed that rln and insl3 were neurohormones that recruited their rxfp1/2-type receptors after the divergence of mammals, a view that is inconsistent with the data presented here and elsewhere [2, 8, 29, 30 ]. the goal of this study was to establish a theoretical background for further experimental work on the rln / insl - rxpf systems in teleosts. although the study was limited because its methodology relied on the known ligand - receptor pairings and expression data from mammals as a reference, our analyses suggest that the orthologs of the four 2r - derived ligand genes (rln, insl3, rln3, and insl5) have similar ligand - receptor pairings in teleosts and mammals (with the exception of the unusual situation with rln - rxfp1). despite these similar patterns, there is also evidence of differential selection on specific amino acids in mammalian versus teleost lineages, suggesting functional divergence in the two lineages. it is interesting that the rln / insl peptides diversified their reproductive functions in mammals, owing to local duplications at the relaxin locus [23, 25, 26, 29 ], while teleosts underwent a massive diversification of the genes believed to be involved in neuroendocrine regulation (rln3/insl5-rxpf3/4). overall, we find evidence that many of these additional receptor genes in teleosts have characteristics of the rln3-rxfp3 system, that is, slow evolution and predominant expression in the brain, while the primary receptors for the two insl5 paralogs in teleosts remain obscure. nevertheless, we find that teleosts greatly expanded and probably subfunctionalized the role of the rxfp3 - 2- and rxfp3 - 3-derived receptors ; their cognate ligands and their physiological functions should be the focus of future experimental work. we obtained sequences and performed an alignment based on the coding sequence for the rln / insl - rxfp genes from 5 teleosts (zebrafish, medaka, fugu, tetraodon, and stickleback) and 11 placental mammals (human, rhesus, cow, pig, horse, dog, guinea pig, mouse, rat, rabbit, and elephant) as described previously. the accession numbers of all genes are listed in tables s4 and s7 in yegorov and good, and the alignment is available upon request. we calculated the proportion of codons in ligand and receptor pairs estimated to be subject to purifying, neutral, or positive selection using the sites model in paml. next, to assess whether teleost ligand or receptor genes have been subject to adaptive divergent selection, we used several methods that examine the ratio of nonsynonymous to synonymous (dn / ds) substitutions. because ds provides an approximation of the neutral rate of substitution, = dn / ds ratios are used to determine selection pressure on genes or codon positions, with > 1 indicative of positive darwinian selection. site modelswe employed models that allow to vary among sites and tested a series of models to look for evidence of positive selection. first, we compared model m7 (beta) versus m8 (beta +) to test for evidence of positive selection and then compared model 8 versus model 8a to assess whether the evidence for positive selection was actually caused by a relaxation of purifying selection (or true positive selection) ; for both comparisons we used the site model tests in paml. likelihood ratio tests (lrts) were constructed to compare model m7 versus m8 and m8a versus m8. twice the log likelihood difference between models was compared with a chi - square distribution with number of degrees of freedom (df) calculated as the difference in the number of estimated parameters between models. model m8 was additionally used to identify codon sites under positive selection using a bayes empirical bayes (beb) criterion. we employed models that allow to vary among sites and tested a series of models to look for evidence of positive selection. first, we compared model m7 (beta) versus m8 (beta +) to test for evidence of positive selection and then compared model 8 versus model 8a to assess whether the evidence for positive selection was actually caused by a relaxation of purifying selection (or true positive selection) ; for both comparisons we used the site model tests in paml. likelihood ratio tests (lrts) were constructed to compare model m7 versus m8 and m8a versus m8. twice the log likelihood difference between models was compared with a chi - square distribution with number of degrees of freedom (df) calculated as the difference in the number of estimated parameters between models. model m8 was additionally used to identify codon sites under positive selection using a bayes empirical bayes (beb) criterion. branch - site modelswe hypothesized that at least some of the receptor genes may have experienced lineage - specific positive selection in mammals versus teleosts. to examine this we used the branch - site model a of zhang., which tests whether the members of a user - defined clade (branch) on a phylogenetic tree exhibit evidence of codon - specific selection relative to the remaining (background) lineages. tests of positive selection were made by comparing the branch - site model a in which (dn / ds) > 1 (alternative hypothesis) to the model a in which dn / ds = 1 fixed (null hypothesis) and by setting the foreground branch to the base of the clade containing the relaxin family ortholog in teleosts and the background to the same ortholog in mammals or tetrapods (depending on the tree structure) or vice versa. analysis of the branch - site model a was done using codeml from the paml package (paml v. 4.2) ; models were compared using the likelihood ratio test with 1 degree of freedom and, where significant, the posterior probability that a codon was under positive selection was estimated using the bayes empirical bayes (beb) procedure. we hypothesized that at least some of the receptor genes may have experienced lineage - specific positive selection in mammals versus teleosts. to examine this we used the branch - site model a of zhang., which tests whether the members of a user - defined clade (branch) on a phylogenetic tree exhibit evidence of codon - specific selection relative to the remaining (background) lineages. tests of positive selection were made by comparing the branch - site model a in which (dn / ds) > 1 (alternative hypothesis) to the model a in which dn / ds = 1 fixed (null hypothesis) and by setting the foreground branch to the base of the clade containing the relaxin family ortholog in teleosts and the background to the same ortholog in mammals or tetrapods (depending on the tree structure) or vice versa. analysis of the branch - site model a was done using codeml from the paml package (paml v. 4.2) ; models were compared using the likelihood ratio test with 1 degree of freedom and, where significant, the posterior probability that a codon was under positive selection was estimated using the bayes empirical bayes (beb) procedure. sexually mature male and female zebrafish (danio rerio) from the tbingen ab strain were used. animal housing and experimentation were consistent with dutch national regulations and were approved by the utrecht university animal use and care committee. rna isolation and cdna synthesisvarious tissues (heart, intestine, testis, ovary, muscle, gill, brain, and eye) were dissected from male and female adult zebrafish and immediately flash frozen in liquid nitrogen.tissue samples from 3 individual zebrafish, for each gender, were combined for each replicate and the rna was isolated using the fastrna pro green kit (bio 101 systems), according to the manufacturer 's recommendations. three independent rna isolations (biological replicates), each containing pooled tissues from 3 individual fish, were performed for each tissue per sex. possible genomic dna contamination was removed from each total rna fraction with the rnase - free dnase treatment & removal kit (ambion), which includes a final step to remove the dnase i from the reaction. next, cdna synthesis was performed with 2 g of each total rna samples, as described previously. various tissues (heart, intestine, testis, ovary, muscle, gill, brain, and eye) were dissected from male and female adult zebrafish and immediately flash frozen in liquid nitrogen.tissue samples from 3 individual zebrafish, for each gender, were combined for each replicate and the rna was isolated using the fastrna pro green kit (bio 101 systems), according to the manufacturer 's recommendations. three independent rna isolations (biological replicates), each containing pooled tissues from 3 individual fish, were performed for each tissue per sex. possible genomic dna contamination was removed from each total rna fraction with the rnase - free dnase treatment & removal kit (ambion), which includes a final step to remove the dnase i from the reaction. next, cdna synthesis was performed with 2 g of each total rna samples, as described previously. real - time, quantitative pcrprimers (table s6) for real - time, quantitative pcr (qpcr) to detect zebrafish rln / insl and rxfp mrnas were designed and validated for specificity and amplification efficiency on serial dilutions of testis cdna using sybr green - based assays (applied biosystems, foster city, ca, usa). all primers were designed on different exons, except for the primers detecting the rxfp3 cdnas, since all rxfp3 genes are single - exon genes. moreover, each qpcr run was followed by a melt curve analyses to exclude potential pcr amplifications from genomic dna contamination. to normalize the data, a taqman gene expression assay was acquired to detect the endogenous control rna, eukaryotic 18s ribosomal rna (applied biosystems). to examine the relative expression of genes across tissues, the relative fold change of the genes of interest was normalized to the 18s ribosomal rna reference gene and to a calibrator (calculated as the mean expression of all genes) (supplementary figures s2 (ligands) and s3 (receptors). all qpcrs and calculations (using the ct method) were performed as described previously [3537 ]. to compare the expression levels of all relaxin family peptide and receptor genes in whole zebrafish tissues, expression levels were additionally corrected for the total rna yield per tissue per sex (figure 8). primers (table s6) for real - time, quantitative pcr (qpcr) to detect zebrafish rln / insl and rxfp mrnas were designed and validated for specificity and amplification efficiency on serial dilutions of testis cdna using sybr green - based assays (applied biosystems, foster city, ca, usa). all primers were designed on different exons, except for the primers detecting the rxfp3 cdnas, since all rxfp3 genes are single - exon genes. moreover, each qpcr run was followed by a melt curve analyses to exclude potential pcr amplifications from genomic dna contamination. to normalize the data, a taqman gene expression assay was acquired to detect the endogenous control rna, eukaryotic 18s ribosomal rna (applied biosystems). to examine the relative expression of genes across tissues, the relative fold change of the genes of interest was normalized to the 18s ribosomal rna reference gene and to a calibrator (calculated as the mean expression of all genes) (supplementary figures s2 (ligands) and s3 (receptors). all qpcrs and calculations (using the ct method) were performed as described previously [3537 ]. to compare the expression levels of all relaxin family peptide and receptor genes in whole zebrafish tissues, expression levels were additionally corrected for the total rna yield per tissue per sex (figure 8). | relaxin - like peptides (rln / insl) play diverse roles in reproductive and neuroendocrine processes in placental mammals and are functionally associated with two distinct types of receptors (rxfp) for each respective function. the diversification of rln / insl and rxfp gene families in vertebrates was predominantly driven by whole genome duplications (2r and 3r). teleosts preferentially retained duplicates of genes putatively involved in neuroendocrine regulation, harboring a total of 10 - 11 receptors and 6 ligand genes, while most mammals have equal numbers of ligands and receptors. to date, the ligand - receptor relationships of teleost rln / insl peptides and their receptors have largely remained unexplored. here, we use selection analyses based on sequence data from 5 teleosts and qpcr expression data from zebrafish to explore possible ligand - receptor pairings in teleosts. we find support for the hypothesis that, with the exception of rln, which has undergone strong positive selection in mammalian lineages, the ligand and receptor genes shared between mammals and teleosts appear to have similar pairings. on the other hand, the teleost - specific receptors show evidence of subfunctionalization. overall, this study underscores the complexity of rln / insl and rxfp ligand - receptor interactions in teleosts and establishes theoretical background for further experimental work in nonmammals. |
the danish colorectal cancer group (dccg) is a subgroup of the danish surgical society. in 1994, dccg founded a national database with the aim to improve the quality of the diagnostic workup and treatment of danish patients with rectal cancer. since may 2001, the database has also included all danish patients with colon cancer.1 in 2006, dccg became a multidisciplinary cancer group under the auspices of the danish multidisciplinary cancer group (dmcg.dk),2 with participation of the following specialties : surgery, radiology, oncology, and pathology. until 2009, only the surgeons entered data into the database, but since the end of 2009, the pathologists have also entered data into the database. from the end of 2009 to 2013, the oncologists entered data manually by registering pre- and postoperative start of oncological treatment, but due to low data completeness, this was abandoned by the end of 2014. the aim of the database is to improve the prognosis for the danish patients with colorectal cancer by the following : 1) standardizing and improving the diagnostic workup of the patients with colorectal cancer, 2) standardizing and improving the surgical and oncological treatments, and 3) standardizing and improving the follow - up of the patients. this is achieved by monitoring the clinical quality indicators, which are published in annual reports, at department, regional, and national levels. the individual departments can monitor their own performance and extract their own data from the database, thus enabling local auditing of performance. the database of the study population is all danish citizens aged at least 18 years, diagnosed with primary colorectal cancer from may 2001 and onward, and diagnosed and/or treated at a surgical department at a public danish hospital. only patients with primary adenocarcinoma, mucinous adenocarcinoma, signet ring cell carcinoma, medullary carcinoma, or undifferentiated carcinoma are included. patients with metachronous colorectal cancer are not included, and data on recurrent disease are not recorded. every person in denmark is registered at birth by a unique identification number in the central civil registration registry.3 this number is also used by health registries, other health authorities (eg, the national patient registry4,5 and the cause of death registry6), and the dccg database giving the opportunity to have a virtually complete registration and follow - up in all contacts to the health system. since 2010, the patient completeness (the rate of patients with a surgical registration to all patients identified in the central registries) has been 99%, and previously, > 95%. to achieve such patient completeness based on the searches for patients with colorectal cancer in the national patient registry, the database provides lists to the surgical and pathology departments of patients who are not yet recorded in the database. since the dccg database does not include metachronous cancer and recurrences, it does not provide a complete picture of all the patients with colorectal cancer. in addition, there may be a small proportion of patients who were not included in the database. these include patients where a diagnosis of colorectal cancer is based on a death certificate only, or who have not been admitted to a surgical department, or for whom no biopsy exists. as of june 8, 2015, the database included 54,326 patients (35,922 with colon cancer and 18,404 with rectal cancer) with a completed surgical proforma. the database comprises a number of clinical, surgical, radiological, and pathological variables. the database uses an array of different data sources, the most important being a web - based proforma with manual online recording in a surgical and pathology proforma by the colorectal cancer surgeons and pathologists, respectively. the other data sources are listed in table 2. the surgeons record data about the patient lifestyle and comorbidity, performance, american society of anesthesiologists score,7 height, weight, and results of the clinical examination including radiology, bowel cancer screening, and multidisciplinary team meetings. variables regarding preoperative surgical and/or oncological treatment, the primary surgical procedure, operative details such as blood loss, division of tumor feeding arteries, tumor location, and operative approach, such as the type of intraoperative complications and postoperative surgical and nonsurgical complications, occurring within 30 days postoperatively, are also recorded. since 2014, dindo classification.8,9 the data includes a number of other important disease characteristics most important being variables about tumor stage according to union for international cancer control and ptnm stage.10 the union for international cancer control stage is calculated using an algorithm involving data from both the surgical and the pathology proforma. the database calculates a charlson comorbidity index11 based on the data from the national patient registry using an algorithm used by the danish multidisciplinary cancer group (dmcg. dk).2 other descriptive variables are 30-day postoperative mortality and overall survival using data from the central civil registration registry.3 the pathologists record data regarding the local excision specimens and the resection specimens (table 3). the pathology proforma includes variables such as histology including results of immunohistochemical staining for expression of mismatch repair proteins, tumor regression after neoadjuvant chemo- or radio- or chemoradiotherapy, surgical margin status, risk factors, such as tumor perforation, serosal involvement, venous invasion, and perineural spread, morphometric data, number of examined nodes and metastases, and the quality of the resection specimens. the pt, pn, and pv stages are calculated from the raw data entered into the pathology proforma. the key variables have been listed in table 3. due to poor data completeness regarding oncological data about adjuvant chemotherapy, the steering committee of the database has decided to abandon manual registration by the oncologists in 2014. in the future, the database will capture data about oncological treatment of the patients with colorectal cancer from the national patient registry. from 2016, the pathology data will come from the danish pathology registry, and the pathologists will no longer need to enter the data manually. the surgical proforma was upgraded in 2014 where new variables were introduced, variables were defined, and a number of old variables were excluded from the database. the proforma will undergo an annual revision in order to adapt to new diagnostic and treatment options. the database has planned a separate proforma to monitor the implementation of a new surgical procedure, transanal minimally invasive surgery. the registration in the database does not include registration of clinical follow - up data besides survival of the patients. thus, the database does not record follow - up data, such as local or distant recurrence, functional outcome, or quality of life. some clinical data might, however, be obtained by linkages to other relevant registries as described earlier. the database provides data to researchers and research groups after a formal application to the database and after evaluation by the dccg s scientific committee. the database has seen an increasing demand for its data, with 20 data sets provided to researchers in 2014. the database has provided data to > 70 publications and scientific reports as described in the annual reports.12 in 2014, the database undertook to analyze survival of danish patients with colorectal cancer, as part of a scientific report published by the danish multidisciplinary cancer group (dmcg.dk) benchmarking consortium.13 examples of research based on data from the database are many. noteworthy is the paper by andersen on intraoperative lesions of the ureter14 and a number of important papers by krarup,16 and bertelsen regarding anastomotic leakage, which is an area of focus in the database. the database has two quality indicators, regarding anastomotic leakage in both rectal and colon cancer surgery. another important publication based on data from the database is the publication by blow on intraoperative perforation of rectal cancer tumors.18 another focus area for the database is emergency surgery, where the risk of postoperative death is > 20%. the database has a steering committee with a chairman appointed by the board of the dccg. the database is funded by the danish regions21 and administered by the danish clinical registries (rkkp).22 rkkp administers all national cancer databases in denmark, including the dccg. the dccg database is a national database covering all newly diagnosed patients with colorectal cancer, with > 95% patient and data completeness. the database has an important positive impact on the quality of colorectal cancer treatment in denmark by monitoring an array of clinical quality indicators, such as postoperative mortality, lymph node yield, and anastomotic leakage. in the near future the database feeds data to an increasing number of researchers, providing important results and information about colorectal cancer treatment in denmark, as was done regarding the benchmarking report on survival after colorectal cancer in denmark.13 | aim of databasethe aim of the database, which has existed for registration of all patients with colorectal cancer in denmark since 2001, is to improve the prognosis for this patient group.study populationall danish patients with newly diagnosed colorectal cancer who are either diagnosed or treated in a surgical department of a public danish hospital.main variablesthe database comprises an array of surgical, radiological, oncological, and pathological variables. the surgeons record data such as diagnostics performed, including type and results of radiological examinations, lifestyle factors, comorbidity and performance, treatment including the surgical procedure, urgency of surgery, and intra- and postoperative complications within 30 days after surgery. the pathologists record data such as tumor type, number of lymph nodes and metastatic lymph nodes, surgical margin status, and other pathological risk factors.descriptive datathe database has had > 95% completeness in including patients with colorectal adenocarcinoma with > 54,000 patients registered so far with approximately one - third rectal cancers and two - third colon cancers and an overrepresentation of men among rectal cancer patients. the stage distribution has been more or less constant until 2014 with a tendency toward a lower rate of stage iv and higher rate of stage i after introduction of the national screening program in 2014. the 30-day mortality rate after elective surgery has been reduced from > 7% in 20012003 to < 2% since 2013.conclusionthe database is a national population - based clinical database with high patient and data completeness for the perioperative period. the resolution of data is high for description of the patient at the time of diagnosis, including comorbidities, and for characterizing diagnosis, surgical interventions, and short - term outcomes. the database does not have high - resolution oncological data and does not register recurrences after primary surgery. the danish colorectal cancer group provides high - quality data and has been documenting an increase in short- and long - term survivals since it started in 2001 for both patients with colon and rectal cancers. |
nearly 23.6 million people will die from cvds and coronary artery disease (cad) will become the leading cause of death by 2030 (1, 2). hyperinsulinemia resulted from insulin resistance is a valuable prediction factor in cad disease (3, 4). insulin resistance can increase blood fibrinogen (5) and is closely associated with salt - sensitive type of hypertension in obese and non - obese patients ; and therefore, can increase individual vulnerability to cardiovascular diseases (6). changes in lifestyle and also consumption of some dietary supplements have a critical role in managing of many chronic diseases (7). for example, diets reduced in total fat can prevent cvd (8) and high levels of monounsaturated fatty acids (mufa) and some polyunsaturated fatty acids (pufa) can reduce the risk of cvd (9). relatively, mediterranean diets rich in fish oil and other marine sources of omega 3 fatty acids can decrease the risk of chd mortality (10). omega 3 supplementation can improve endothelial function and decrease serum endothelial dysfunction markers such as e - selectin, i - cam and v - cam (11, 12). in some studies, consumption of omega 3 fatty acids was associated to increased risk of type 2 diabetes (13, 14) ; however, other studies did not show such an association (15, 16). omega 3 supplementation has resulted in a significant decrease in insulin resistance in several studies (17). this decrease seems to be linked to changes in adiponectin (18) and also activation of ampk pathway (19). vitamin e is an important nutrient and can develop beneficial effects in patients with heart diseases. vitamin e also is an anti - inflammatory agent and can decrease inflammatory factors such as il-1 and il-6 (20, 21). effects of omega 3 and vitamin e co - administration on insulin resistance and glucose homeostasis have been less described in previous studies. therefore, the current study was carried out to assess possible effects of omega 3 alone and combined omega 3 and vitamin e supplementations on serum lipid profile and glucose homeostasis in cad patients. sixty - five non - smoker male patients with cad were participated in this randomized double - blind placebo - controlled clinical trial. all participants had at least 50% stenosis in one coronary artery and were selected from the heart medical center of tehran, tehran, iran. furthermore, this study was registered in www.clinicaltrial.org under the registry number of nct02011906 and approved by the ethical committee of tehran university of medical sciences (i d : 23605). all patients had bmi 30 and none of them had history of kidney and liver disorders, diabetes and thyroid malfunction. patients were divided randomly into three groups including op group received 4 g / day of omega-3 fatty acids and vitamin e placebo, oe group received 4 g / day of omega-3 fatty acid soft gels and 400 iu of vitamin e, and pp group received omega-3 fatty acids and vitamin e placebos. the contents of dha and epa in each gram of omega 3 soft gels were respectively 120 and 180 mg. supplements and placebos used in this study were supplied by minoo pharmaceutical, cosmetic and hygienic company, iran. weight was measured closely to the nearest 0.1 kg with minimal clothing and no shoes. bmi was calculated as the weight in kg divided by the square of height in meter. waist to hip ratio (whr) was calculated by dividing the circumference of waist to the circumference of hip. dietary information of the patients was obtained using a 2-day food recall filled before the beginning and after finishing off the intervention. ten milliliters of blood were collected from the patients after 12 to14 h of overnight fasting. then, serum samples were separated and stored at 80 c until use. fasting serum glucose, total cholesterol, hdl - c, ldlc and triglyceride concentrations were assessed using commercial kits (pars azmoon, iran). serum insulin was assessed using elisa method (diametra, italy) with the sensitivity of 0.25 mciu / ml and homa - ir was calculated by fasting glucose (mg / dl) multiplied by fasting insulin (iu / ml)/405 (22). dietary data were analyzed using nutritionist iv software and statistical analysis was carried out using spss software v18. oneway analysis - of - variance (anova) test and paired t - test were used respectively to compare the mean of variables between and within the groups before and after the intervention. at the beginning of the study, 65 male cad patients were participated in the clinical trial. however, three patients were hospitalized for heart surgery during the intervention and two patients consumed less than 90% of the total supplements and hence, were excluded from the study. therefore, the study was carried out with 60 patients, as 20, 21 and 19 patients were included in oe, op and pp groups, respectively. patients in these groups were not statistically different from each other in mean ages and disease duration at the baseline of the study (p= 0.079 and p= 0.299, respectively). table 1 describes the baseline anthropometric measurements of the three groups at the beginning of intervention. no significant differences were seen between the means of weight, bmi and other anthropometric parameters of the study groups at the baseline. food recall analysis of the patients revealed that no significant differences existed between energy and macronutrient intakes in op, oe and pp groups at the baseline and at the end of the intervention. furthermore, the intakes of vitamin e and different fatty acids were not different between study groups and they were not changed their routine dietary patterns during the intervention. basic anthropometric parameters of the study groups before the intervention op, omega-3 fatty acid and placebo ; oe, omega-3 fatty acid and vitamin e ; pp, placebo and placebo ; bmi, body mass index ; whr ; waist to hip ratio ; dietary intakes of the study groups before and after the intervention oe, omega-3 fatty acid and vitamin e ; op, omega-3 fatty acid and placebo ; pp, placebo and placebo ; table 3 demonstrates fasting serum levels and homa - ir index in the study groups at the baseline and at the end of the supplementation. the patients were not statistically different in mean fbs at the end of the intervention. oe and op groups showed a significant decrease in fasting serum tg (p=0.020 and p=0.001, respectively). serum level of insulin and homa - ir decreased significantly in oe group (p=0.044 and p=0.039, respectively), but not in op or pp group. furthermore, the means of serum insulin level and homa - ir were significantly different between the study groups at the end of the intervention (p=0.032 and p=0.023, respectively). post - hoc analysis (tukey test) showed no significant differences between the mean of serum insulin level and homa - ir in oe and op groups (p=0.029 and p=0.019, respectively). serum levels of ldl, hdl and tc did not change significantly within or between the study groups. serum biochemical characteristics in the study groups before and after the intervention oe, omega-3 fatty acid and vitamin e ; op, omega-3 fatty acid and placebo ; pp, placebo and placebo ; hscrp, high - sensitivity c - reactive protein ; in the current study, omega 3 supplementation alone or in combination with vitamin e significantly decreased serum tg levels in oe and op groups. supplementation with omega 3 fatty acids can include a dose - dependent effect on serum triglycerides and reduce its concentration up to 30% (23, 24). these fatty acids seem to be able to inhibit the production of tg and apolipoprotein b by the liver (25). furthermore, these fatty acids can increase fatty acid oxidation by mitochondria (23). serum glucose levels have increased significantly in patients receiving omega 3 supplements alone ; however, the level was in normal range. this increase was not seen in oe group. therefore, the question was if omega 3 fatty acid supplements could include adverse effects on glucose homeostasis. effects of omega 3 fatty acids on serum glucose level have been conflicting in various studies. while, omega 3 supplementation was previously shown to adversely influence glucose homeostasis (26, 27), these fatty acids can increase glucose uptake and improve insulin sensitivity (28, 29). an experimental study on rats has demonstrated that consumption of fish oil can prevent insulin resistance induced by high - fat feeding (30). in contrast, results of the current study revealed that serum insulin level and homa - ir index did not change significantly in patients receiving omega 3 supplements only. however, the mechanism for this is not clearly explained, it is possibly associated to use of vitamin e. vitamin e can improve glucose homeostasis because it can influence insulin sensitivity (31, 32). indeed, oxidative stress is a key factor in development of insulin resistance and vitamin e with its ros scavenger ability can positively affect insulin action and glycemic control (33). another mechanism, which links vitamin e to insulin resistance, is the anti - inflammatory property of this vitamin. tnf- can increase adipocyte lipolysis and serine / threonine phosphorylation of irs-1 and il-1 can increase crp level via elevating il-6 and impair signaling of insulin in peripheral tissues which can result in reduced insulin sensitivity and impair insulin secretion (34, 35). both and -tocopherol forms of vitamin e include anti - inflammatory effects and can reduce serum level of crp (36). supplementation with combined omega 3 fatty acids and vitamin e includes beneficial effects on serum insulin level and homa - ir and co - administration of these supplements can be a good strategy against development of insulin resistance seen in cad patients. ethical issues (including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc.) have been completely observed by the authors. | background : omega 3 and vitamin e are two critical nutrients which include beneficial effects in coronary artery disease (cad). the aim of this study was to assess the effects of omega 3 alone supplementation or in combination with vitamin e on serum glucose and lipid levels and insulin resistance in cad patients.methods:participants of this clinical trial included 60 male patients with cad who selected from tehran heart center in tehran, iran in 2014. they received 4 g / day omega 3 plus 400 iu / day vitamin e (oe), 4 g / day omega 3 with vitamin e placebo (op), or omega 3 and vitamin e placebo (pp) for two months. serum glucose, lipids and insulin were assessed and homa - ir was calculated before and after the trial and effects of these nutrients on the highlighted parameters were compared within the study groups.results:serum glucose level increased significantly in op group (p=0.004), but not in oe group. oe and op groups showed a significant decrease in fasting serum tg (p=0.020 and p=0.001, respectively). serum insulin and homa - ir decreased significantly in oe group (p=0.044 and p=0.039, respectively) but did not change significantly in op group.conclusion:although, omega 3 supplementation may include adverse effects on serum glucose level, co - administration of omega 3 and vitamin e can beneficially decrease serum insulin and insulin resistance in cad patients. |
inflammation is a complex defense response of immune system, attempted to neutralize an insult and reestablish normal tissue structure and function. inflammation is characterized by redness, swelling, and pain and sometimes failure of function. proinflammatory cytokines are concerned with the upgrading of inflammatory reactions while anti - inflammatory cytokines dampen the proinflammatory cytokine response. chronic inflammation has been associated with different diseases, such as cancer, diabetes, cardiovascular disorders, pulmonary, and neurological diseases [26 ]. chronic inflammation is now well acknowledged as a threat feature for major types of cancer [711 ]. about 25% of all cancers are connected to chronic inflammation, which is linked to various stages of tumorigenesis including cellular transformation, tumor progression, endurance, propagation, invasion, angiogenesis, and metastasis [1214 ]. proinflammatory cytokines like chemokines, adhesion molecules, and inflammatory enzymes are known to cause chronic inflammation. several proinflammatory genes, for example, tumor necrosis factor (tnf) and members of its superfamily, il-1a, il-1b, il-6, il-8, il-18, chemokines, vegf, mmp-9, 5-lox, and cox-2, play critical role in the control of apoptosis, angiogenesis, proliferation, invasion, and metastasis. overexpression of transcription factors like nf-b, which becomes constitutively active in most tumors, is principally responsible for the expression of these genes [1517 ]. many inflammatory mediators such as interleukins, eicosanoids, and chemokines are able to motivate the propagation of both normal and cancer cells. clinical and epidemiological studies have suggested a strong connection between cancer, inflammation, and chronic infection [12, 1922 ]. proinflammatory mediators can contribute to tumor promotion and progression when produced intolerably and persistently because of events like aberrant epigenetic changes [12, 18, 23, 24 ]. epigenetic gene silencing is the transcriptional repression of specific genes throughout growth and cellular differentiation [26, 27 ]. the active or silent gene states are controlled by the processes of addition or removal of chemical modifications in the chromatin [28, 29 ]. these modifications include dna methylation and a variety of posttranslational histone modifications (acetylation, methylation, phosphorylation, etc.). these modifications are recognized by protein complexes that decide the fate of gene expression. in mammals, dna methylation mainly involves the attachment of methyl groups (ch3) to cytosine residues present at the cpg sites. hypermethylation of promoter regions of genes is typically associated with transcriptional silencing while hypomethylation facilitates gene expression. a number of reviews have been published on diverse aspects of cancer epigenetics, inflammation and the relationship between the chronic inflammation and cancer. the current review is focused on epigenetic regulation of different inflammatory cytokines involved in various human malignances with emphasis on aberrant methylation. epigenetic regulation is the mechanism by which chromatin undergoes multiple types of alterations including histone modifications and dna methylation. these modifications are important for chromatin remodeling and accessibility of transcriptional machinery to facilitate and regulate the transfer of genetic information. histone modifications (like acetylation and methylation, etc.) are important regulators of gene transcription. these can activate transcription by acetylation and are depending on the level of histone methylation and repress or inactivate transcription. these modifications have not only the ability to regulate the binding of effector molecules essential to dna processes including transcription, repair, and replication, but also the ability to regulate higher order chromatin structure and stability. therefore, it is not surprising that many chromatin - modifying enzymes are deranged during malignant transformation. histone acetylation of the lysine residues is regulated by enzymes that catalyze the addition or removal of acetyl group at numerous positions in histones. histone acetyltransferases (hats) and histone deacetyltransferases (hdats) are the enzymes that acetylate or deacetylate histones, respectively, and thus activate or suppress the transcriptional activity. however, exact mechanism of this gene regulation by histone acetylation is not well understood. it is believed that acetylation process neutralizes positively charged histones and reduces their interaction with the negatively charged dna thus loosening the chromatin structure (figure 1). histone methyltransferases (hmts) are the most recently discovered histone - modifying enzymes that regulate gene expression by histone methylation at specific sites. these enzymes catalyze the transfer of methyl groups to lysine and arginine residues of histones (h3 and h4). depending upon the number of methyl groups transferred and the histone residues involved, transcription of a gene can be suppressed or activated. dna methylation is the most widely studied mechanism of epigenetic modifications meant to regulate gene expression. it involves covalent attachment of a methyl group to the cytosine residue by the dna methyltransferase enzymes (dnmts). in dna methylation, cytosine is converted to methyl - cytosine at cpg (cytosine - phosphate - guanine) site. these cpg sites can span at an area of more than 200 bp (called cpg islands), are present in about 70% of human gene promoters, and are important modulators of gene transcription (figure 1) [3739 ]. the cpg islands are methylated on the inactive female x - chromosome and in certain other conditions like tissue specific and age - related genes. the aberrant methylation of the cpg islands especially in the promoter region of tumor suppressor genes are responsible for inducing cancer and related human diseases. along with being epigenetically regulated in malignancies, cytokines also affect the regulation of other genes. few studies report certain cytokines as epigenetic regulators of other genes having role in cancer initiation and progression. tgf- is an inflammatory cytokine having role in organ development, cellular differentiation apoptosis, and fibrosis. it regulates the expression of cd133 (gene symbol : prom1) in cancer stem cells (cscs) by dna methylation (figure 2). cd133 is used extensively as a stem cell marker for the identification of normal and cancer cells especially in liver. the high expression of cd133 by promoter demethylation adds to the resistance of tumor cells to chemotherapy and apoptosis. tgfb1 itself and its receptors (tgfbr1 and tgfgr2) are aberrantly regulated by promoter methylation in various cancers including gastric, breast, lung, and ovarian. downregulation of tgfb1 has also been linked with the paclitaxel resistance, a mitotic inhibitor used in cancer chemotherapy [4551 ]. suppressor of cytokine signaling 1 (socs1), a negative regulator of cytokine signaling and suppressor of inflammation related diseases, is also regulated epigenetically. socs1 promoter hypermethylation is one of the best - categorized epigenetic changes in macrophages and hepatocellular carcinoma [5254 ]. a recent study provides evidence that loss of socs1 expression inside tumor cells via promoter hypermethylation is strongly associated with overproduction of inflammatory cytokines like tnf- and il-6.. cxcl1/gro decreases the expression of extracellular matrix and plasma protein, fibulin-1d (gene symbol : fbln1) in prostate cancer cells. in highly invasive castration - resistant prostate cancer (crpc), cxcl1/gro signaling results in nuclear factor - kappa b (nf-b) activation that interacts with histone deacetylase 1 (hdac1) and form gene - silencing complex. this complex represses the expression of fibulin-1d by deacetylation of histones h3 and h4 on the nf-b - binding site of the fibulin-1d promoter. epigenetic changes can alter the pattern of normal gene expression thus resulting in pathological conditions including cancer.. these changes typically result in silencing of tumor suppressor genes or activation of tumor inducing genes. the transcriptional silencing usually involves hypermethylation of gene promoters while overexpression is a result of dna hypomethylation. various inflammatory cytokines are regulated aberrantly in human cancer via epigenetic mechanisms [5860 ]. although epigenetic regulation, especially aberrant dna methylation, is considered a common mechanism in tumorigenesis, there are only few studies regarding the role of epigenetic regulations of cytokines in malignancies. it is evident that inflammatory cytokines and chemokines, for example, tumor necrosis factor - alpha (tnf-), il-1 and il-6, and interferon gamma (ifn-), which can be produced by the tumor cells and/or tumor - associated leukocytes and platelets, may add directly to the development of malignancy. cytokines can also mediate the activities of immune cells in the fight against malignant cells. recent evidences show that chemokines and their receptors also play a critical role in neoplastic transformations, cancer progression, and angiogenesis, in addition to their role in development and inflammatory responses. cxcl14 it acts as a chemoattractant and serves immunity by stimulating trafficking of natural killer cells to the sites of inflammation or malignancy. several parallel studies show that cxcl14 mrna and protein are universally expressed in normal tissues but are absent in tumor cell lines and primary tumors [64, 65 ]. cxcl14 was found to be transcriptionally inactivated by promoter cpg hypermethylation in human prostate cancer, in vitro demethylation of the promoter in pc3 cells reexpressed the chemokine. aberrant methylation of cpg islands in promoter region and the first exon of the gene was associated with its downregulation in gastric cancer. cxcl14 is also known to control colorectal cancer by inhibiting migration and invasion by suppressing nf-b signaling. apart from gastric cancer cxcl14 is silenced in colorectal cancer by frequent methylation of the promoter region. another member of the same family, cxcl12, and its receptor cxcr4 are also associated with tumorigenesis. interaction between chemokines cxcl12 and cxcr4 promotes cellular adhesion, survival, proliferation, and migration. its upregulation is reported in skin, lung, pancreas, brain, and breast cancer, while, in pancreatic cancer and melanoma, cxcr4 is downregulated by promoter aberrant methylation [6971 ]. breast cancer cells, with upregulated cxcr4 genes, have been found engrossed to cxcl12 expressing cells in the lymph nodes, liver, and lungs, thus causing metastasis of disconnected tumor cells. epigenetic mechanisms like demethylation of cxcr4 and hypermethylation of cxcl12 and esr1 are a characteristic of tumor stage, size, metastasis, and poor overall survival. knowing the methylation status of both these genes can serve as a biomarker for diagnosis and prognosis in breast cancer. many proinflammatory cytokines including interleukins are frequently claimed to be epigenetically regulated in cancers, especially lung cancer [7577 ]. dna methylation at the promoters of il-1b, il-6, and il-8 genes and their consequential downregulation is reported in lung cancer. among them, il-1b promoter has the highest methylation status. in addition, il-23, a member of the il-6 superfamily that plays a key role in cancer, is also epigenetically modified in malignancies. it promotes inflammatory responses in the defense against pathogenic infection and upregulation of angiogenic factors and mmps. recently, interleukin-23 (il-23) is claimed to be epigenetically regulated in non - small - cell lung cancer (nsclc) by both histone acetylation and dna methylation. il-23a together with its receptor il-23r mediates inflammatory pathways ; the receptor is also regulated by epigenetic mechanisms in lung cancer. another member of the same family, il-12 is known to inhibit directly the growth of human lung adenocarcinoma. it holds a powerful antitumor potential, due to activation of combined immune stimulatory and antiangiogenic mechanisms [8284 ]. il-12 facilitates cytotoxic natural killer cells and induces the production of ifn- from nk and t cells. in addition, il-12 downregulates the production of the proangiogenic factors vegf and fgf-2 [8588 ]. il12rb2 gene encodes il-12r chain essential for the il-12 signal transduction [82, 90 ]. aberrant methylation of this gene sounds like a useful forecaster of long - standing result for adenocarcinoma of lung. il12rb2 methylation is also reported to be a more frequent in the patients suffering from both chronic obstructive pulmonary diseases (copd) and non - small - cell lung cancer (nsclc). ifn- is a pleiotropic cytokine secreted by type-1 helper (th1) t cells, cytotoxic t cells, and stimulated natural killer. production of ifn- is related to the induction of reaction in t lymphocytes, which contributes to enhancement of an immune response against malignant cells. ifn- (gene symbol : ifng) stimulates antitumor immune activity by inhibiting cell proliferation and sensitizes tumor cells to apoptosis [93, 94 ]. downregulation of ifng mediated by hypermethylation has been observed in lung and cervical cancer [95, 96 ]. human papillomavirus (hpv) is now a well - known risk factor involved in the progression of cervical cancer targeting keratinocytes which produces ifn-. a recent study reported that ifn- is suppressed in the presence of e6, a hpv protein, signifying the involvement of e6 in ifn- de novo methylation followed by transcriptional silencing. epigenetic therapy is emerging as an exciting, novel approach to treat a variety of diseases, particularly cancer. this therapy consists of using dna methylation inhibitors and hadc inhibitors for the reversal of the epigenetic aberrations inside the diseased genome. the reversal of aberrant gene methylation is more considerable to reversal of gene mutations or deletions. epigenetic drugs, whether demethylating agent or hdac inhibitor, target aberrantly heterochromatic regions, leading to reactivation of tumor suppressor genes and/or other genes that are vital for the normal cells. dna methylation inhibitors include 5-azacytidine (5-aza - cr), zebularine, 5-aza-2-deoxycytidine (5-aza - cdr), and 5-fluoro-2 deoxycytidine (5-f - cdr), and so forth. 5-aza - cr and 5-aza - cdr have been widely studied for the treatment of hematological diseases. hdac inhibitors include short chain fatty acids, hydroxamic acids, cyclic tetrapeptides and benzamides, each of them possesses different functional groups. many histone acetylases and deacetylases have been identified but their specific inhibitors still need to be investigated. these agents inhibit histone deacetylase enzymes, so histones remain acetylated and tailed by changes in cellular processes that were malfunctioning in malignant cells. unlike chemotherapeutic drugs, inhibition of dna methylation and consequently reactivation of genes, including apoptotic genes and cell - cycle regulators, finally lead the transformed cell to death and cell - cycle arrest. many investigators have combined dna - methylation inhibitors with hdac inhibitors and have shown synergistic tumour - cell - growth inhibition and gene reexpression [102, 103 ]. it might be advantageous to combine both drugs for the treatment of solid tumors because treatment with dna - methylation inhibitors alone in solid tumors is not sufficient. epigenetic targeting of different misregulated genes inside human malignancies is currently in phases i, ii, and iii clinical trials, to restore them back. considering the great potential of epigenetic therapy, there is a hope that in near future it will be possible to target the aberrant regulation of inflammatory cytokines including interleukins, interferons, and chemokines that are facilitating the malignancies. complete understanding of these epigenetic modifiers and their specific target cytokine will make development of the most effective therapies possible, not only to treat but also to prevent cancer. the literature cited in this review demonstrates that aberrant epigenetic regulation of diverse members of inflammatory cytokines inside different cancers is linked to tumor initiation, endurance, invasion, and progression in one way or the other. epigenetically regulated cytokines mediate the expression of tumor - associated genes and manipulate their biological role in cancer. targeting the reversal of aberrantly regulated cytokines could be a good potential target for cancer therapy and advanced research in this field could enable us to develop more efficient methods for cancer treatment. | inflammation is a multifaceted defense response of immune system against infection. chronic inflammation has been implicated as an imminent threat for major human malignancies and is directly linked to various steps involved in tumorigenesis. inflammatory cytokines, interleukins, interferons, transforming growth factors, chemokines, and adhesion molecules have been associated with chronic inflammation. numerous cytokines are reported to be aberrantly regulated by different epigenetic mechanisms like dna methylation and histone modifications in tumor tissues, contributing to pathogenesis of tumor in multiple ways. some of these cytokines also work as epigenetic regulators of other crucial genes in tumor biology, either directly or indirectly. such regulations are reported in lung, breast, cervical, gastric, colorectal, pancreatic, prostate, and head and neck cancers. epigenetics of inflammatory mediators in cancer is currently subject of extensive research. these investigations may help in understanding cancer biology and to develop effective therapeutic strategies. the purpose of this paper is to have a brief view of the aberrant regulation of inflammatory cytokines in human malignancies. |
mastocytosis is a heterogeneous group of myeloid neoplasms displaying abnormal proliferation and accumulation of mast cells in one or more organ systems including the skin, bone marrow, liver, spleen, lymph nodes and gastrointestinal tract. it may be associated with a mutation in the gene encoding the c - kit receptor. the cutaneous forms include urticaria pigmentosa (up), mastocytomas, diffuse cutaneous and telangiectasia macularis eruptiva perstans in their order of their frequency. it is difficult to determine the incidence of cutaneous mastocytosis (cm) due to its rarity, self - limiting nature in many cases and reluctance in reporting. some authors have extrapolated a frequency of about 5 - 10 new cases per million population and about one in every 1000 - 8000 dermatology out - patients. bullous mastocytosis is even rarer form of diffuse cm, most often associated with its generalized form. herein we report a rare case of bullous mastocytosis in a 3-month - old infant. this exclusively breastfed male child presented with multiple asymptomatic, brownish macules, plaques and vesico - pustular lesions over the whole body. the baby was asymptomatic until 10 days of life, when his mother noted fluid filled and crusted papules over the scalp. he was given oral and topical antibiotics without any improvement, and new lesions appeared in a generalized distribution over the next few weeks. furthermore, multiple brownish macules and plaques were observed de novo on the trunk and also at the sites of healed vesicular lesions [figures 1 and 2 ]. many of these plaques showed episodes of renewed activity in the form of bullous lesions that sometimes turned hemorrhagic over time [figure 3 ]. the parents did not observe any change in the lesions associated with rubbing of the skin or with temperature change. the child was not irritable and there were no symptoms suggestive of gastrointestinal upset, breathing difficulty, flushing, hemodynamic disturbance or failure to thrive. there was no significant antenatal history except that of oligamnios at 36 weeks necessitating cesarean section with a slightly low birth weight baby. the child had normal weight gain after birth and was weighed 8 kg at the time of presentation, normal for his age. he was the only sibling and the parents or other family members had no similar illness ever. brownish macules, tiny pustules and post - inflammatory changes generalized involvement seen on trunk typical hemorrhagic blisters on examination, the child was comfortable and alert, general physical and systemic examination revealed no apparent abnormality clinically. the mucocutaneous examination showed generalized involvement in the form of multiple, discrete, brownish macules and slightly raised plaques with velvety or nevoid surface of various shapes and size ranging from 0.5 to 3 cm. these lesions were interspersed with multiple vesicular, bullous, pustular or erosion - crusts, few of them surmounting the brownish background macules or plaques. there was no scarring, alopecia, milia formation, nail dystrophy or mucosal involvement. we considered cm as a strong possibility owing to typical morphology and positive darier 's sign. however, because of absence of itching and unusual widespread involvement, congenital epidermolysis bullosa was considered as a differential diagnosis despite there being no predilection for the appearance of lesions at trauma - prone sites. congenital syphilis and non - langerhans cell histocytosis a punch biopsy taken from a plaque over the forearm revealed a subepidermal split and dense infiltrate of monomorphic mast cells throughout the papillary, upper and mid reticular dermis [figure 4a and b ]. (a) subepidermal split in scanning view. (h and e, 20) (b) mast cells densely filling the dermis below the blister (h and e, 200) (a) positive giemsa stain. it is important to differentiate between cm, systemic mastocytosis (sm) and localized mastocytomas as their clinical behaviors and long - term outcome are diverse. the recent world health organization (who) classification (2008) defines major categories as cm, sm (indolent, aggressive and associated with clonal hematological non - mast cell lineage disease), mast cell leukemia (mcl), mast cell sarcoma and an extremely rare third major category of localized extracutaneous mastocytomas [table 1 ]. the diagnosis of cm is based on the clinical and histological findings in the skin together with the absence of criteria that would allow the diagnosis of sm. the definitive who diagnosis of sm requires the presence of one major and one minor criteria ; or three minor criteria. these are described in table 2. who classification (2008) of mastocytosis variants who criteria for systemic mastocytosis peripheral total and differential blood counts were in normal range in our patient. to confirm mastocytosis in bone marrow or in blood, the mast cell count should be more than 20% of the nucleated cells in the bone marrow or > 10% peripheral blood leukocytes. however, systemic investigations such as bone marrow aspiration / biopsy or serum total tryptase level could not be performed because of reluctance of the parents. however, they have been adhering to periodic follow - up evaluations for past six months and in the meantime, the patient could be successfully maintained on conservative treatment alone without the appearance of any systemic symptoms. parents were counseled in detail about the preventive measures related to baby care and drug avoidance. symptomatic control could be achieved by use of antihistamines and topical mid - potent corticosteroid creams. the prognosis and complications of cutaneous or indolent sm is definitely much better than aggressive systemic or leukemic variants. the important issues encountered in systemic variants, i.e. cytopenias, ascites, gastrointestinal disturbances, malabsorption, organomegaly, osteolysis, hemodynamic instability and malignant (leukemic) transformation are rarely associated with the cutaneous form. however, there have been reports of internal organ involvement in disease apparently limited to skin only. these can range from mild derangements like disturbed liver function tests to potentially life - threatening hemodynamic complications. the treating physician should therefore actively search for any probable systemic associations based on clinical clues in the individual case. extensive blistering, large surface area involved and high serum total tryptase level could be important markers for systemic involvement. these subtypes are also more prone for sudden death due to risk of sudden and massive mast cell degranulation which can lead to serious complications such as anaphylaxis, bronchoconstriction or cardiovascular collapse even after a small provocating factor like an insect bite or exposure to histamine releasing anesthetic agent. management of a patient with mastocytosis include alleviation of the symptoms and avoidance of potential mast cell degranulating stimuli such as heat, friction, sunlight, narcotics, alcohol, anticholinergic preparations, aspirin and other non - steroidal anti - inflammatory drugs, polymyxin b, local or systemic anesthetics and poisons like hymenoptera venom or bee bite. for control of symptoms, antihistamines, sodium chromoglycate, acetyl salicylic acid and ketotifen are used as needed in individual case. topical steroids, photochemotherapy, topical tacrolimus or pimecrolimus, topical miltefosine or intralesional injections of corticosteroids may also be useful. second - generation and newer tyrosine kinase inhibitors (e.g. dasatinib and midostaurin [pkc412 ]) are potential therapeutic options, which might have promising activity in sm cases. hematopoietic stem cell transplantation may induce remission in selected cases with advanced sm (i.e. aggressive sm and mcl). our patient responded well to oral antihistamines and topical steroid and had gradual reduction in blistering and erosions. however, since most of the case of pediatric mastocytosis resolve over time partially or completely, only the persistent disease may justify repeated bone marrow examination and aggressive systemic therapy. it could be concluded that pediatricians and dermatologists should remain aware of varied forms of cm because of its rarity and the distinctive management of each individual case. | mastocytosis is a rare myeloid neoplasm characterized by abnormal proliferation and accumulation of mast cells in one or more organ systems including the skin, bone marrow, liver, spleen, lymph nodes and gastrointestinal tract. an infant presenting with bullous lesions is an even rarer clinical presentation of cutaneous mastocytosis. the symptoms and complications are mostly in proportion to the mast cell degranulation in tissues. management is focused on preventing and treating this event. we report a three - month - old infant with bullous mastocytosis to enhance awareness about this rare diagnosis. |
the authors have attempted to measure the activity concentration of cs-137 in the natural environment of the gorce mts. the distribution of contamination by radioactive cs-137 in poland (south poland in particular) (cf. section deposition of cs-137 and [1, 1013 ]) and in its immediate vicinity (e.g. [2, 3 ]) is well known, but there is a lack of detailed data pertaining to the level and distribution of this contamination in smaller areas, covering only several hundred square kilometres. such investigations provide, on the one hand, information on the current distribution of the activity concentration, and on the other hand, may establish a point of reference in the future when dealing with the changes of this distribution in the course of time. moreover, baseline studies of this type are necessary because of the location of nuclear power plants in the immediate vicinity of poland and the anticipated construction of such a plant in poland around the year 2025. the gorce mts. situated in southern poland (fig., approximately 44 km in length, a width of approximately 15 km and an area of approximately 530 km. turbacz (1,310 m asl). in 1981, the central and north - eastern parts of the gorce mts. were declared the gorce national park, which presently has an area of around 70 km. the geological structure of the gorce mts. is made of folded and thrusted flysch formations : mainly the magura quartz sandstones cemented with silica (silicon oxide) or calcite (calcium carbonate), minor conglomerates and shales. the soils of the study area belong mainly to various brown soil sub - types, in the lower subalpine zone and various podzoilic soil sub - types in the upper subalpine zone. the cs-137 deposition values have been presented in numerical and graphic form and rounded up to 0.5 kbq / m. the border shown on the map (solid bold line) separates two areas of different cs-137 deposition deposition of cs-137 in the selected sites of the gorce mts. the cs-137 deposition values have been presented in numerical and graphic form and rounded up to 0.5 kbq / m. the border shown on the map (solid bold line) separates two areas of different cs-137 deposition the gorce mts. have been selected for the study as it is an area which has been practically unaffected by human economic activity, it is reflected, among others, in the presence of an intact surface soil layer and low level of contamination of the natural environment. additionally, as a large part of the study area is located within the gorce national park, it is most probable that this part of the gorce mts. will not be significantly affected by human economic activity in the future. therefore, it is a very good testing ground for long - term measurements of the dynamics of radioactive contamination. the study included : soil;lichens, because this species is commonly used as bioindicators of air pollution (cf.). the lichen hypogymnia physodes, often used in environmental studies, was chosen in our measurements. lichens, because this species is commonly used as bioindicators of air pollution (cf.). the lichen hypogymnia physodes, often used in environmental studies, was chosen in our measurements. sampling was carried out in the years 20012005 over the whole area of the gorce mts., in sites of dense forest complexes to minimise interference by human activity. in particular, the selected sampling sites differed in their absolute altitude (between 530 and 1,250 m asl.), slope inclination, morphology (valleys, slopes, ridges, etc.), and so on. they had to be representative of a study area and to cover it with possible regularity. a total of 74 samples of soil (49 samples) and lichen (25 samples) were collected. soil samples were taken from the top 10-cm layer in the form of cores with a diameter of 10 cm and a length of 10.0 cm (uncertainty 0.5 cm), using a pvc cutter. the core was next divided into three layers, according to the soil horizon types distinguished (organic, mineral). the samples were further prepared for radiometric measurements in accordance with iaea recommendations, applicable to environmental samples. larger rock fragments, exceeding a diameter of 3 mm, were discarded, the remaining material was dried to a constant weight at 105 c and ground to a grain size below 1 mm. the ground material was placed in cylindrical measuring vessels with a volume of 121 cm, the activity concentration of cs-137 (bq / kg) in the soil samples (layers) distinguished was measured, and the cs-137 deposition per 1 m (its activity concentration in the top 10-cm soil layer ; kbq / m) calculated. the lichen samples were collected from the bark of european beech and norwegian spruce trees at a height of between 0.5 and 1.5 m above the soil surface. this procedure should minimise the effect of dependence of the cs-137 concentration upon the height of the sampling site (cf.). the area in which samples were taken did not exceed 1 ha, while the mass of lichen samples ranged between single grams and several tens of grams. in the laboratory the next stage of cleaning was carried out under a magnifying glass and involved final removal of other lichen species and splitting the thallus of the lichen from the tree bark. for lichens the material collected was dried to a constant mass at around 70 c, and next, depending on the amount of the material, either ground to a grain size below 1 mm, or broken up by hand into fragments below 5 mm. the material was placed in cylindrical measuring vessels of a volume of 48 or 121 cm. the activity concentration was determined using a semiconductor hpge detector (canberra gx4020) with 42 % relative efficiency and resolution of 1.9 kev, placed in lead housing with walls 10 cm thick. the standard solution of gamma emitting radionuclides, was used as a calibration source ; the uncertainty of radionuclide activities should not exceed 2 %. the duration of measurements was generally chosen so that the relative uncertainty of the net count for 662 kev line (cs-137) was less than 3 %. in further calculation a detailed description of the methodology is set out elsewhere [7, 8 ]. the activity concentrations of cs-137 originating from global fallout and the chernobyl fallout were decay corrected to 1st july 2005. results for all sampling sites are presented in table 1 ; comprehensive results are contained in the final report of the study.table description of sampling sites, cs-137 deposition, cs-137 activity concentration in the soil and lichen h. physodes site nosite location coordinates altitude (m asl)gorce mts. part cs-137 deposition (kbq / m) cs-137 activity concentration (bq / kg d.m.)soil layer lichen 1st2nd3rd10 cm1maciejowa (f)493455n 200035e830nw10.537556910131443.01amaciejowa (g)493458n 200007e830nw5.11858690872rdzawka ponice (f)493312n 195921e770nw9.41259259821423stare wierchy (f)493344n 200256e1,030nw9.675323882721041.25redni wierch (f)493244n 200420e1,110nw15.83708484125116koninki / valley (f)493442n 200522e700nw7.04198118521048kocurka glade (f)493323n 200517e1,080nw11.02516761143069turbacz (f)493236n 200712e1,240nw13.33939472435399turbacz (f)493232n 200717e1,180nw7.6623226814919752.69ahala duga glade (g)493247n 200755e1,200se3.44697461679bhala duga glade (g)493242n 200746e1,200se4.5216038166321turbaczyk (f)493444n 200655e1,050nw7.1420113299687.722konina valley (f)493449n 200801e680nw17.137233220828632.923mostownica (f)493352n 200755e950se3.1613325134624lubomierz (f)493531n 201008e840nw8.6534360610525kosarzysko glade (f)493445n 201013e1,020se3.641655675126kudo (f)493418n 201035e1,250se6.0729425011719527podskale (f)493504n 201133e970nw11.914069531233327.729kiczora kamien. (f)493252n 200948e1,250se5.2834723514720543kamienica valley (f)493344n 201115e970se2.474238333650.745jaszcze due (f)493211n 201201e800se2.25872363047skaka (f)493323n 201343e1,050se5.944282031410948gorc (f)493353n 201453e1,110se3.10314106278173.149gorcowe (f)493245n 201614e750se7.27349833311361bukowina obidow. (f)493125n 200556e1,050se4.203336256865may kowaniec (f)493038n 200347e780se3.08161107123541.466jankwki (f)493133n 200814e1,050se7.392873945020445.868opuszna / valley (f)493026n 200750e730se2.18721569910627.269fordwki (f)493105n 201136e870se2.788430315154.980knurowska pass (f)492950n 201046e800se6.173791923512481studzionki (f)492941n 201260e890se3.5811120227720082szlembark (f)492919n 201248e780se4.61268119127464.683runek (f)492952n 201549e980se6.211994187331.784morgi glade (f)492931n 201800e1,000se2.344952324285mizerna / valley (f)492843n 201746e760se4.31187100439113.487lubanskie (f)493035n 202014e640se4.5118745157088luba (f)492922n 202029e1,180se7.593612889519845.488aluba (g)492918n 202029e1,190se4.493384836889kotelnica (f)492747n 202337e660se3.154939353969.891ochotnica stasichy (f)493014n 201431e730se6.402851846013092ochotnica kudows. (f)492858n 202340e530se4.93236781876 (f)forest, (g)glade ; kamien.kamieniecka, obidow.obidowska, waksm.waksmundzka, ochotn. ochotnickie, kudows.kudowskie, ziem.ziemianki uncertainty of the site location identification amounts to 80 m two contaminated gorce mts. parts distinguished by the authors : nw north - western part, se south - eastern part relative uncertainty of less than 10 % ; uncertainty of core height was included activity concentration in 1st, 2nd, 3rd soil layer and in a 10 cm soil layer (whole core) respectively ; relative uncertainty less than 5 % relative uncertainty less than 10 % results of the cs-137 analysis in the gorce mts. : description of sampling sites, cs-137 deposition, cs-137 activity concentration in the soil and lichen h. physodes (f)forest, (g)glade ; kamien.kamieniecka, obidow.obidowska, waksm.waksmundzka, ochotn. ochotnickie, kudows.kudowskie, ziem.ziemianki uncertainty of the site location identification amounts to 80 m two contaminated gorce mts. parts distinguished by the authors : nw north - western part, se south - eastern part relative uncertainty of less than 10 % ; uncertainty of core height was included activity concentration in 1st, 2nd, 3rd soil layer and in a 10 cm soil layer (whole core) respectively ; relative uncertainty less than 5 % relative uncertainty less than 10 % as the result of measurements, cs-137 activity concentration was determined in individual soil layers, and then the vertical distribution of cs-137 (depth profile). statistical parameters for each of the three layers are presented in table 2 ; due to the different cs-137 deposition in the gorce mts. next chapter), the data is presented separately for the two distinguished parts of the study area. the values presented in table 2 only provide information on the mean concentrations in individual layers, thus they should not be the used as a basis for estimation of concentration variability in soil profile.table 2cs-137 activity concentration in soil layerslayerrangemean / medianstandard deviationcvgorce mts.north-western part (bq / kg)(bq / kg)(bq / kg)(%)1. layer 6412154/11113386mean 87540253/21014859gorce mts.south-eastern part (bq / kg)(bq / kg)(bq / kg)(%)1 layer 327743/3153125mean 3020591/755358gorce mts.whole area (%) (%) (%) (%) 1 layer 167738/3314373. layer 26423/181670 number of samples : nw gorce mts. 34, gorce mts.whole area 49 cs-137 activity concentration in a successive soil layer mean activity concentration in a 10 cm soil layer percentage of a successive soil layer in cs-137 total activity in 10 cm soil layer cs-137 activity concentration in soil layers number of samples : nw gorce mts. 34, gorce mts.whole area 49 cs-137 activity concentration in a successive soil layer mean activity concentration in a 10 cm soil layer percentage of a successive soil layer in cs-137 total activity in 10 cm soil layer the change of cs-137 activity concentration in soil profile is not uniform. in the entire gorce mts., the profiles, whose concentration rapidly falls with depth (69 %) dominate, there are profiles in which the maximum concentration appears in the second layer (16 %), and also profiles in which concentration is more or less constant in all the layers (10 %). the total activity of cs-137 (bq) in individual layers was calculated based on the cs-137 activity concentrations in individual soil layers (bq / kg) ; results show that, on average, in the gorce : in the first layer there is approx. 39 % of cs-137 deposited per 10 cm soil layer, in the second 38 %, and in the third 23 %. based on the analysis of deposition of cs-137 in the selected sampling points, the authors distinguished two areas of the gorce mts. (cf. fig. 1 ; tables 1, 3) : south - eastern and north - western ; one has double the caesium contamination of the other. the deposition in the north - western part, which has a higher level of contamination, is in the range 4.5 - 17.1 kbq / m with an average of 9.9 kbq / m, while in the less contaminated south - eastern part deposition ranges between 2.2 and 7.6 kbq / m with an average of 4.4 kbq / m. the border of the two parts runs approximately along the line nowy targ mt. 1).table 3deposition of cs-137 and cs-137 activity concentration in h. physodes in the gorce mts.arearangeaverage/medianstandard deviationcvcs-137 deposition(kbq / m)(kbq / m)(kbq / m)(%)gorce mts.north-western part 4.517.19.9/9.73.636gorce mts.south-eastern part 2.27.64.4/4.41.535gorce mts.whole area 2.217.16.1/4.93.557mt. mostownica local variability 2.28.24.8/3.91.940 hypogymnia physodes (bq / kg)(bq / kg)(bq mostownica 19 number of samples : 25 deposition of cs-137 and cs-137 activity concentration in h. physodes in the gorce mts. mostownica 19 number of samples : 25 it seems that the reason for such significant differences in contamination in the two distinguished parts may be possibly due to the various meteorological conditions when radioactive contamination (radioactive cloud) from the chernobyl disaster spread in the atmosphere. however, the polish institute of meteorology and water management (imgw) data indicates that the impact of rainfall differences should probably be excluded. it was found that in the period when the chernobyl cloud was spreading over southern poland, i.e., between 28th april and mid - may 1986, the rainfall in the gorce mts. area was minimal (up to fifteen or so mm daily ; mainly on 30th april and between 8th may and 11th may) and no differences in the rainfall level between the two parts of the gorce mts. 2. as data is scarce, it is difficult to determine the nature of these distributions. nevertheless, it is characteristic that in each of the two distinguished parts of the gorce mts. se part has been presented twice : once at the bin width being the same as in the nw part (for comparison of the two parts), and again at the bin width being half of that in the nw part statistical distribution of cs-137 deposition in the gorce mts. se part has been presented twice : once at the bin width being the same as in the nw part (for comparison of the two parts), and again at the bin width being half of that in the nw part the relationship between the cs-137 deposition values and the absolute altitude of the sampling sites is shown for the south - eastern part of the study area (fig. 3) as more data was available just for this part of the gorce mts. the analysis of the graphs indicates that there is no basis to assume any dependence between cs-137 deposition and the absolute altitude.fig. each entry shows : the mean value and its uncertainty (longer horizontal line) and variability range (shorter horizontal line) dependence between the cs-137 deposition and the altitude of the sampling site. each entry shows : the mean value and its uncertainty (longer horizontal line) and variability range (shorter horizontal line) in order to compare the results obtained with those of other authors, the literature data has been decay - corrected to 1st july 2005. according to mietelski., the gorce mts. are situated on the border between the deposition areas 1.54.4 and 4.47.3 kbq / m. the radioecological map of poland elaborated using the in situ method assigns the gorce mts. to the area with a deposition of 05.9 kbq / m, while the average deposition in the then nowy scz voivodeship that encompassed the area of the gorce mts. the central laboratory for radiological protection (clor) carried out measurements in five villages situated in the gorce mts.. the deposition of cs-137 ranged between 0.7 and 2.0 kbq / m, while the average deposition in the maopolska voivodeship encompassing at that time the gorce mts. 30 km from gorce mts.) and obtained results between 0.2 and 17.5 kbq / m with an average of 5.6 kbq / m. comparison of the results obtained with the previous data is justified because the cs-137 deposition decreases only slightly faster than cs-137 decays (cs-137 t1/2 = 30 years). the average deposition of 9.9 kbq / m measured by the present authors in the north - western part of the gorce mts. is significantly higher than the values presented on the maps of cs-137 deposition in poland [1012 ]. this difference is probably mainly due to averaging the local values on these maps as the maps were constructed to reflect regional variations. therefore, they do not show small areas with a higher deposition of cs-137. when interpreting the cs-137 deposition data, its significant local variability should be taken into account. it is defined as the coefficient of variation (cv) of statistical distribution of the deposition in the case of multiple sampling points around one site. the author determined local variability of cs-137 deposition in 19 points on the 400 m slope located in the mt. (cf. fig. 4 and table 3). the deposition value varied in the interval 2.59.2 kbq / m, the mean value was 5.3 kbq / m, and cv 40 %. the cv value is slightly above the variability interval of the literature data oscillating (for an area of approximately 0.11 km) in the range 1030 % (cf. the cv value slightly higher than quoted by other authors, can be explained by the undulations of the gorce mts. area and the possibility of cs-137 accumulation in surface irregularities of the area ; as a result, there are points of significantly higher deposition and the cv value increases. an analysis of the data presented in table 3 indicates that the variability in the north - western and south - eastern parts of the gorce mts. 35 %) is comparable with the local variability (40 %). therefore, it may be assumed that each of the two parts is contaminated evenly, and the spreads of deposition values obtained in these parts for various sampling points (cf. 4cs-137 deposition value in 19 sampling points located on 400 m long slope profile of mt. values on the x - axis represent the distance of a sampling point from the centre of the investigated area ; the + and symbols, indicate that the point is located accordingly above or below the centre of the area cs-137 deposition value in 19 sampling points located on 400 m long slope profile of mt. values on the x - axis represent the distance of a sampling point from the centre of the investigated area ; the + and symbols, indicate that the point is located accordingly above or below the centre of the area the results of measurements of cs-137 activity concentration in h. physodes from selected sites in the gorce mts. the cs-137 activity concentration in lichens is in the range 1388 bq / kg d.m. the cs-137 activity concentration values measured are comparable to the literature data of around 100 bq / kg d.m. for samples of pseudevernia furfuracea and parmeliaceae family from south and south - eastern poland [17, 18 ]. the local variability (cf. turbacz (site no 9), where lichen samples were collected from ten trees (five beech and five spruce specimens). 50 % being comparable to the variability established over the whole of the gorce mts. area therefore, it may be accepted that the spreads of cs-137 activity concentrations in lichens, obtained for various sampling points in the gorce mts., one of the reasons for the high local variability of the cs-137 activity concentration in the lichen is the growth of the lichen resulting in two co - occurring types of the thallus in a single sample : the thallus highly contaminated shortly after the chernobyl disaster in 1986 (direct contamination) or the thallus contaminated by the former due to cs-137 leaching (indirect contamination),the thallus formed much later, i.e., in the period when the activity concentration of cs-137 in the air was lower by several orders of magnitude. the thallus highly contaminated shortly after the chernobyl disaster in 1986 (direct contamination) or the thallus contaminated by the former due to cs-137 leaching (indirect contamination), the thallus formed much later, i.e., in the period when the activity concentration of cs-137 in the air was lower by several orders of magnitude. to sum up, the cs-137 activity concentration in the lichen is currently due first of all to the contribution of the thallus contaminated shortly after the chernobyl disaster, to the total thallus volume sampled. as the result of measurements, cs-137 activity concentration was determined in individual soil layers, and then the vertical distribution of cs-137 (depth profile). statistical parameters for each of the three layers are presented in table 2 ; due to the different cs-137 deposition in the gorce mts. next chapter), the data is presented separately for the two distinguished parts of the study area. the values presented in table 2 only provide information on the mean concentrations in individual layers, thus they should not be the used as a basis for estimation of concentration variability in soil profile.table 2cs-137 activity concentration in soil layerslayerrangemean / medianstandard deviationcvgorce mts.north-western part (bq / kg)(bq / kg)(bq / kg)(%)1. layer 85533286/259122432. layer 60947405/332283703. layer 6412154/11113386mean 87540253/21014859gorce mts.south-eastern part (bq / kg)(bq / kg)(bq / kg)(%)1. layer 42428207/187113542. layer 327743/3153125mean 3020591/755358gorce mts.whole area (%) (%) (%) (%) 1. 34, gorce mts.whole area 49 cs-137 activity concentration in a successive soil layer mean activity concentration in a 10 cm soil layer percentage of a successive soil layer in cs-137 total activity in 10 cm soil layer cs-137 activity concentration in soil layers number of samples : nw gorce mts. 34, gorce mts.whole area 49 cs-137 activity concentration in a successive soil layer mean activity concentration in a 10 cm soil layer percentage of a successive soil layer in cs-137 total activity in 10 cm soil layer the change of cs-137 activity concentration in soil profile is not uniform. in the entire gorce mts., the profiles, whose concentration rapidly falls with depth (69 %) dominate, there are profiles in which the maximum concentration appears in the second layer (16 %), and also profiles in which concentration is more or less constant in all the layers (10 %). the total activity of cs-137 (bq) in individual layers was calculated based on the cs-137 activity concentrations in individual soil layers (bq / kg) ; results show that, on average, in the gorce : in the first layer there is approx. 39 % of cs-137 deposited per 10 cm soil layer, in the second 38 %, and in the third 23 %. based on the analysis of deposition of cs-137 in the selected sampling points, the authors distinguished two areas of the gorce mts. (cf. fig. 1 ; tables 1, 3) : south - eastern and north - western ; one has double the caesium contamination of the other. the deposition in the north - western part, which has a higher level of contamination, is in the range 4.5 - 17.1 kbq / m with an average of 9.9 kbq / m, while in the less contaminated south - eastern part deposition ranges between 2.2 and 7.6 kbq / m with an average of 4.4 kbq / m. the border of the two parts runs approximately along the line nowy targ mt. turbacz 1).table 3deposition of cs-137 and cs-137 activity concentration in h. physodes in the gorce mts.arearangeaverage/medianstandard deviationcvcs-137 deposition(kbq / m)(kbq / m)(kbq / m)(%)gorce mts.north-western part 4.517.19.9/9.73.636gorce mts.south-eastern part 2.27.64.4/4.41.535gorce mts.whole area 2.217.16.1/4.93.557mt. mostownica 19 number of samples : 25 deposition of cs-137 and cs-137 activity concentration in h. physodes in the gorce mts. mostownica 19 number of samples : 25 it seems that the reason for such significant differences in contamination in the two distinguished parts may be possibly due to the various meteorological conditions when radioactive contamination (radioactive cloud) from the chernobyl disaster spread in the atmosphere. however, the polish institute of meteorology and water management (imgw) data indicates that the impact of rainfall differences should probably be excluded. it was found that in the period when the chernobyl cloud was spreading over southern poland, i.e., between 28th april and mid - may 1986, the rainfall in the gorce mts. area was minimal (up to fifteen or so mm daily ; mainly on 30th april and between 8th may and 11th may) and no differences in the rainfall level between the two parts of the gorce mts. were identified. the statistical distribution of the cs-137 deposition in the whole gorce mts. and 2. as data is scarce, it is difficult to determine the nature of these distributions. nevertheless, it is characteristic that in each of the two distinguished parts of the gorce mts. se part has been presented twice : once at the bin width being the same as in the nw part (for comparison of the two parts), and again at the bin width being half of that in the nw part statistical distribution of cs-137 deposition in the gorce mts. se part has been presented twice : once at the bin width being the same as in the nw part (for comparison of the two parts), and again at the bin width being half of that in the nw part the relationship between the cs-137 deposition values and the absolute altitude of the sampling sites is shown for the south - eastern part of the study area (fig. 3) as more data was available just for this part of the gorce mts. the analysis of the graphs indicates that there is no basis to assume any dependence between cs-137 deposition and the absolute altitude.fig. each entry shows : the mean value and its uncertainty (longer horizontal line) and variability range (shorter horizontal line) dependence between the cs-137 deposition and the altitude of the sampling site. each entry shows : the mean value and its uncertainty (longer horizontal line) and variability range (shorter horizontal line) in order to compare the results obtained with those of other authors, the literature data has been decay - corrected to 1st july 2005. according to mietelski., the gorce mts. are situated on the border between the deposition areas 1.54.4 and 4.47.3 kbq / m. the radioecological map of poland elaborated using the in situ method assigns the gorce mts. to the area with a deposition of 05.9 kbq / m, while the average deposition in the then nowy scz voivodeship that encompassed the area of the gorce mts. the central laboratory for radiological protection (clor) carried out measurements in five villages situated in the gorce mts.. the deposition of cs-137 ranged between 0.7 and 2.0 kbq / m, while the average deposition in the maopolska voivodeship encompassing at that time the gorce mts. 30 km from gorce mts.) and obtained results between 0.2 and 17.5 kbq / m with an average of 5.6 kbq / m. comparison of the results obtained with the previous data is justified because the cs-137 deposition decreases only slightly faster than cs-137 decays (cs-137 t1/2 = 30 years). the average deposition of 9.9 kbq / m measured by the present authors in the north - western part of the gorce mts. is significantly higher than the values presented on the maps of cs-137 deposition in poland [1012 ]. this difference is probably mainly due to averaging the local values on these maps as the maps were constructed to reflect regional variations. therefore, they do not show small areas with a higher deposition of cs-137. when interpreting the cs-137 deposition data, its significant local variability should be taken into account it is defined as the coefficient of variation (cv) of statistical distribution of the deposition in the case of multiple sampling points around one site. the author determined local variability of cs-137 deposition in 19 points on the 400 m slope located in the mt. (cf. fig. 4 and table 3). the deposition value varied in the interval 2.59.2 kbq / m, the mean value was 5.3 kbq / m, and cv 40 %. the cv value is slightly above the variability interval of the literature data oscillating (for an area of approximately 0.11 km) in the range 1030 % (cf. the cv value slightly higher than quoted by other authors, can be explained by the undulations of the gorce mts. area and the possibility of cs-137 accumulation in surface irregularities of the area ; as a result, there are points of significantly higher deposition and the cv value increases. an analysis of the data presented in table 3 indicates that the variability in the north - western and south - eastern parts of the gorce mts. 35 %) is comparable with the local variability (40 %). therefore, it may be assumed that each of the two parts is contaminated evenly, and the spreads of deposition values obtained in these parts for various sampling points (cf. fig. 1), are associated with the local variability of the deposition.fig. 4cs-137 deposition value in 19 sampling points located on 400 m long slope profile of mt. values on the x - axis represent the distance of a sampling point from the centre of the investigated area ; the + and symbols, indicate that the point is located accordingly above or below the centre of the area cs-137 deposition value in 19 sampling points located on 400 m long slope profile of mt. values on the x - axis represent the distance of a sampling point from the centre of the investigated area ; the + and symbols, indicate that the point is located accordingly above or below the centre of the area the results of measurements of cs-137 activity concentration in h. physodes from selected sites in the gorce mts. are presented in table 3. the cs-137 activity concentration in lichens is in the range 1388 bq / kg d.m. the cs-137 activity concentration values measured are comparable to the literature data of around 100 bq / kg d.m. for samples of pseudevernia furfuracea and parmeliaceae family from south and south - eastern poland [17, 18 ]. the local variability (cf.) has been estimated in the sampling site (an area of approx turbacz (site no 9), where lichen samples were collected from ten trees (five beech and five spruce specimens). 50 % being comparable to the variability established over the whole of the gorce mts. area (cv = 38 %). therefore, it may be accepted that the spreads of cs-137 activity concentrations in lichens, obtained for various sampling points in the gorce mts., are associated with the local variability. in the opinion of the present authors, one of the reasons for the high local variability of the cs-137 activity concentration in the lichen is the growth of the lichen resulting in two co - occurring types of the thallus in a single sample : the thallus highly contaminated shortly after the chernobyl disaster in 1986 (direct contamination) or the thallus contaminated by the former due to cs-137 leaching (indirect contamination),the thallus formed much later, i.e., in the period when the activity concentration of cs-137 in the air was lower by several orders of magnitude. the thallus highly contaminated shortly after the chernobyl disaster in 1986 (direct contamination) or the thallus contaminated by the former due to cs-137 leaching (indirect contamination), the thallus formed much later, i.e., in the period when the activity concentration of cs-137 in the air was lower by several orders of magnitude. to sum up, the cs-137 activity concentration in the lichen is currently due first of all to the contribution of the thallus contaminated shortly after the chernobyl disaster, to the total thallus volume sampled. (several hundred km), the authors established in detail the activity concentration of cs-137 and its spatial distribution in soils and lichen. such investigations provide, on the one hand, information on the current distribution of the caesium contamination, and on the other hand, may establish a point of reference in future changes of this distribution in the course of time. two parts of the gorce mts. have been distinguished on the basis of different deposition of cs-137. the respective average values are 4.4 and 9.9 kbq / m as at 1st july 2005, providing a relative difference of around 2. the value 9.9 kbq / m is much higher than the values given in the maps of cs-137 deposition in poland. the average cs-137 activity concentration in the h. physodes from the gorce mts. is 47 bq / kg d.m. when interpreting this data, significant local variability of measured quantities (cs-137 deposition, cs-137 activity concentration in lichen) should be taken into account. this local variability amounts to several dozen percent and is comparable with the variability calculated for the whole area of the gorce mts. thus, there are no grounds to draw isolines of measured quantities on the map of the gorce mts. | concentration of activity of cs-137 and its spatial distribution in soils and lichen hypogymnia physodes were determined in the gorce mts. (several hundred km2) in s poland. the authors distinguished two areas of the gorce mts. on the basis of markedly different cs-137 depositions, whose respective average values are 4.4 and 9.9 kbq / m2 as at 1st july 2005. the average cs-137 activity concentration in the lichen h. physodes from the gorce is 47 bq / kg d.m. a significant local variability of quantities measured amounts to a few dozen percent was found. |
over the past 15 years, u.s. has witnessed a general decline in overall rates of youth homicide. nonetheless, youth violence rates remain high in this country, with homicide being the third leading cause of death among persons aged 1024 years. youth violence can affect communities by substantially increasing the cost of health care, reducing productivity, and diminishing property values. in 2000, it was estimated that the medical care and lost productivity costs associated with youth violence were more than $ 70 billion. youth violence has been linked to a variety of factors including individual, family, community, and societal characteristics. community - level risk can have negative influences even on youth who are not exposed to individual- or family - level risk factors. major community risk factors for violence include high density of alcohol outlets, community norms favorable toward violence, residential instability, transitions and mobility, low neighborhood attachment, social disorganization, the presence of gangs, and extreme economic deprivation [5, 6 ]. although much research has been conducted on interventions to change the characteristics of individuals and families, less has focused on evaluating interventions and policies designed to change community economic conditions or characteristics of the physical environment. studies have been conducted on the impact of interventions on individual - level variables ; however, evaluations at the community level have been sparse. some research is emerging that highlights the promise of community- and policy - level strategies in preventing youth violence. for example, modifications to the built environment such as improvements to the pedestrian environment and architectural changes can contribute to relative reductions in 911 calls and crimes, possibly through increases in social capital and intolerance of criminal activity [7, 8 ]. it is theorized that large - scale economic developments, such as sports and entertainment arenas and casinos, improve the living conditions, economics, public health, and overall wellbeing of area residents and may influence rates of violence within communities [9, 10 ]. local government and business developers often suggest that building an arena or a casino in a neighborhood may provide potential societal benefits as a result of the construction and the business drawn in ; thus, many new arenas are placed in areas of needed economic growth. however, scientific research to support these ideas is scarce, and there is much debate about the true benefits of economic developments. research consistently suggests that economically disadvantaged neighborhoods have poorer public health outcomes, including higher rates of violent crime, chronic disease, and risky behavior, and that there is large heterogeneity with regard to these factors across neighborhoods within a city [11, 12 ]. according to siegfried and zimbalist, independent work on the economic impact of stadiums and arenas has uniformly found that there is no statistically significant positive correlation between sports facility construction and economic development. whether casino development causes economic growth in surrounding neighborhoods is a complex question, and the literature is not consistent in its conclusions [9, 10 ]. the few studies that have assessed economic growth looked at large geographic areas such as the county or state. for example, describes that after the licensing of casinos in monaco, nevada, and atlantic city, these cities grew dramatically economically and became destinations for tourism. also, few studies have looked at the effects of casinos on crime and delinquency, but, of those that have, the findings have been inconsistent [15, 16 ]. there is a need for a more focused, community - level survey of resident perceptions regarding the effect of these major community development projects on perceived safety, violence, and economic benefits. a consistent literature suggests that the perception of crime is often different from actual crime. for instance, in a study conducted in australia, results suggested that people often exaggerated the risks of becoming a victim of crime. this was corroborated in two subsequent studies. in another study conducted in new zealand, crime in an individual 's own neighborhood influenced fear of crime, but crime occurring in neighboring communities had little effect on perceived safety. this difference in perception is referred to as the paradox of fear [20, 21 ]. age, gender, and race have been demonstrated to affect the differences between the perception of crime and actual crime. for example, the elderly, women, and racial and ethnic minorities have been shown as having higher perceptions of crime than actual risk [23, 24 ]. in the current study, we have a greater percent of elderly female minorities, explaining in part this discrepancy. when taken into context with other individual factors, this has been referred to as the vulnerability perspective. this idea emphasizes that fear is highest when individuals perceive themselves to be vulnerable. in a 2010 study, investigators examined resident 's perception of crime based on the neighborhood in which they live. the results suggested that perceived disorder of neighborhood structure, including social cohesion, was strongly associated with perception of crime even after controlling for race, age, and gender. in pittsburgh, pennsylvania, two large economic developments were recently constructed in two historically disadvantaged minority neighborhoods. in august 2009, gambling was legalized in pittsburgh, and a casino was opened in the north side neighborhood. the consol energy center (cec), an indoor sports and entertainment facility, was opened in the hill district neighborhood in august 2010. these community - level changes provide a unique opportunity to study the potential effect of two different community economic development efforts to examine whether the economic benefits directly have an effect on perceptions and rates of community violence. specifically, the current study has three objectives : describing residents ' perceptions of the effect of the arena and casino on neighborhood violence, safety, and economic benefits.describing residents ' perceptions of change in neighborhood violence, safety, and economic benefits after the opening of the arena and casino.comparing the above residents ' perceptions with census and police data over the same time.results from this study will fill some of the existing gaps in the field around the relationship between community economic development efforts and community violence while highlighting some of the potential mechanisms through which they may have an effect (e.g., job availability for community residents). describing residents ' perceptions of the effect of the arena and casino on neighborhood violence, safety, and economic benefits. describing residents ' perceptions of change in neighborhood violence, safety, and economic benefits after the opening of the arena and casino. comparing the above residents ' perceptions with census and police data over the same time. we conducted a telephone survey in 2011 using a listed sample of randomly selected telephone numbers in each of six neighborhoods of pittsburgh (figure 1). the neighborhoods included the north side, where the casino was built in 2009, as well as the hill district, where the entertainment arena was built in 2010. one adjacent neighborhood for each of these communities was also included to assess whether any potential benefits spilled over into nearby areas. north oakland was the neighboring community for the hill district, and an area consisting of spring garden, fineview, spring hill - city view, and perry south (hereafter collectively referred to as spring garden) was the neighboring community for the north side. two additional neighborhoods were also examined and were intended to serve as comparisons to the economic development communities : squirrel hill is a neighborhood of high socioeconomic status (ses) and low violent crime rates, and homewood is a neighborhood of lower ses and higher levels of violent crime. interviews were conducted between july and december 2011 and lasted for an average of 25 minutes in length. survey questions gauged demographic variables, employment history, neighborhood factors, and relationships / interactions with both the casino and the arena. respondents were categorized by the neighborhood in which they resided based on self - report. in addition, respondents were asked to identify the closest intersection to validate neighborhood assignment. respondents were asked if they felt safe in their neighborhood both during the day and at night. respondents were asked whether, compared with 2006, they thought there was more violent crime. employed respondents were identified as those who were employed for wages / salary, self - employed, student, or a combination of those ; unemployed respondents were identified as those who responded respondents were also asked if their household income had changed since 2006, whether the rivers casino affected businesses in their neighborhood, and whether they thought either development affected employment or income in their neighborhood. the analyses were conducted using spss, version 21, software (spss inc., we conducted descriptive analyses to examine the various measures of perceived violence and safety and economic benefit. pearson 's chi - square tests determined significant differences in proportions for all neighborhoods. if significant at the p < 0.05 level, we then ran pairwise comparisons of column proportions using the bonferroni correction. we only present comparisons for both the hill and the north side compared with each other and the other neighborhoods given that these were our a priori comparisons of interest. table 1 shows the participants ' demographic data. in general, the participants were older than the average age of the surveyed communities, with a mean of 64 15 years of age. the participants were primarily female (69.8%) and white (62.1%), though this varied by neighborhood. two notable exceptions are the hill district and homewood, where around 90% of participants were identified as black (see table 2 for census figures). in table 3, we show statistically significant differences between the hill district and the north side. squirrel hill residents were significantly more likely to report feeling safe during the day compared with the hill and the north side (99.5% versus 90.6% and 92.4% resp., the percentage of participants in squirrel hill who felt safe at night was higher (91.7%, p < 0.05), while the percentage in homewood was lower (48.3%, p < 0.05) compared with both the hill and the north side. participants from the hill district, the north side, and spring garden agreed with the statement violence is common in my neighborhood at similar rates (ranging from 32.1 to 42.9%) while far fewer respondents from north oakland and squirrel hill (8.6% and 1.5%, resp., p < 0.05) agreed with the statement compared with the hill and the north side, and 62.1% of participants from homewood agreed (p < 0.05). similarly, participants from north oakland and squirrel hill agreed with the statement compared with 2006, there is more violent crime in my neighborhood now at the lowest rates (13.7% and 2.4%, resp., p < 0.05) compared with the hill and the north side and participants from the remaining four neighborhoods agreed at rates ranging from 29.3 to 41.8%. squirrel hill had the least amount of recorded violence in 2011 (0.6 violent crimes per 1000 residents), followed by oakland (2.6), spring garden (10.5), hill district (12.1), north side (15.1), and homewood with the most amount of recorded violence (18.1) when looking at actual violent crime rates in these neighborhoods (see figure 2). all neighborhoods except spring garden experienced less violent crime in 2011 than in 2006 ; spring garden saw a slight increase (+ 0.4). participants in the north side stated more often that the rivers casino has had a positive impact on their life (14.1%) compared with 7.2% of participants from the north side who responded that the casino has had a negative impact. participants in squirrel hill were more likely to not report positive effects compared with the north side (p < 0.05). participants from both north oakland and squirrel hill were statistically much less likely to report an effect on economic issues compared with the north side (p < 0.05, table 3). when asked, how has the rivers casino affected crime in your neighborhood ?, the only statistical difference was for squirrel hill, with no one reporting that the casino had a positive effect on crime in his or her neighborhood (p < 0.05). in summary, the north side appears to have benefitted more from the casino than any other neighborhoods, but this benefit was only reported by a minority of north side participants. this is reflected in responses to the question overall, how has the rivers casino affected your neighborhood ? the highest rate of responses indicating a positive effect was observed in the north side, at 24.5%, while the majority of participants in each neighborhood responded neither positively nor negatively the less disadvantaged neighborhoods, north oakland and squirrel hill, were the only neighborhoods with significant differences. when asked, how has the consol energy center impacted your life ?, the majority of participants in each neighborhood answered neither positively nor negatively (69.6% of participants from the hill district, where the arena is located, and around 80% of participants in all other neighborhoods). when asked, how has the consol energy center affected income in your neighborhood ?, the most common answer across all neighborhoods was neither positively nor negatively 31.9% of participants from the hill district reported that the consol energy center had a positive effect on employment in their neighborhood, and 24.6% of participants from the hill district reported that the consol energy center had a positive effect on local businesses in their neighborhood. participants from every other neighborhood reported statistically significantly lower proportions of positive effects on the economic questions compared to the hill (p < 0.05). in response to the question how has the consol energy center affected crime in your neighborhood ?, the majority of participants in each neighborhood responded neither positively nor negatively (68.6% of participants from the hill district), followed by the response do not know / unsure (18.8% of participants from the hill district). responses to the question how has the consol energy center affected violence in your neighborhood ? followed a similar pattern, with 68.6% of participants from the hill district responding neither positively nor negatively and 17.4% of participants from the hill district responding do not know / unsure. in summary, participants from the hill district reported benefitting the most from the consol energy center. when asked, overall, how has the consol energy center affected your neighborhood ?, 32.9% of participants from the hill district reported that the arena had a positive effect while 46.4% of respondents reported that the arena had neither a positive nor a negative effect, and 9.7% reported a negative effect. participants in all other neighborhoods reported statistically significantly lower levels of positive effects in comparison (p < 0.05). community economic development is one strategy that may lead to more resources and opportunities within neighborhoods. this, in turn, may result in reductions in community - level rates of violence. this study addressed the relationship between two large economic development efforts within the city of pittsburgh a casino and a sports arena on perceptions of economic opportunities and community safety. overall, we found that residents in neighborhoods with the large - scale economic developments reported more development in specific economic benefits than did residents from other neighborhoods. a large proportion of hill district respondents thought violent crime had increased since 2006 even though it actually decreased more in the hill district than in any other survey neighborhoods. in addition, hill district respondents felt less safe than north side respondents did, even though the north side experiences more crime than the hill district. if more crime in the north side is being committed by nonresidents in the entertainment districts than crime in the hill district being committed by nonresidents coming into the hill, the north side residents may actually be experiencing less violence near their homes than hill district residents. when participants ' perceptions are compared with the actual violent crime rates for their neighborhoods, they often matched. participants from squirrel hill were most likely to say they felt safe during the day or night and were least likely to believe that violence is common in their neighborhood while participants from homewood were least likely to say they felt safe during the day or night and were most likely to believe that violence is common in their neighborhood. accordingly, the 2011 violent crime rates indicate that squirrel hill experienced the least amount of violence (0.6 violent crimes per 1000 residents) of the six neighborhoods survey while homewood experienced the most (18.1 violent crimes per 1000 residents). it should be noted, however, that although the majority of participants from homewood agreed that violence is common in their neighborhood and indeed it is thirty times more common in homewood than squirrel hill, the majority of participants did not feel unsafe, even at night. participants were also mostly correct in their perceptions about the change in violence since 2006. the majority from every neighborhood did not believe that there was more violence now than in 2006, and indeed violent crime rates have decreased in every neighborhood except spring garden, which saw a slight increase. interestingly, however, the rate of violent crime in spring garden in 2011 was at its lowest since 2006 due to a spike in 2007, so it may have been difficult for participants in that neighborhood to differentiate between the slight increase in violence compared with 2006 and the decrease in violence seen almost every year since 2007. even though homewood and the hill district had the greatest declines in violence from 2006 to 2011 of all survey neighborhoods, this was not evident in the participant responses in these neighborhoods when the perception of crime is taken into context with other individual factors, it has been referred to as the vulnerability perspective. this idea emphasizes that fear is highest when individuals perceive themselves to be vulnerable. in a 2010 study, investigators examined resident 's perception of crime based on the neighborhood in which they live. the results suggested that perceived disorder of neighborhood structure, including social cohesion, was strongly associated with perception of crime even after controlling for race, age, and gender. two major criminological theories on crime, the broken windows hypothesis and the collective efficacy perspective [29, 30 ], are related to both actual crime and the perception of crime. social cohesion measures mutual trust among residents and is thought to reduce community problems including fear of crime. previous studies have shown that concentrated disadvantage within communities, as measured by poverty, unemployment, and family disruption, is associated with fear of crime [26, 29, 31 ]. as in our study, low levels of social cohesion may lead to more crime [28, 32 ], which may in turn reduce cohesion among residents. there are likely differences in our results between perception and reality of crime because of individual characteristics, including age, gender, and race, as well as neighborhood context, which has been shown to affect individuals ' perception of crime. surprisingly, there has been a dearth of research examining whether casinos positively benefit community residents. the few studies that have assessed economic growth looked at large geographic areas, such as county or state, rather than areas directly surrounding the developments. an evaluation of the economic impact of casinos by eadington used historical perspective to demonstrate the potential economic benefits. the author cited monaco, nevada, and atlantic city as places that, before the commercialization of legalized gambling, were experiencing economic deterioration. however, after the licensing of casinos in these areas, they economically prospered and became destinations for tourism. other groups studied the causal impact of casino gambling profits and per capita income and found that there was no evidence for causation of casino profits impacting per capita income in two studies [10, 35 ]. both evaluations were conducted at a state level where data were aggregated for all 11 states that allowed casino gambling on any level. our results begin to fill this gap in the literature by examining residents ' specific perceptions of the impact of area developments and providing a glimpse into how the developments affect individuals as well as communities. however, others theorize that economic growth often leaks outside of the community as profits and jobs are exported out of the community [9, 10 ]. we found that the highest rate of respondents stating that the rivers casino has had a positive impact on their life was in the neighborhood where the casino is located ; a similar pattern was observed for the neighborhood in which the arena is located. additionally, the results of the economic benefit questions underlined these findings. residents of the neighborhoods in which the casino and arena are located reported the greatest positive effects on their income. there are a large number of studies that have assessed the effect of large - scale developments on violence. however, these are mostly related to casinos, and the results are varied. in a study by grinols and mustard, casino and noncasino counties property crime levels were evaluated for two years before the opening and five years following the opening. the results of the study showed that there was an increase in crime after the opening of a casino. further, park and stokowski compared four types of counties based on predominant type of recreation / tourism attraction offered, including casinos, and found that total arrest rate was highest in casino counties but this was not statistically significant. stokowski found that gaming counties in colorado had higher rates of property crimes but not violent crimes. research has also indicated that economic crimes and public order crimes increased in biloxi, ms, after introduction of casino gaming [15, 37 ], and disorderly conducted arrests increased after introduction of casino gaming in davenport, iowa. our results, while limited to residents ' perceptions of violence, further support the finding that violence increases after the opening of large - scale developments, in this case a casino. we found that residents in the neighborhoods with the developments reported greater changes in violence. the differences in results across studies are likely caused by differences in measures of violence and size of the geographic area studied. more research is needed in this area with particular emphasis on using consistent definitions, measures, and methodology. interestingly, in the grinols and mustard study, the increase in violent crime occurred most noticeably after a three - year lag. first, in the initial years following the opening of a casino, more community resources, such as funding for labor of police, are at their highest and this in turn reduces crime. additionally, the investigators cite data concerning the effects of addictive gamblers. our results do not contain data after 2 years for the casino and 1 year for the arena, but these findings provide important implications to consider in future research. there is a large amount of heterogeneity between communities which demonstrates the need to assess how casinos may affect different types of neighborhoods differently. for example, kang. studied residents ' perceptions of the impact of limited - stakes community - based casino gambling in colorado in 2006. perceived positive economic impacts from the casino influenced residents ' support of the casino, while perceived negative social impacts did not. our sample consisted largely of older women, and our findings may not be representative of all age groups, particularly young men and women who may be more likely to visit, attend events at, and work at the casino and arena, likely due in part to only including landlines. further research should examine objective data in combination with subjective data regarding residents ' perceptions. this initial work is important in identifying how neighborhood residents perceive the large - scale economic developments in these neighborhoods. the potential benefits to a community are directly related to public health outcomes as well as public policy. more in - depth studies should be conducted to assess long - term effects as well as the social pathways through which these effects occur. | background. emerging research highlights the promise of community- and policy - level strategies in preventing youth violence. large - scale economic developments, such as sports and entertainment arenas and casinos, may improve the living conditions, economics, public health, and overall wellbeing of area residents and may influence rates of violence within communities. objective. to assess the effect of community economic development efforts on neighborhood residents ' perceptions on violence, safety, and economic benefits. methods. telephone survey in 2011 using a listed sample of randomly selected numbers in six pittsburgh neighborhoods. descriptive analyses examined measures of perceived violence and safety and economic benefit. responses were compared across neighborhoods using chi - square tests for multiple comparisons. survey results were compared to census and police data. results. residents in neighborhoods with the large - scale economic developments reported more casino - specific and arena - specific economic benefits. however, 42% of participants in the neighborhood with the entertainment arena felt there was an increase in crime, and 29% of respondents from the neighborhood with the casino felt there was an increase. in contrast, crime decreased in both neighborhoods. conclusions. large - scale economic developments have a direct influence on the perception of violence, despite actual violence rates. |
paraumbilical hernia occurs as a result of congenital incomplete closure of the fetal umbilical defect. they present as a lump and/or swelling commonly associated with abdominal discomfort and the majority are reducible ; however, strangulation is not uncommon. the most common content of the sac is omentum, bowel loop and surgical repair remains the only definitive treatment [1, 2 ]. appendicitis within the hernia sac has been reported in the inguinal (amyand hernia) and femoral region (de garengeot hernia) ; however, their presence in the paraumbilical hernia is extremely rare and a search of the literature demonstrated three previous perorated cases. herein, we would like to report a case of a successfully treated acute appendicitis presenting in a paraumbilical hernia in an 84-year - old lady with 6-month follow - up. an 84-year - old lady with a past medical history of hypertension, ischemic heart disease, congestive heart failure and hypercholesterolemia presented with 2-day history of abdominal pain and nausea. on examination she was found to have a raised c - reactive protein of 97.5 mg / dl and a white cell count of 8.9 10 with otherwise normal hematologic markers. computed tomography (ct) of the abdomen demonstrated cecum and appendix in the hernia sac. this was associated with mild degrees of edema, stranding and presence of mesentery (figs 1 and 2). figure 1:sagittal slice of the ct demonstrating the paraumbilical sac containing cecum and appendix along with fat stranding and air loculation and narrow neck (red arrow). figure 2:coronal and axial slice of the ct showing the paraumbilical hernia (within marked areas). sagittal slice of the ct demonstrating the paraumbilical sac containing cecum and appendix along with fat stranding and air loculation and narrow neck (red arrow). coronal and axial slice of the ct showing the paraumbilical hernia (within marked areas). the decision was made to operate and the patient was taken to the operating room for the repair of the paraumbilical hernia under general anesthesia. a midline incision (mini laparotomy) therefore, a mayo repair with full reduction of the viable cecum back into the abdominal cavity following sac excision and appendectomy was performed. the patient made an uneventful recovery and was discharged 5 days later with no postoperative complication on 6-month follow - up. acute appendicitis remains the most common surgical emergency to date and their presentation can vary, however, the majority present with the classical symptoms of central to right iliac fossa abdominal pain, nausea, anorexia and vomiting. this is related to the anatomical position of the appendix from developmental phase of intestinal rotation. review of n = 8692 cases of acute appendicitis, demonstrated that 0.13% of all appendicitis could present within the incisional, inguinal, femoral and obturator hernia. the incidence of appendicitis in a paraumbilical hernia remains unknown and an extensive search of literature in the english language highlighted only three prior cases that presented with peroration and pus collection. it remains unclear how the cecum and appendix can freely mobilize to the midline and present themselves within the sac of a paraumbilical hernia. it has been suggested that in 10% of the population, there might be an anatomical variation and abnormal cecum mobility otherwise referred to as mobile cecum syndrome. in such individuals, the lateral peritoneal attachment of cecum is either absent or so mobile that terminal ileum and cecum can be found in any quadrant of the abdomen depending on the position and activity of the patient. in addition, acute inflammatory response (appendicitis) and subsequent intraabdominal events (localization, adhesion and abscess) and the presence of a prior defect (paraumbilical) could explain such presentation. another explanation in such circumstances is appendicitis as a result of extrinsic pressure, strangulation and necrosis within the hernia defect. however, it is difficult to establish which pathophysiological process (or perhaps both) could have attributed to this rare presentation. clinical examination remains vital in such cases (irreducible hernia) and arguably ct remains the best investigative modality of the choice in doubtful and complicated circumstances specially when there is a suspicion of malignancy due to old age. however, the only definitive treatment is surgery for both appendicitis and strangulated hernia, and detailed examination of the sac contents remains crucial before their reduction into the abdominal cavity. following an appendectomy, primary repair over mesh repair due to later incidences of mesh infection is highly recommended. | paraumbilical hernia sac usually contains omentum, bowel loop and rarely appendicular epiploicae, metastatic deposits and vermiform appendix. presentation of acute appendicitis in a paraumbilical hernia is rare and limited to few case reports in the literature. herein, we would like to report a case of a successfully treated acute appendicitis presenting in a paraumbilical hernia in an 84-year - old lady with 6-month follow - up. |
intraocular pressure (iop) is still one of the most important modifiable risk factors for glaucoma progression, even in glaucomas where the pressure is normal or low. a single iop measurement in the clinic is not an accurate depiction of the pressure profile of glaucoma patients, as it does not account for the influence of circadian rhythm and nocturnal posturing on iop. various studies have documented iop fluctuations throughout the day and night using intermittent iop measurements taken a few hours apart but it is still uncertain whether it is the frequency of iop peaks or the range of iop fluctuation that leads to glaucoma progression. selective laser trabeculoplasty (slt) is a non - invasive, repeatable, and effective modality of iop reduction for open angle glaucoma. it has a similar efficacy to anti - glaucoma eye drops and the former argon laser trabeculoplasty and a recent study has affirmed slt 's efficacy in normal tension glaucoma (ntg), reducing the iop by an additional 20% while using 27% less medication at 6 months compared with pre - treatment levels. in terms of iop fluctuation, kothy reported reduced iop fluctuation over a 24-hour period after slt and nagar found that slt 's ability to reduced diurnal iop variation was inferior to latanoprost. prasad showed that there was less inter - visit iop fluctuation after 360 slt treatment compared to 180 slt treatment. the majority of studies investigating the effects of iop reduction after slt only measured iop over a few sampling periods or during clinical visits. the sensimed triggerfish (sensimed ag, lausanne, switzerland) is based on a wireless silicon contact lens sensor (cls). the device allows for the recording of the iop related pattern over a 24-hour period with minimal disturbance to one 's daily routines and sleep cycle. the cls records iop - related changes through the detection of biodimensional changes in the corneoscleral area, for 30 seconds every 5 minutes over 24 hours. each recording burst represents 300 data points, of which the median is plotted as a single graph of the 24-hour iop - related profile. it has been shown to be safe and well tolerated during 24-hour recording of iop - related patterns in healthy subjects, glaucoma suspects, and glaucoma patients. the purpose of the study was to analyze the iop - related fluctuations using the cls before and after adjuvant slt in subjects with ntg who were treated with topical anti - glaucoma medications. informed patient consent and approval by the institutional review board were obtained prior to study commencement. this study was supported by the provision of sensimed triggerfish cls and other device supporting items by sensimed. this was a prospective cohort study from july 2012 to june 2013, conducted at a queen mary hospital, a university hospital in hong kong special administrative region, china. the study recruited consenting adults (age > 18 years old) with unilateral or bilateral ntg who were currently on topical anti - glaucoma medications. ntg was defined by open angle on gonioscopy, progressive thinning of the retinal nerve fiber layer (rfnl) on the spectralis (heidelberg engineering gmbh, heidelberg, germany) optical coherence tomography, and an iop 90 mveq.sleep-to-wake and wake - to - sleep slopes. the calculation of sleep - to - wake and wake - to - sleep slopes has been described previously by mansouri reduction in iop after slt was calculated based on the gat iop measured at baseline, 1 month, and 3 months after slt. these gat iop readings were taken at approximately 3 pm each visit and taken before the cls wear at the baseline and 1-month post - slt visits. cls increase and decrease rates (change in cls units / change in time) : maximum, minimum, median, and mean. number of peaks : over 24 hours, 90 mveq. sleep - to - wake and wake - to - sleep slopes. the calculation of sleep - to - wake and wake - to - sleep slopes has been described previously by mansouri the reduction in iop after slt was calculated based on the gat iop measured at baseline, 1 month, and 3 months after slt. these gat iop readings were taken at approximately 3 pm each visit and taken before the cls wear at the baseline and 1-month post - slt visits. the differences between the measured parameters detailed above, were compared before and after slt using the paired t - test or wilcoxon matched pairs test depending on the normality of the variable distribution. statistical significance was taken as p 0.05 and all means were expressed as mean standard deviation. local variability : cls iop - related variability was measured over 24 hours, diurnally, and nocturnally. this was a measure of local variability of the raw cls data from the smoothed function obtained using a locally weighted polynomial regression method (shown below). this parameter reflects the error of the smoothed function, or the amount of information that is missed by the smoothed values. where t is the number of cls measurements over the recording period, tfo is the observed cls signal, and tfp is the predicted cls signal based on the smoothing function selected.global variability : cosinor modeling of 24-hour cls patterns was performed using the below formula. the cosinor model represents the actual amplitude of iop - related fluctuation over a 24-hour period and is most representative of iop - related changes where y is the observed signal in mveq, t is the time, and b0, b1, and b2 are the regression coefficients, estimated from the data.slt success rates : gat iop reductions 20% from the pre - slt levels while on the same anti - glaucoma regimen at 1-month post - slt. local variability : cls iop - related variability was measured over 24 hours, diurnally, and nocturnally. this was a measure of local variability of the raw cls data from the smoothed function obtained using a locally weighted polynomial regression method (shown below). this parameter reflects the error of the smoothed function, or the amount of information that is missed by the smoothed values. where t is the number of cls measurements over the recording period, tfo is the observed cls signal, and tfp is the predicted cls signal based on the smoothing function selected. global variability : cosinor modeling of 24-hour cls patterns was performed using the below formula. the cosinor model represents the actual amplitude of iop - related fluctuation over a 24-hour period and is most representative of iop - related changes where y is the observed signal in mveq, t is the time, and b0, b1, and b2 are the regression coefficients, estimated from the data. slt success rates : gat iop reductions 20% from the pre - slt levels while on the same anti - glaucoma regimen at 1-month post - slt. cls increase and decrease rates (change in cls units / change in time) : maximum, minimum, median, and mean.number of peaks : over 24 hours, 90 mveq.sleep-to-wake and wake - to - sleep slopes. the calculation of sleep - to - wake and wake - to - sleep slopes has been described previously by mansouri reduction in iop after slt was calculated based on the gat iop measured at baseline, 1 month, and 3 months after slt. these gat iop readings were taken at approximately 3 pm each visit and taken before the cls wear at the baseline and 1-month post - slt visits. cls increase and decrease rates (change in cls units / change in time) : maximum, minimum, median, and mean. number of peaks : over 24 hours, 90 mveq. sleep - to - wake and wake - to - sleep slopes. the calculation of sleep - to - wake and wake - to - sleep slopes has been described previously by mansouri the reduction in iop after slt was calculated based on the gat iop measured at baseline, 1 month, and 3 months after slt. these gat iop readings were taken at approximately 3 pm each visit and taken before the cls wear at the baseline and 1-month post - slt visits. all statistical calculations were done using spss version 18.0 (spss, inc., chicago, il). the differences between the measured parameters detailed above, were compared before and after slt using the paired t - test or wilcoxon matched pairs test depending on the normality of the variable distribution. statistical significance was taken as p 0.05 and all means were expressed as mean standard deviation. in 18 subjects that were enrolled in the study, there were 7 males and 11 females. the mean of the average rnfl thickness was 72.9 9.5 micrometers (m). one subject did not accurately record his sleeping and waking times, thus, to ensure the accuracy of the data, this subject was excluded from analyses that required the input of sleeping and waking times. the baseline (pre - slt) gat iop was 15.3 2.2 mm hg while on 1.7 0.7 types of anti - glaucoma eye drops. all subjects received a single session of slt with a mean of 198.2 22.9 shots with a mean energy of 0.9 0.09 mj. the distributions of anti - glaucoma eye drops were : -blockers (32.0%), prostaglandins (20.0%), fixed combination prostaglandin--blocker (20.0%), brimonidine (12.0%), and topical carbonic anhydrase inhibitors (8.0%). the iop - related pattern of the 18 subjects was unique in terms of their peaks and slopes. this personal iop - related pattern remains similar in shape before and after slt and it is only the cls pattern amplitude and steepness of slopes that change. twenty - four - hour iop - related pattern before and after slt. at 1 month after slt, the mean gat iop measured before the cls wear was 12.7 1.8 mm hg while on the same anti - glaucoma medication regimen as before laser, representing a 17.0% reduction in iop after slt (p = 0.001). at 3 months post - slt, the mean gat iop was 11.4 1.7 mm hg while on 1.4 1.2 types of anti - glaucoma eye drops, representing a 25.5% (p = 0.0007) iop reduction in addition to a 17.6% medication reduction compared to pre - slt levels. eight out of 18 (44.4%) subjects fulfilled the criteria of a successful slt outcome. the measured pre- and post - slt parameters are summarized in table 1 to 4. from initially similar levels, the mean acrophase amplitude of the fitted cosinor function (global variability) was reduced after slt by 24.6% in the success subjects (table 1). in subjects for whom the slt was unsuccessful, the global variability increased by 19.2% post - slt, indicating greater 24-hour iop - related fluctuation after slt (figure 2a and b). mean global variability using the cosinor model before and after slt (a) twenty - four - hour iop - related pattern (global) variability in success group. (b) twenty - four - hour iop - related pattern (global) variability in non - success group. for the local variability in the non - success group, the 24-hour variability increased by 21.9% (p = 0.001), driven by the magnified 34.1% increase in diurnal variability (p = 0.002), while the nocturnal variability remained unchanged (p = 0.8). for the success group, there was no significant difference in local variability over 24-hours, nocturnal, or diurnal (p > 0.7). for the mean sleep - to - wake slope, the slope was negative in both groups signifying a decrease in cls output with waking. in the non - success group, the sleep - to - wake slope was flatter after slt (p = 0.04). no significant changes in variability or sleep - to - wake slope were observed in the slt success group (p = 0.2) (table 2). mean local variability and sleep - to - wake slope before and after slt for the overall study population, local diurnal (22.5%, p = 0.01) and 24-hour (12.1%, p = 0.04) variability was higher post - slt, mainly attributed by the increases in the non - success group detailed above (table 2). the number of peaks > 90 mveq increased (p = 0.04) and the number of diurnal troughs decreased (p = 0.01) after slt, independent of slt success (table 3). the mean number of peaks over the 24-hour period as well as the number of peaks 90 mveq after slt. previous studies in rats have demonstrated that rapid iop spikes can result in greater damage to retinal ganglion cells than persistently elevated iop levels although the effect on humans is yet to be determined. nagar previously reported a 41% reduction in iop fluctuation in 50% of subjects treated with slt. however, their study subjects consisted of subjects with primary open angle glaucoma (poag) or ocular hypertension with a pre - slt iop of 26.0 mm hg and iop was only measured during 4 time intervals at 08:00, 11:00, 14:00, and 18:00 hours. in contrast, our population consisted of only ntg subjects with a pre - slt iop of 15.3 2.2 mm hg while on 1.7 0.7 types of anti - glaucoma eye drops and our iop - related pattern recording was on a continuous basis for 24 hours. thus, the timing and frequency of iop measurement is important in accurately assessing the effects of slt on iop control. kothy reported in their poag series that after slt, 5 eyes had iop reduction > 20% during office hour (08:0012:00 hours) but none of the 26 eyes had a mean iop reduction of 20% when the assessment period was extended from 08:00 to 00:00 hours. the influence of slt on diurnal and nocturnal iop fluctuation is also diversified in the literature and probably related to whether or not anti - glaucoma medications were used. kothy reported a significant reduction in diurnal iop fluctuation of 4.3 1.7 and 5.0 1.7 mm hg at 3 and 6 months after slt, respectively, in their poag population after a 4-week washout period prior to slt. in contrast, lee reported in their medically treated poag patients that after slt, iop range was significantly reduced nocturnally but not diurnally. both of these studies however, only measured iop at intervals rather than continuously over 24 hours. therefore, the influence of slt on iop fluctuation can not be fully evaluated using interval or daytime iop measurements alone. in this study, slt was offered as an adjuvant therapy for ntg with the aim of further lowering iop or to reduce the anti - glaucoma medication requirement. the authors decided not to washout anti - glaucoma medication prior to slt in order to simulate the realistic clinical scenario where adjuvant slt is offered to medically controlled ntg patients to reduce medication load or for those with poor adherence or intolerant to topical anti - glaucoma medications. in addition, there is no definite consensus from the literature on the negative influence of topical prostaglandin analogs on slt outcome. in a retrospective study by scherer, a greater mean iop reduction was demonstrated following slt in poag subjects being treated with topical prostaglandin analogs. alvarado on the other hand, showed that prostaglandin analogs might dampen the iop - lowering effects of slt while singh reported no significant difference in slt outcome with prostaglandin analog use. the findings of our study suggest that adjuvant and successful slt may offer an additional benefit reducing 24-hour iop - related fluctuation for ntg patients who are already on anti - glaucoma medication. the majority of our patients (40%) was on a once nightly topical prostaglandin analog or fixed combination prostaglandin--blocker and 32% were on a topical -blocker. slt and prostaglandin analogs have been postulated to share a common iop - lowering pathway by opening up the intercellular junction and increasing conductivity at level of the schlemm canal endothelial cells. we postulate that after the prostaglandin analogs reach their maximal iop - lowering effect at 8 to 12 hours after instillation, this leads to the rebound in diurnal iop fluctuation. for those subjects who were responsive to slt (the success group), their diurnal variability was kept low by the continued effects of the slt, accounting for the significantly lower diurnal variability as compared to the non - success group. in all 18 subjects, both before and after slt, the wake - to - sleep slope was positive and the sleep - to - wake slope was negative, signifying that the supine - posture induced iop increases are still preserved after adjuvant slt. there was no significant difference in the wake - to - sleep slope before and after slt (p = 0.9). however, the sleep - to - wake slope was dampened by 3.9 times, following slt (p = 0.02) signifying a more gradual drop in iop upon waking after slt. it has been demonstrated in animal models that slt may increase the expression of aqueous endothelin-1 (et-1) in the early post - laser periods, which can reduce aqueous outflow, explaining the slower drop in iop upon waking. the magnitude and duration of et-1 influence following slt has not been well established in human subjects. however, we do not have an account for why only the sleep - to - wake slope was affected as the daytime iop was reduced after slt signifying an increase in aqueous outflow overall. although it has been established that the post - slt iop values as early as 2 weeks were predictive of future iop control, future studies monitoring 24-hour continuous iop changes at a longer time frame after slt would provide more long - term results. secondly, at present, the cls can only measure iop - related fluctuations in mveq. there are no effective formulae to convert these readings into the gold standard unit of iop measurement (mm hg). further developments in this area would popularize the use of this device in clinical practice. thirdly, due to the high cost and single use nature of the cls, the sample size was relatively small due to the constraints of resources. further studies involving larger samples should be carried out to further strength the preliminary conclusions drawn from this study. fourthly, the observations from our study are only applicable to medically treated ntg patients and may not be generalizeable to other glaucoma patients or to those without baseline anti - glaucoma medication prior to slt. nevertheless, this study has served to provide objective evidence for the controversies around iop fluctuations after slt by evaluating iop related changes in a 24-hour, continuous manner. it seems that slt was able to produce a significant iop reduction measured at a single time point during the day, accompanied by dampened amplitude of the nycthemeral rhythm in patients for whom slt was successful but an increased in those where slt was not successful. our findings confirm the relevance of continuous 24-hour iop - related pattern monitoring in the accurate evaluation of glaucoma treatments. further trials involving larger samples, different glaucoma subtypes, and groups with and without baseline anti - glaucoma medications would provide us with a clearer understanding of the influence of slt on iop fluctuation. | abstractto investigate intraocular pressure (iop) related patterns before and after selective laser trabeculoplasty (slt) for normal tension glaucoma (ntg).in this prospective cohort study, 18 ntg patients underwent slt. success was defined as iop reduction 20% by goldmann applanation tonometry. 24-hour iop - related pattern recording with a contact lens sensor (cls) (sensimed triggerfish, sensimed, switzerland) was done before (baseline) and 1 month after slt. a cosine function was fitted to the mean cls patterns for each individual in the slt success and non - success groups and the amplitude before and after slt was calculated. diurnal, nocturnal, and 24-hour cls pattern local variability was determined for pre- and post - slt sessions. cosine amplitude and variability were compared before and after slt by group using paired t - tests, with = 0.05.patients (11 women, 7 men) had a mean age of 65.1 13.7 years. mean iop was 15.3 2.2 mm hg at baseline and was reduced by 17.0% to 12.7 1.8 mm hg 1 month after slt (p = 0.001). slt was successful in 8 patients (44%). the amplitude of the fitted cosine was reduced by 24.6% in the success group, but displayed an amplitude increase of 19.2% post - slt in the non - success group. higher diurnal local variability of the cls pattern was observed after slt in non - success subjects (p = 0.002), while nocturnal variability showed no significant change. the increase in diurnal variability in the non - success group led to an increase in 24-hour variability in this group (p = 0.001). no change in local variability (diurnal, nocturnal, and 24-hour) was seen in the success group.the iop - related pattern cosinor amplitude was reduced in ntg patients with a successful slt treatment whereas the non - success group exhibited an increase of cosine amplitude. higher diurnal and 24-hour cls pattern variability was observed in non - success patients 1 month post - slt. |
the classification of the fluid as exudates or transudate is the first step in the evaluation of its etiology. the liquid is generally a transudate in patients with acute or chronic liver failure, massive liver metastases, hypoalbuminemia, or congestive heart failure, while in cases such as carcinomatosis, bacterial peritonitis, or pancreatitis, it is an exudate. the diagnostic paracentesis should be routinely performed in patients with recent - onset ascites, those requiring hospitalization due to stroke and those with ascites and unexplained clinical deterioration. traditionally, the exudate - transudate concept was based on the protein concentration of ascitic fluid (af) for classifying peritoneal exudates. different levels of protein in the af have been suggested as cutoffs for identifying exudates, ranging from 25 to 30 the use of this single biochemical parameter erroneously classifies many exudates originating in infectious or tumors as transudates, while some transudates in cirrhosis and congestive heart failure may be classified as exudates due to high protein levels. boyer used an adaptation of light criteria [5, 6 ] for differentiating between transudates and exudates in af using the concentration of protein and lactic dehydrogenase (ldh) levels as well as the ratio of these values between the serum and af. the difference between the albumin concentration between serum and af, called serum - ascites albumin gradient (saag), reflects directly and indirectly the colloid osmotic pressure and the degree of portal hypertension. pare suggested that saag is a better discriminator of portal hypertension than protein concentration in af. patients with ascites related to portal hypertension have saag 1.1 g / dl (transudates) and in those with ascites whose portal pressure is normal, saag is 1.1 g / dl (exudates) [4, 9 - 11 ]. moreover, with regard to cellularity, in the literature, a cutoff of > 1,000 cells / mm is used to classify af as exudates. the mechanisms that contribute to the malignant ascites include obstruction related to tumor lymphatic drainage, increasing the capillary permeability and activation of the renin - angiotensin - aldosterone system, producing neoplastic fluids and metalloproteinases which degrade the extracellular matrix. the cytology has a high specificity for the detection of neoplastic cells in effusions ; the sensitivity varies from 40% to 90% [13, 14 ]. the aim of this study was to evaluate the usefulness of different cutoffs applied to the cellularity and biochemical markers (metabolic and enzymatic markers) in the differential diagnosis of af. to the best of our knowledge, it is the first report about the value of aspartate aminotransferase (ast) and the cellular counting for differentiating between exudates and transudates. we studied 191 samples from patients with af, who were admitted to the hospital from january 01, 2009 to december 31, 2014. one hundred fifty - two of them were included in the analysis, and the remaining 39 where excluded because they had more than one associated pathology, clotted or hemolyzed. cell count was performed by duplicate as total cells / mm in a hemocytometer, directly or diluted with saline or turk solution for liquids with plenty of cells or bleeding smears. the differential count was performed with the giemsa method, counting a minimum of 200 cells ; when the smears were suspicious for malignant cells, the papanicolaou (pap) stain was used to confirm the diagnosis of malignancy. in cases of inconclusive cytology, simultaneous determinations were performed in af and serum (s) of the following biochemical parameters : glucose (glu), proteins (pt), albumin (alb), cholesterol (col), triglycerides (tg), ldh, creatine kinase (ck), alanine aminotransferase (alt), ast, alkaline phosphatase (alp), amylase (ami) and total bilirubin (tb). spontaneous bacterial peritonitis was diagnosed by the presence of a counting of neutrophils > 250/mm or by microbiological positive cultures. the diagnosis of tuberculous peritonitis required positive culture of the af for mycobacterium tuberculosis or the presence of granulomas in peritoneal biopsy in the absence of other causes for granulomatosis. liver cirrhosis was diagnosed by the combination of impaired liver function tests, evidence of portal hypertension or liver biopsy. the diagnosis of congestive heart failure was performed using clinical criteria and echocardiographic evidence of ventricular dysfunction. our study was carried out according to the principles of declaration of helsinki for research in human subjects. the usefulness of cellularity and each biochemical parameter for identifying exudates was evaluated in terms of sensitivity, specificity and efficiency. yates - corrected chi - square () analyses and fisher method were used to detect statistically significant differences. yates - corrected chi - square () analyses and fisher method were used to detect statistically significant differences. a value of p 0.05 was considered statistically significant. of the 152 samples studied, 83 (55%) were exudates and 69 (45%) were transudates. the more frequent etiologies in exudate ascites were infectious (22%) and malignant (19%) ; from these last groups, 12% corresponded to paraneoplastic effusions and 7% to malignant effusions. the malignant cells were detected in the fluid (pap and giemsa stains) in 7/10 neoplastic af (70%). the most frequent metastatic cancers were lymphoma (fig. 1) and stomach adenocarcinoma (figs. 2 and 3). in two patients (one stomach adenocarcinoma and one breast carcinoma), the results of the giemsa and pap stains were inconclusive but the application of the agnor technique confirmed the diagnosis of malignancy, reaching a value > 14 (values of number of nor dots for malignant cells in body fluids : 13.78 3.89), increasing the sensitivity to 90%. other metastases detected were one testicular germ cell carcinoma and one renal clear cell carcinoma. the most common cause of transudates was cirrhosis (29%) ; 4% of the transudates were related to heart failure, and other pathologies reached 12% of the total of patients. the percentages of patients with transudates and exudates related to cellularity and biochemical parameters are shown in table 2, and sensitivity, specificity and efficiency for each biochemical parameter are shown in table 3. t : transudate ; e : exudate ; saag : serum - ascites albumin gradient. forty - seven of the 83 exudates (57%) were detected using the traditional cutoff for cell count greater than 500/mm, but using the cutoff proposed in the present paper (300 cells / mm), the detection increased to 65/83 (78%). of the biochemical parameters studied, the ast ratio af / s (> 0.5) detected the greater number of exudates correctly classified 66/83 (80%), while 10 of 69 (15%) transudates were falsely classified. the af / s of ldh (> 0.6), pt (> 0.5), col (> 0.4), and alt (> 0.5) correctly detected 78%, 72%, 70%, and 70% of the exudates, respectively. the saag (1.1 g / dl) detected correctly 60/68 (88%) transudates. iu / l) and glu (500 cells / mm to classify af as exudate. this paper proposes to reduce the cutoff to 300/mm in afs, since the detection of exudates is increased by 21% without affecting the number of incorrectly classified transudates, obtaining a sensitivity of 78% and an efficiency of 97%. the sensitivity of cytology to detect malignant cells in serous effusions varies between 40% and 90% according to the recent literature [12 - 14 ]. the high frequency of false negatives (over 70%) is mainly due to sampling error, and to a lesser extent, to a misinterpretation of study material. in this study, 10% of the afs were of malignant origin and the sensitivity of cytology in the detection of neoplastic cells was high (70%). however, no false positives were detected, consistent with the low frequency described in the literature, in which reports are produced by an excessive interpretation of a reactive atypia. spontaneous bacterial peritonitis (sbp) is defined by a cell count of more than 250 neutrophil cells / mm, as by a positive culture of the af. however, the role that biochemical parameters play for such differentiation is controversial. in this paper, the af / s > 0.5 for ast and > 0.6 for ldh were the most helpful in the diagnosis of exudate, with a sensitivity of 80% and 78% and efficiency of 85% and 90%, respectively for the both parameters. there are no references in the literature concerning the ast as a marker of differentiation between exudates and transudates. we suggest that the observed increase of ast in exudate ascites could be attributed to an increase in the number of cells in these processes, together with an increased cell destruction which would bring about the release of the enzyme from an intracellular localization. another parameter that was useful in the differentiation between exudates and transudates was the af / s ratio of pt > 0.5, with sensitivity and efficiency of 72% and 85%, respectively ; this parameter erroneously classified the fluids as transudates in a low percentage of the cases (15%). moreover, the af / s ratio for alt (> 0.5) and col (> 0.4) could also be useful in the differentiation of the fluids, though less than the aforementioned parameters, as detected 70% of the exudates with an efficiency of 81% and 85%, respectively. concerning col and pt, although there are controversies in the literature, some authors have described that af / s for pt 90% [11, 18, 20, 21 ]. tb was not useful in characterizing the af unlike some authors that reported a sensitivity and specificity of 72% and 86%, respectively. glu values 300/mm) to improve detection of exudate ascites. it is proposed to incorporate the ratio af / s for transaminases and especially for ast, with the purpose of optimizing the differentiation and characterization of the exudate ascites, together with the ratios for ldh and pt. we suggested the utilization of a new cutoff of cellular counting, major of 300/mm, since it would allow improving the detection of exudate ascites, without including the cases of transudate ascites. of the biochemical parameters studied, ast af / s showed major usefulness in the differentiation and characterization of af ; ldh, pt, col, and alt might be also acceptable in the above mentioned differentiation. the saag turned out to be a good marker of portal hypertension associated with cirrhotic processes. the glu and ami did not show usefulness in the discrimination between transudates and exudates. we suggested the utilization of a new cutoff of cellular counting, major of 300/mm, since it would allow improving the detection of exudate ascites, without including the cases of transudate ascites. of the biochemical parameters studied, ast af / s showed major usefulness in the differentiation and characterization of af ; ldh, pt, col, and alt might be also acceptable in the above mentioned differentiation. the saag turned out to be a good marker of portal hypertension associated with cirrhotic processes. the glu and ami did not show usefulness in the discrimination between transudates and exudates. | backgroundin the cases of ascitis, it is essential to determine their origin using the parameters obtained by the cytological and biochemical examinations. the aim of this study was to evaluate the usefulness of different biochemical markers and the number of cells in the differential diagnosis of ascitic fluid (af).methodsone hundred ninety - one cases of af were studied, who were admitted to the hospital from january 01, 2009 to december 31, 2014. one hundred fifty - two of them were included in the analysis, and the remaining 39 were excluded because they had more than one associated pathology, clotted or hemolyzed.resultsthe more frequent etiologies of af were the cirrhosis (29%), the infections (22%) and the neoplasies (19%). other pathologies reached 16%. cutoff > 300 cells / mm3 detected the 78% of exudates. the af / serum (s) of aspartate aminotransferase (ast) (> 0.5), lactate dehydrogenase (ldh) (> 0.6), proteins (pt) (> 0.5), cholesterol (col) (> 0.4), and alanine aminotransferase (alt) (> 0.5) correctly detected 80%, 78%, 72%, 70% and 70% of the exudates, respectively.conclusionwe proposed the utilization of a new cutoff of cellular counting, major of 300/mm3, since it would allow improving the detection of exudate ascites, without including the transudate ascites. ast af / serum ratio (af / s) showed the major usefulness in the differentiation and characterization of af ; ldh, proteins, cholesterol and alt might be also acceptable in the above mentioned differentiation. the serum - ascites albumin gradient (saag) turned out to be a good marker of portal hypertension associated with cirrhotic processes. creatine kinase (ck), alkaline phosphatase (alp), amylase (ami), total bilirubin (tb), triglycerides (tg) and glucose (glu) did not allow differentiating exudates from transudates. |
a 46-year - old man was referred for ophthalmologic examination due to gradually decreasing visual acuity. he had suffered from disturbances of ocular movements, ptosis and weakness of the extremities for two years. through polymerase chain reaction (pcr), the patient was found to have amplification of ctg repeats on chromosome 9, which is compatible with myotonic dystrophy type 1. on ophthalmologic examination, intraocular pressure was 13 mmhg in the right eye and 15 mmhg in the left eye. the levator function was 0 mm in the right eye and 1 mm in the left eye. fundus examination showed geographic depigmentation of the retinal pigment epithelium along the vascular arcade and pigment clumps in the peripheral area (fig. fourteen months later, the patient 's best - corrected visual acuity had decreased to 0.3 in both eyes. phacoemulsification and intraocular lens implantation were sequentially performed in both eyes and his best - corrected visual acuity improved to 0.7. myotonic dystrophy type i is caused by a genetic defect composed of an expansion of cytosine - thymidine - guanine (ctg) repeats, resultingin a defect of the dystrophia myotonica protein kinase (dmpk) gene and a reduction of dmpk messenger rna expression. this genetic defect affects zinc finger protein 9 expression.6 the most common ocular complications associated with myotonic dystrophy are cataracts that may be induced by a defect of the lens epithelium.7 as in the present case, they generally have the appearance of iridescent dust and fine points with colored crystals.3 various retinal degenerations associated with myotonic dystrophy include a butterfly pattern in the macular area, peripheral reticular pigmentary changes, and polymorphic atrophy.8 peripheral reticular pigmentary change is the most common form of retinal degeneration and was observed in the present case. this pigmentary change causes decreased b - wave amplitude on electroretinography which we also observed.3 kimizuka.9 report on atrophy of the inner retinal layers with preservation of the photoreceptors in the peripheral retina and increased pigmentation with granular, striate or satellite patternin the macula. the reduced saccadic peak velocity and impaired smooth pursuit gain in the present case were almost identical with those observed and described in a previous report.10 lessell s.10 reported that limitation of ocular movement and ptosis may be due to one or all of three possible mechanisms : supranuclear control, peripheral motor apparatus and extraocular muscle atrophy. previously reported histological findings showed pathological changes with a shredded appearance and an abundance of lipofuscin in muscle fibers.11 in conclusion, the visual disturbances associated with myotonic dystrophy may be caused not only by cataracts but also by retinal degeneration. therefore, fundus examination is essential and should be performed carefully in patients with myotonic dystrophy. | a 46-year - old man presented with visual disturbances in both eyes. his best corrected visual acuity was 0.7 (both eyes). ptosis and limitation of ocular movement in every direction were observed. slit lamp examination showed a bilateral iridescent cataract. fundus examination showed peripheral depigmentation of the retinal pigment epithelium and pigmentary clumping in both eyes that agreed with blocked fluorescence and widow defects on fluorescein angiography. the amplitude of b - wave was decreased on electroretinography. fourteen months later, the patient 's best corrected visual acuity decreased to 0.3 due to increased lens opacity. phacoemulsification and intraocular lens implantation were performed on both eyes. at the patient 's final visit, retinal findings were stable with a best corrected visual acuity of 0.7 in both eyes. in conclusion, the visual disturbance could have been caused by both cataracts and retinal degeneration, meaning the fundus should be examined carefully in patients with myotonic dystrophy. |
aneurysmal dilatation of the left auricle by fasseas, and an early autopsy report of a huge left atrium (la) was published by kronzon. elsewhere, a case of the massive enlargement of the la was reported by schwartzman. the reported incidence of the huge la in rheumatic mitral valve disease was 3 in each 1000 operations according to piccoli. huge enlargement of the la is usually associated with rheumatic mitral stenosis or regurgitation, left ventricular failure, chronic atrial fibrillation, and left - to - right shunts such as those occurring with patent ductus arteriosus and ventricular septal defects. piccoli. stated that enlargement of the la may create compression of the surrounding structures such as the esophagus, pulmonary veins, trachea, left main bronchus, middle and lower lobes of the right lung, inferior vena cava, recurrent laryngeal nerve, and thoracic vertebrae, leading to dysphagia respiratory dysfunction, peripheral edema, hoarse voice, or back pain. johnson stated that paradoxical movement of the left ventricular posterobasal wall occurs in the giant la and might exert a negative and depressive effect on the patient s hemodynamic. the giant la is defined as an la measuring larger than 8 cm, and it is typically found in patients who have rheumatic mitral valve disease with severe regurgitation. our thorough literature search did not yield any reports of the giant la (2022 cm) in normal prosthetic valve function in adults. a 46-year - old woman with a past medical history of rheumatic heart disease, mitral valve replacement, and chronic atrial fibrillation was admitted to our hospital with a chief complaint of cough and shortness of breath, worsened in the last month. she was referred by a cardiologist from rural areas and, despite persistent symptoms, she had not seen a physician for many years. physical examination showed elevated jugular venous pressure, respiratory distress, cardiac cachexia, symptoms of progressive heart failure, hepatomegaly, and severe edema in the legs. she had undergone the bjork - shiley mitral valve prosthesis replacement for severe mitral stenosis when she was 21 years old, in 1987. in the emergency ward, a plain chest x - ray study showed a marked cardiomegaly and nearly complete opacification of the lower, middle, and upper lung fields (figure 1). she suffered from voice hoarseness, but there were no complaints of dysphagia or any other gastrointestinal symptoms. transthoracic echocardiographic examinations in four chamber view (figure 2) revealed a moderate decrease of systolic function and severe regurgitation of the tricuspid valve. this examination also unexpectedly demonstrated a massively enlarged la with a maximum diameter of 20 cm and a transverse diameter of 21 cm with huge thrombosis, marked enlargement of the right ventricles, and severe dilatation of the pulmonary artery, superior vena cava, inferior vena cava, and pulmonary artery. there was also evidence of dilated right - side heart chambers, severe tricuspid valve regurgitation, and pulmonary artery systolic pressure of 100 mmhg. laboratory examinations revealed abnormal renal function (creatinine > 2.5 mg / dl), abnormal liver function (alanine transferase > 300u), and iron deficiency anemia (hemoglobin = 10 mg / dl). chest x - ray in posteroanterior view, showing a huge left atrium cardiomegaly (arrows) echocardiography in the four - chamber view, showing a gigantic left atrium (arrows) the gigantic la is uncommon and is defined according to the chest x - ray study appearance, in which either the la forms the right margin of the heart shadow and approximates the right chest with a cardio - thoracic ratio greater than 60% or the la on echocardiography has an anteroposterior diameter larger than 8 cm. a normal la is located in the middle of the chest, is the most posterior chamber of the heart, and is not located on the left ; nevertheless, when it enlarges, it moves rightward and approximates the right chest margin. in our patient, the la measured 20 22 cm, representing one of the largest las reported in an adult. the patient s systolic pulmonary artery pressure was 100 mm hg, which was consistent with severe pulmonary hypertension. the entire heart mass was tightly adherent through relatively firm and edematous adhesions to the collapsed left and right lungs and the sternal plate. it was very difficult to find a dissection plane to reach the aorta for cannulation in consequence of severe main pulmonary artery dilatation and the enlargement of the right atrium and the right ventricular outflow tract. in order to assess the small aorta, embedded in fibrous adhesion between a huge superior vena cava and a huge main pulmonary artery, cardiopulmonary bypass (cpb) was commenced by instituting femoral artery and vein cannulation. instituting cpb and unloading the cardiac chamber revealed the small aorta, embedded between the large superior vena cava and the main pulmonary artery. the small aorta was then released from the fibrous adhesion and prepared for aortic cross - clamping. the aorta was thereafter clamped, and cardiac arrest was induced with 1.4 liters of antegrade cold blood cardioplegia. next, tricuspid valve repair without an annuloplasty ring was undertaken, and a classic de vega procedure was performed (single ethibond suture from the anteroseptal commissure to the posteroseptal commissure with a pledget at each end). the aorta was cross - clamped for a total of 100 minutes, and the heart function was resumed after multiple cardioversion attempts. after 20 minutes, partial bypass was resumed for a further 30 minutes because of major bleeding from the traumatic rupture of the right pulmonary artery by aortic cross - clamping. seven hours after the start of sternotomy, the patient was returned to the intensive care unit (icu) while she was hemodynamically unstable and had an unresponsive low cardiac output. the most common causes of the enlarged la in adults are mitral stenosis and severe mitral regurgitation with pulmonary hypertension. a few case reports have described concomitant huge dilatation of three heart chambers in adults. in a case report on a patient with a normally functioning prosthetic mitral valve, rahimtoola and colleagues stated that the size of the left and right atria as well as that of the right ventricle increases substantially in pulmonary hypertension with non - regression of pulmonary vascular resistance and concluded that activation of the frank - starling mechanism occurs in both right chambers. the authors attributed these chamber enlargements to valve prosthesis - patient mismatch, underscored the problem of valve prosthesis - patient mismatch in the first generation of prosthetic valves, and asserted that la dilatation results from the following two factors. first, the in vivo effective orifice valve area of almost all types of old prosthetic valves that can be inserted in most patients is less than that of the normal human valve. the in vivo effective prosthetic valve area is even further reduced by organized clot, pannus formation, tissue ingrowths, and endothelialization and, therefore, these valves can be considered stenotic. second, in some patients, the problem is complicated because the size of the prosthesis that can be inserted is limited by the size of the annulus, which is small compared with the size of the patient, and also by the size of the cavity in which the prosthesis must lie. castrillo and colleagues reported a patient with a huge la (8 cm) with a normally functioning mitral valve without explaining the probable causes of the enlargement. in the funk. case reports, a female patient with a mitral starr - edwards valve prosthesis, which had been implanted 35 years previously for rheumatic mitral valve disease, was reported. transthoracic echocardiography showed a giant la (12cm13 cm ; area of 127 cm) with hyperechogenic walls, mostly occupied by thrombus. she was treated conservatively ; her symptoms improved mildly on diuretics, digoxin, and anticoagulants and she was discharged 8 days after admission. plaschkes and colleagues reported the case of a 56-year - old man with an la diameter of 17 cm as measured by echocardiography and an la size of 18 20 17 cm according to magnetic resonance imaging. an la diameter of 18.5 cm, reported by ates and collogues, was the largest diameter that we managed to find in the medical literature. castrillo and colleagues demonstrated that la enlargement is not solely due to mitral regurgitation but it is also correlated with the quality of the la wall. plaschkes and colleagues described partial auto transplantation for the reduction of the size of the la and thus prevention of thromboembolism. in this technique, after cross - clamping, the superior vena cava, aorta, and pulmonary artery are detached, and the heart is easily moved upward. according to piccolli and colleagues, paradoxical movement of the left ventricular posterobasal wall occurs in the giant la and may affect hemodynamic in a negative manner and also cause atrial fibrillation and thromboembolism. in the lee - roux and colleagues study, radiographic evidence of asymmetrical enlargement of the la without atrial infarction the authors stated that giant atrial enlargement is rarely symmetrical, the atrial appendage can contribute to the enlargement, and the giant atrium can be effectively trimmed. in their study, all patients with an enlarged la (more than 6 cm), irrespective of the cause and mechanism of the enlargement, were evaluated. in the ates and colleaguesstudy, postoperative clinical and hemodynamic parameters showed a positive response to mitral valve replacement in patients with a huge la. a direct correlation between early or late thromboembolism and the gigantic la was not found. inadequate control of the anticoagulation level was a major risk factor for thromboembolism, which was encountered during the follow - up, especially in patients dwelling in rural areas. johnson found that para - annular plication, posterior wall plication, ligation of the appendix of the la, and partial resection using auto transplantation are the possible modes of diminishing size and preventing stagnation. goldberg and colleagues described a 67-year - old woman with a history of rheumatic heart disease admitted due to dyspnea and anasarca. the patient expired during hospitalization. in the case of our patient, we were initially unable to define the role of atrial enlargement in determining or contributing to the patient s congestive heart failure. be that as it may, the role of atrial enlargement in la thrombosis formation is clear. with an apparently normal mitral prosthetic function and echocardiographic evidence of a preserved left ventricular systolic function with some diastolic restraining, johnson and colleagues hypothesized that pericardial release could increase the compliance of the left ventricle and eliminate a possible cause of limited left ventricular filling and cardiac output. we believe that in the absence of discrete constrictive pericarditis, this maneuver would not have improved our patient s cardiac output but instead would have aggravated respiratory dysfunction by causing total left lung atelectasis and worsening the patient s congestive heart failure symptoms. the role of atrial reduction in the setting of mitral valve replacement has been widely discussed in the literature both in regard to its ventilatory effect as well as its hemodynamic effects but a consensus has not yet been reached. parinnelo and colleagues stated that la compensation due to volume or pressure overload in mitral valve disease is, frequently, dilatation. proposing that la structural remodeling is independent of the atrial pressure value and effective orifice area of the mitral valve opening, and pulmonary or arterial capillary pressure values, the investigators stated that the amount of mitral regurgitation causes the enlargement of the la. finally, severe la enlargement is more commonly associated with mitral regurgitation than with mitral stenosis and more commonly with the rheumatic rather than with the non - rheumatic causes of mitral regurgitation. this report raises some questions vis - - vis patients with a gigantic la and normal prosthetic function. indeed, does an underlying disease beget a gigantic la or, as parinnelo and colleagues proposed, does the patient develop a giant la in consequence of many years of mitral malfunction after valve replacement in combination with la wall remodeling change ? we believe the patient - valve mismatch in the old generation of prosthetic valves and chronic volume / pressure overload of many years are allied to the gigantic la and that an increased trans - prosthetic mean pressure gradient and unrelieved pulmonary hypertension generate the dilated la. in addition, the pre - existent rheumatic myocardial disease may, in all likelihood, facilitate muscular remodeling with subsequent severe atrial enlargement. this case report is an attractive model for understanding the nearly unlimited la compliance and enlargement during rheumatic valve disease. the novelty of the report appears to be the association between a huge la (largest la reported to date in the literature), normal prosthetic function, huge la thrombosis associated with a gigantic superior vena cava, huge right atrium, and pulmonary artery and tricuspid valve regurgitation. the present report is also unique inasmuch as the surgeon was unable to institute cardiopulmonary bypass (cpb) via median sternotomy because the exposure of the aorta was impossible without unloading the anteriorly placed and enlarged pulmonary artery and superior vena cava. in our patient, congestive heart failure and respiratory failure concomitant with la thrombosis occurred with moderately reduced left ventricular dysfunction, severe right ventricular dysfunction, and a normally functioning mitral prosthesis. we were, therefore, compelled to postulate that the pathophysiology involved was created by la enlargement in concert with pressure and volume overload and unrelieved pulmonary hypertension. in retrospect, we were faced with a patient condemned by a natural history that could not possibly be addressed by a less invasive operation. clot removal procedure would have been the sole chance of success if the huge cardiac chambers and adhesion severity had allowed a reasonable and usual operative time. we would believe that such tragic results make a case for the prevention of this severe complication during conventional mitral valve surgery via primary plication of the enlarged la, even at the expense of a prolonged aortic - cross clamping time. | abstractgiant left atria are defined as those measuring larger than 8 cm and are typically found in patients who have rheumatic mitral valve disease with severe regurgitation. enlargement of the left atrium may create compression of the surrounding structures such as the esophagus, pulmonary veins, respiratory tract, lung, inferior vena cava, recurrent laryngeal nerve, and thoracic vertebrae and lead to dysphagia, respiratory dysfunction, peripheral edema, hoarse voice, or back pain. however, a huge left atrium is usually associated with rheumatic mitral valve disease but is very rare in a normally functioning prosthetic mitral valve, as was the case in our patient. a 46-year - old woman with a past medical history of mitral valve replacement and chronic atrial fibrillation was admitted to our hospital with a chief complaint of cough and shortness of breath, worsened in the last month. physical examination showed elevated jugular venous pressure, respiratory distress, cardiac cachexia, heart failure, hepatomegaly, and severe edema in the legs. chest radiography revealed an inconceivably huge cardiac sell - out. transthoracic echocardiography demonstrated a huge left atrium, associated with thrombosis, and normal function of the prosthetic mitral valve. cardiac surgery with left atrial exploration for the extraction of the huge thrombosis and de vega annuloplasty for tricuspid regurgitation were carried out. the postoperative course was eventful due to right ventricular failure and low cardiac output syndrome ; and after two days, the patient expired with multiple organ failure. thorough literature review showed that our case was the largest left atrium (20 22 cm) reported thus far in adults with a normal prosthetic mitral valve function. |
retinoblastoma is the most common primary intraocular malignancy of infancy and childhood, with an incidence of 1 per 15,000 to 1 per 20,000 live births. intravenous chemotherapy (ivc) is an effective way of treating the disease when there is no vitreous or subretinal seeding. according to the international classification of retinoblastoma, the success rate of ivc combined with local therapy was 90% to 100% for groups a, b, and c ; 47% for group d ; and 25% for group e retinoblastoma, though these articles rarely stated the percent of d or e eyes that were primarily enucleated [2, 3 ]. although there was no randomized study which compared the outcome of ophthalmic artery chemosurgery (oac) and ivc, single institution retrospective case series seemed to suggest that oac may have a higher success rate and less systemic side effects than ivc for d or e eyes [4, 5 ]. despite the dramatic increase in ocular salvage with oac, vitreous seeding is still one of the main reasons for subsequent enucleation in treated eyes. vitreous seeds respond poorly to chemotherapeutical drugs delivered via intravenous, intra - arterial, or periocular route. more than 50 years ago, ericson and his group reported on the intravitreal delivery of chemotherapeutical drugs targeting vitreous seeds. however, this method was not employed in routine use due to the concern on possible extraocular spread of tumor cells and inconsistent successes. half a century later, several groups revisited the chemotherapeutical drugs and intravitreal drug delivery methods for treating vitreous seeds. in 2011, suzuki and kaneko reported the results on intravitreal delivery of melphalan to treat 237 eyes of 227 patients with vitreous seeds. so it is difficult to know the contribution of the intravitreal injection to overall success. other groups in europe and america then showed that intravitreal chemotherapy with melphalan is an effective and safe modality for eliminating vitreous seeds from retinoblastoma when the dose was increased to 2030 ug [1012 ]. here, we report our experience on intravitreal melphalan in treating vitreous seeds in 17 chinese retinoblastoma patients. the study followed the tenets of the declaration of helsinki and was undertaken with the understanding of each guardian of participating patient. informed written consent was obtained from each guardian. seventeen consecutive chinese retinoblastoma patients with active vitreous seeds who received intravitreal injection of melphalan between november 2011 and august 2013 were included. there were 6 eyes with resistant vitreous seeds and 13 eyes with recurrent vitreous seeds after completion of 2 - 3 sessions of oac and/or 46 sessions of ivc., the location of retinal tumor and vitreous seeds was identified using retcam and indirect ophthalmoscope. in order to minimize reflux, hypotony was induced by aspirating 0.1 ml of anterior chamber fluid using a 27-gauge needle prior to intravitreal injection. scleral entry site was selected at 2.53.0 mm away from the limbus to avoid touching the tumor tissue, vitreous seeds, and detached retina. a 30-gauge needle attached to a tuberculin syringe twenty - microgram melphalan in 0.1 ml solution was injected slowly and continuously within 3 seconds. after pulling out the needle, forty - microgram melphalan in 0.2 ml was administered subconjunctivally around the scleral injection site. retinal tumors were treated with oac, ivc, and focal consolidation such as transpupillary thermotherapy, laser coagulation, and cryotherapy if necessary. we assessed response to treatment and complications under anesthesia every 24 weeks with retcam and indirect ophthalmoscope. according to munier 's study, complete response is established if the seeds present completely disappear (vitreous seeds regression type 0), refringent and/or calcified residues (type i), amorphous often nonspherical inactive residues (type ii), or a combination of the latter two (type iii). multiple injections were performed to control vitreous seeds. when complete response is noted, intravitreal injection would be stopped. data was analyzed by one - way anova using statistical package for the social sciences (spss version 19) after levene 's test for the equity of variance. nine patients with bilateral and 8 with unilateral retinoblastoma (10 boys and 7 girls) were included in the study. based on the international classification of retinoblastoma, these eyes were classified as group b (n = 1), c (n = 5), d (n = 11), or e (n = 2). a total of 19 eyes were treated, which included 11 eyes (58%) with localised vitreous seeds (confined to one quadrant) and 8 (42%) with extensive vitreous seeds (more than one quadrant). based on morphologic features, vitreous seeds were classified as dust (n = 5), spheres (n = 8), and cloud (n = 6). the median age at the first injection was 27 months (range 11109 months)., these cases received 123 (median, 6 times ; range, 115 times) intravitreal injections delivered every 24 weeks. two patients received bilateral injection and others received unilateral treatment. in this study, vitreous seeds in all cases regressed after the completion of intravitreal injection with type 0 (n = 10), type i (n = 3), and type iii (n = 6) (figures 1 and 2). there is a significant difference in response to intravitreal melphalan for cloud, spheres, and dust seeds with a median number of injections of 9, 6, and 3, respectively (p = 0.003). no significant difference was noted in the number of injections to control diffuse and localised seeds, and between recurrent and resistant seeds. overall, vitreous seeds were successfully controlled in 16 out of 19 eyes (84.21%). the interval between the end of intravitreal injection and recurrence was 2, 3, and 7 months, respectively. no recurrence of retinal tumor was noted in the 3 eyes. except for the 3 eyes, 2 more eyes were removed due to retinal tumor recurrence (n = 1) and hypotony after vitrectomy (n = 1). the patients were followed up for 27 months on average (median : 26 ; range : 1742 months). cytopathological examination of the anterior chamber fluid was negative for malignant cells in each case. a localised peripheral salt - and - pepper retinopathy was found in 8 eyes near the site of injection. there was no case of extraocular extension or metastasis within the period of follow - up. this study summarises our experience performing intravitreal injection of melphalan to treat vitreous seeds from retinoblastoma. it showed that intravitreal injection with melphalan could achieve high control rate with 84% (16/19) for vitreous seeds.. showed an unprecedented success rate of tumor control in the presence of vitreous seeds with intravitreal melphalan. according to the work of japanese group, 68% of eyes treated with intravitreal melphalan achieved complete vitreous seed remission in the long follow - up. in this study, although 3 patients (cases 3, 14, and 9) received additional ivc and/or oac during the period of intravitreal melphalan, we think that regression of vitreous seeds in these cases was mainly due to the intravitreal melphalan. case 3 received 4 ivc and case 13 received 4 ivc and 2 oac before intravitreal melphalan. case 9 received additional 1 ivc and intravitreal melphalan for vitreous seeds, but the treatments failed to control vitreous seeds that resulted in enucleation. francis. found that eyes with dust seeds received fewer injections and a lower cumulative dose of melphalan, whereas eyes with clouds seeds received more injections and a higher cumulative dose of melphalan. in this study, we also found that eyes with dust seeds have the best response to intravitreal melphalan, while eyes with cloud seeds have the worst response. recent reports by ghassemi. showed that the combination of intravitreal melphalan and topotecan injection was effective for refractory vitreous seeds from retinoblastoma. so combination of multiple chemotherapeutical agents may be needed to maximize the therapeutical power for cloud vitreous seeds. one of the major concerns regarding intravitreal injection for vitreous seeds was the risk of having cancer cell spread extraocularly. different techniques have been employed to minimize the risk, such as the employment of repetitive freeze and thaw cycles at the injection site when pulling out needle. francis. pointed out that irrigation with sterile distilled water submersion on the surface of the eye for at least 3 minutes could further reduce the risk in addition to freeze - thaw cryotherapy. we took the following measures : (1) choosing the entry site far away from tumor and vitreous seeds, (2) performing paracentesis to soften the globe before injection to prevent retroflex of intraocular fluid when pulling out the needle, (3) pressing the scleral injection site for about 5 seconds after retracting the needle, and (4) injecting 40 ug of melphalan in 0.2 ml subconjunctivally around the scleral injection site. the dose of melphalan for intravitreal injection was another important issue. in this study, we used the dose of 20 ug in order to minimize the damage to the retina. ghassemi and shields reported that 50 ug melphalan could lead to the severe complications such as subretinal hemorrhage, severe hypotonia, and phthisis. in another study, the results showed no changes in the a and b waves of bright - flash electroretinograms. however, it is generally accepted that melphalan of less than 30 ug is safer for the retina. except for electroretinograms, other various modalities such as vision acuity test, visual evoked potentials, fluorescein angiography, and optic coherence tomography our study confirmed that the intravitreal delivery of melphalan is both an effective and a safe approach in controlling vitreous seeds from retinoblastoma in chinese patients. | purpose. to evaluate the efficacy of intravitreal melphalan for vitreous seeds from retinoblastoma in chinese patients. methods. this is a retrospective review of 17 consecutive chinese patients (19 eyes) with viable vitreous seeds from retinoblastoma. the patients received multiple intravitreal injections of 20 ug melphalan. results. the international classification of retinoblastoma groups were b in 1 eye, c in 5 eyes, d in 11 eyes, and e in 2 eyes. on average, 6 injections (range : 115) were given to each eye at the interval of 24 weeks. successful control of vitreous seeds was achieved in 16 of 19 eyes (84.21%). globe retention was achieved in 73.68% (14/19) eyes. the patients were followed up for 27 months on average (median : 26 ; range : 1742 months). there is a significant difference in response to intravitreal melphalan for cloud, spheres, and dust seeds with a median number of injections of 9, 6, and 3, respectively (p = 0.003). complications related to intravitreal melphalan included vitreous hemorrhage, cataract, salt - and - pepper retinopathy, and pupil posterior synechia. there was no case of epibulbar extension or systemic metastasis within the period of follow - up. conclusion. intravitreal melphalan achieved a high local control rate for vitreous seeds without extraocular extension and with acceptable toxicity in chinese retinoblastoma patients. |
at the end of april 2009, a physician from a private clinic in port - louis, the capital city, reported having seen several patients with fever, malaise, diarrhea, increased levels of liver enzymes, and marked thrombocytopenia. rash and arthralgia were not mentioned, and no test was requested for dengue or chikungunya viruses. on june 1, another physician requested dengue serologic testing for a patient who had fever for the past 10 days, rigors, generalized aches and pains, and a petechial rash. the patient had lived in malaysia several years previously but had no history of recent travel. his liver enzyme levels were increased, and his thrombocyte count was 15,000 cells/l. at the central health laboratory (chl) in mauritius, the patient s serum was positive for immunoglobulin (ig) g and igm against dengue with the hexagon dengue rapid immunochromatography test (human gmbh, wiesbaden, germany) and negative for platelia dengue nonstructural protein 1 (bio - rad laboratories, marnes - la - coquette, france). the next day, 6 patients who had previously been admitted with fever at the above - mentioned private clinic were traced by the department of public health of the ministry of health and quality of life (mohql). the mohql immediately initiated an action plan which included mosquito control measures by fogging and larviciding, environmental cleaning, and a public awareness campaign on how to eliminate mosquito breeding sites. the first 10 positive serum specimens, all of which had dengue antibodies were sent for confirmation to the national health laboratory services in south africa, where all were subsequently found to be positive for dengue igg and igm by hemagglutination - inhibition test and elisa, respectively. on june 5, four days later, serum samples from 11 patients that were positive for dengue antigen were sent to tan tock seng hospital in singapore for reverse transcription pcr (rt - pcr) testing. approximately 24 hours later, the laboratory reported that denv rna was detected in 7 of the samples by real - time rt - pcr, with previously described primers using sybr green and gel electrophoresis (6). the serum samples were later found to be positive for denv-2 by multiplex rt - pcr with serotype - specific primers, and detection with serotype - specific probes by using a luminex xmap - based assay (luminex, austin, tx, usa) (unpub. technique). subsequently, nucleotide sequencing and phylogenetic analysis of the envelope gene from the pcr products of the 7 positive serum specimens showed that, although all the viruses belonged to the cosmopolitan genotype, 2 separate clades were present. four samples clustered with isolates from india, and the remaining 3 were most closely related to an isolate from sri lanka (figure). overall, during june, dengue ns1 antigen was detected in the serum specimens of 194 patients. in 40 other cases, the serum specimens tested positive for dengue igm by immunochromatography or capture elisa (panbio, brisbane, queensland, australia) but negative for ns1 antigen. only 5 and 3 new cases of dengue were diagnosed in july and august, respectively, and no case was reported in september. the case - patients ranged in age from1 to 91 years ; median age was 36 years, and 52.5% were male. phylogenetic relationships of dengue virus isolates from mauritius inferred by envelope (e) gene sequence by using the maximum likelihood method as implemented in paup version 4.0b10 (http://paup.csit.fsu.edu/about.html). primers used for amplification of product for sequencing were 5-aatccagatgtcatcaggaaac-3 and 5-cctatagatgtgaacactcctcc-3. the e gene sequences were consolidated from overlapping, bidirectional sequences. dengue has reemerged in mauritius after > 30 years, but the outbreak was short - lived because of the institution of control measures and the arrival of cooler and drier weather. in the affected areas, monthly mean maximum temperature dropped from 28.3c in june to 26.4c in august, and total monthly rainfall amount fell from 126.4 mm in may to 44.8 mm in august. the outbreak was also restricted to some suburbs of the capital city, possibly because of relatively warm temperatures and high population density. the high bootstrap value of 94% in the phylogenetic analysis suggests at least 2 separate importations of denv-2 occurred. in 2008, an imported case of dengue was diagnosed in a child returning from india, but control measures were rapidly instituted and no local transmission occurred. no case of dengue hemorrhagic fever was recorded in this outbreak, probably because the population has not been exposed previously to another serotype. albopictus mosquitoes, which are widely distributed in mauritius (ae. aegypti was eradicated from the country in the early 1950s as a result of a ddt indoor - spraying campaign in 19491951 to control malaria) (7). however, the rapid increase in the number of observed cases in june is more consistent with an ae. borne dengue outbreak, and a new comprehensive entomologic study is needed to exclude the possibility that ae. aegypti has recently been reintroduced into mauritius. whether denv-2 will persist in mauritius throughout the winter and lead to more cases next summer, despite maintenance of intensive mosquito control programs, is uncertain however, all practical measures must be taken to prevent introduction and transmission of another denv serotype in mauritius to minimize the risk for dengue hemorrhagic fever. in particular, surveillance of travelers from dengue - endemic regions should be instituted. the thermal scanner, recently installed at mauritius only airport, could be used to screen passengers for dengue fever because a study from taiwan suggested that fever screening at airports was a cost - effective means of identifying many imported dengue cases (8). moreover, the present policy of monitoring all persons arriving from malaria - endemic areas for fever and parasitemia could be extended to include testing for dengue in febrile travelers arriving from dengue - endemic areas. the recently opened chl molecular biology unit needs to be ready by next summer to detect and serotype dengue viruses to enable prompt diagnosis and epidemiologic evaluation of any new case. | dengue reemerged in mauritius in 2009 after an absence of > 30 years, and > 200 cases were confirmed serologically. molecular studies showed that the outbreak was caused by dengue virus type 2. phylogenetic analysis of the envelope gene identified 2 clades of the virus. no case of hemorrhagic fever was recorded. |
the mortality of cervical cancer has decreased since cervical cancer screening tests, such as papanicolaou smear, were introduced, but it remains a significant cause of death. about 7,000 - 15,000 new patients are diagnosed with cervical cancer every year and 4,600 - 6,800 patients die of cervical cancer every year worldwide [2 - 4 ]. the treatment of recurrent disease may differ by the site of recurrence, prior treatment, and degree of recurrence. the lung is commonly affected by hematogenous spread, often detected as a solitary pulmonary nodule or multiple metastases. especially in cases of a solitary pulmonary nodule, it is important to discriminate between pulmonary metastasis, primary lung cancer, and carcinoid tumors. the literature has led many investigators to focus on surgery upon a solitary pulmonary nodule and its outcome., we reviewed the medical records of cervical cancer patients who were diagnosed with lung metastases (solitary or multiple) during or after primary treatment and analyzed their clinicopathological characteristics. we analyzed 56 patients with cervical cancer who were treated with radical hysterectomy, concurrent chemoradiation, or combination chemotherapy, and developed pulmonary metastases during or after primary treatment between january 1990 and march 2014. the records include demographic characteristics, the initial stage, symptoms at the diagnosis of pulmonary metastasis, event - free duration (efd), and survival time. all statistical analyses were done using the wilcoxon sign rank, chi - square, log - rank, and anova tests. 22.0 (ibm co., armonk, ny) was used for all statistical analyses. the median age was 55 years, with a range of 36 to 61 years. ten patients (17.9%) were at stage i (4 patients were at stage ib1 and 6 patients were at stage ib2), 29 patients (51.8%) at stage ii, six patients (10.1%) at stage iii, 10 patients (17.9%) at stage iv, and one patient s (1.8%) stage was unknown. of the six patients at stage ib2, four underwent concurrent chemoradiation therapy (ccrt) for lymph node metastasis and two with suspicious lymphatic invasion who refused treatment were tracked for outcomes. forty - six patients, excluding patients at stage i, underwent combination chemotherapy every 3 weeks as a primary treatment or after ccrt depending on tumor stage : carboplatin (300 mg / m)+vp16 (100 mg / m), carboplatin (area under the curve 5 - 6 mg / m)+paclitaxel (175 mg / m), or cisplatin (50 mg / m)+5-fluorouracil (1,000 mg / m). when lung metastasis was suspected during exam at the outpatient clinic, immunohistochemistry staining of the resected or biopsied specimens for cytokeratin 7, cd56a, chromogranin, thyroid transcription factor-1, and p16 were conducted to rule out primary lung cancer. based on the immunohistochemical staining results, specimens that were diffusely stained and strongly positive for p16 diffuse and strong positive were included in the study. although a significant portion of the patients (40 patients, 71.4%) did not have pulmonary symptoms at recurrence, their pulmonary recurrence or metastasis was incidentally detected by chest x - ray or pelvic computed tomography (ct). four patients (7.1%) complained of dyspnea, four patients (7.1%) of general ache or weakness, and eight patients (14.4%) of dry cough. the histological findings of primary cervical cancer showed 45 patients with squamous cell carcinoma (scc), seven patients with adenocarcinoma, three patients with adenosquamous cell carcinoma, and one patient with small cell carcinoma. the pulmonary lesions were noticed in the right lung (n=10, 17.9%), the left lung (n=5, 8.9%), and both lungs (n=37, 66.1%). twelve patients underwent complete removal of lung metastasis : six had one metastatic nodule, four had two metastatic nodules, and two had three metastatic nodules. of the remaining 44 patients, four with malignant pleural effusion underwent paracentesis to relieve dyspnea, and 40 underwent biopsy (bronchoscopic biopsy, ct guided biopsy, open lung biopsy) because the pulmonary metastatic lesions were small and multiple. the mean efd (duration from the diagnosis of initial cervical cancer to lung metastasis) was 12 months. efd did not differ significantly by initial stage, but os decreased by initial stage (p 4 nodules (p=0.034 and p=0.045, respectively). of 22 patients who had 3 nodules, 12 were treated with surgical resection (10 patients, mass excision ; 2 patients, lobectomy). no surgical treatment was conducted on seven patients : two with the lesions in the hilar area, in one with a poor general condition and low operability, and in four with extrapulmonary metastatic lesions. os was longer in patients who were treated with surgical resection than in those who were not (p=0.006). the relationship between the incidence of pulmonary metastasis and the initial stage is extremely weak. in our study, 17.9% of the patients were at stage i, 51.8% at stage ii, 10.1% at stage iii, 17.9% at stage iv, and 1.8% at an unknown stage. recurrence occurred most frequently in patients at stage ii and decreased in those at stages iii and iv. barter. also reported that pulmonary metastasis is not associated with the initial stage. by their report, 36% of the patients were at stage i, 33% at stage ii, 11% at stage iii, and 15% at stage iv. in contrast, imachi. demonstrated that the incidence of pulmonary metastasis is high when the initial stage is advanced and that the size of primary cancer is related to the incidence of pulmonary metastasis. in our study pulmonary lesions were detected on routine follow - up chest x - ray or pelvic ct. in previous studies, the mean efd was 24 months. since many patients with pulmonary metastasis have no specific symptoms, they should receive regular and long - term chest exams. serological tumor markers, such as scc and carcinoembryonic antigen (cea), can detect tumor recurrence. rose. and ngan. indicated that scc levels are higher in the squamous cell tumors than in the non - squamous cell tumors and decreased after treatment. in our study, we investigated the association between tumor markers at the diagnosis of metastasis and survival, using the spearman correlation coefficient, suggesting that as scc or cea levels are increased, os becomes shorter (p=0.008 and p=0.006, respectively). in this study, the decision to perform surgery should be based on the location and number of metastatic lesions, and general condition. many investigators treat metastatic lesions with resection and adjuvant chemotherapy, especially for a solitary pulmonary nodule [1,7,14 - 16 ]. their criteria for surgical treatment include the following : (1) complete removal or control of primary lesions, (2) no evidence of extrapulmonary lesions, (3) patient tolerance of surgery, (4) sufficient pulmonary reserve after resection, and (5) no treatment better than surgery [1,14 - 17 ]. in our study, the mean number of the resected lesions was 1.8, the mean size was 3.3 cm, and negative resection margins were observed in all patients. when pulmonary nodules are found on chest x - ray or ct, it is important to discriminate between primary and metastatic lesions. discrimination between primary pulmonary scc and metastatic pulmonary scc from the cervix is important in therapeutic strategy for scc. to differentiate between primary pulmonary scc and lung metastatic pulmonary scc from the cervix, p16 immunohistochemical the marker p16 is a member of the ink4a family and acts as a cyclin dependent inhibitor. human papillomavirus (hpv) infection changes cell cycle regulation through degradation of prb and causes the development of carcinoma. wang. reported that immunohistochemical staining was positive for p16 in 98% of patients with metastatic pulmonary scc from cervix, which was diffusely and strongly stained. however, in 21% of patients with primary pulmonary scc, immunohistochemical staining was also positive for p16, which was focally or weakly stained. distinguished these disease entities using reverse transcription in situ polymerase chain reaction to detect hpv. in our study, we also used immunohistochemical staining for p16. pulmonary metastasis is related to prognostic factors, including efd, the number of lung masses, radiographic patterns of pulmonary metastasis, and cell types (table 3). observe that long efd (> 12 months) is associated with good prognosis. in our study, os was more prolonged in patients with longer efd. report that the number of resection mass does not influence prognosis, whereas resectability of pulmonary masses is associated with a good prognosis. in our study, os was significantly longer in patients treated with surgical resection than in those who were not (fig.. suggest that the right lung is involved more frequently than the left lung and that mediastinal and hilar involvements lead to poorer prognosis than parenchymal involvement. report that scc shows a better prognosis than non - scc, such as adenocarcinoma and adenosquamous cell carcinoma. imachi. also report that the incidence of pulmonary metastasis and positivity in peritoneal cytology are higher in the adenocarcinoma group than in the scc group. in our study, however, os and efd did not differ significantly by the histologic type. the reason for this may be that the number of non - squamous cell tumor cases was smaller than that of squamous cell tumor cases. the response rates to cytoxan and adriamycin are reported to be 65% and 16%-40%, respectively. in our study, patients were treated with platinum - based combination chemotherapy, with a response rate of 69.6%. although hematogenous spread of primary tumors more often occurs in the lung rather than the brain and liver, pulmonary metastases from cervical cancer is rare. we reviewed the medical records of patients with cervical cancer with pulmonary metastases during or after treatment and analyzed clinical and histopathological characteristics. | purposethe purpose of this study was to investigate the clinicopathological features of pulmonary metastasis from cervical cancer.materials and methodswe reviewed the medical records of 56 patients with cervical cancer who developed pulmonary metastasis after radical hysterectomy, postoperative concurrent chemoradiation or systemic chemotherapy between january 1990 and march 2014.resultsfifty-six patients were diagnosed with pulmonary metastasis from cervical cancer. the prevalence of pulmonary metastasis was 3.6%. the mean event - free duration was 12 months. twelve patients underwent surgical removal of metastatic lesions. the overall survival (os) of patients with 3 metastatic lung lesions was 40.7 months, longer than those with > 4 lesions (25 months, p=0.034). the os of patients who underwent surgical resection was 53.8 months, longer than that of those who did not (p=0.006). in addition, the os of patients with adjuvant platinum - based chemotherapy was 32.6 months (p=0.027).conclusionin this study, we found that the number of metastatic nodules, surgical resection, and postoperative platinum - based chemotherapy can influence clinical outcome. further studies on prognostic factors and successful treatment modalities are warranted. |
patients with chronic schizophrenia have higher rates of overweight and obesity than population comparisons.1 sedentary behavior is prevalent, while physical activity is low.2,3 other studies also report reduced health - related quality of life (qol) in relation to body weight that is mostly experienced as a physical problem, irrespective of age and sex.4,5 concurrently, the physical fitness of patients with chronic schizophrenia is low and is inversely related to their body weight and especially body fat,6 which renders them vulnerable to cardiovascular disease (cvd) and other adverse health outcomes,2 prospectively worsening qol further.7 however, while the relation of body composition, physical activity, and sedentary behavior with qol has been studied extensively in healthy subjects,8 there is far less corresponding data for the schizophrenia population. because dimensions of qol have been found to be associated with long - term outcome in schizophrenia,7,9,10 we decided to explore potential relationships with body composition, physical activity, and sedentary behavior in a group of patients with early schizophrenia. this would determine any correlations among body composition (especially body fat), physical activity, sedentary behavior, and qol, identifying modifiable behavioral risk factors for targeted intervention to improve qol early in the course of schizophrenia and potentially reduce cvd morbidity and mortality. a group of 36 subjects with early schizophrenia took part in a comprehensive metabolic assessment after written informed consent was obtained in accordance with procedures approved by the university of toronto research ethics board. body weight and height were measured in kilograms (kg) and meters (m), and body mass index (bmi) was calculated (kg / m). subjects were classified as normal weight if bmi was below 25, overweight if bmi was between 25 and 29.9, and obese if bmi was 30 or above, in accordance with national heart, lung, and blood institute guidelines.11 the subjects were all on stable doses of antipsychotics for at least 2 weeks and were not actively psychotic at the time of the study. subjects provided sociodemographic information and completed the rand sf 36-item short form health survey.12 the rand sf-36 qol questionnaire is a widely used and freely accessible measure of health - related qol13 that has also been validated in schizophrenia.14 the questionnaire yields eight different items of functioning physical functioning, role limitations due to physical problems, vitality, bodily pain, social functioning, role limitations due to emotional problems, mental health, and general health13 which are summarized into a physical component score and a mental component score. these two summary scores alone have been shown to account for 85% of reliable variance of the eight sf-36 subscores, without losing significant information.15 whole - body dual - energy x - ray absorptiometry (dxa) scans (hologic qdr-4500a densitometer ; hologic inc, bedford, ma) were completed to determine body composition, including total body mass and fat ; total truncal mass and fat ; and a truncal / le ratio (proxy measure of body fat distribution). the short form international physical activity questionnaire (sf - ipaq), a structured screening instrument previously validated for the measurement of physical activity in schizophrenia,16 was used to ascertain habitual physical activity ; the questionnaire aims to elicit the amount of time in minutes spent daily in three different physical activity categories (expressed as metabolic equivalents per minute, or metmin / week) and in idle time time spent sitting or lying (not sleeping), ie, sedentary behavior for 1 week. the ipaq has shown good correlation with acceler - ometry in schizophrenia.16 spss software (for windows, version 18 ; spss inc, chicago, il) was employed for data analysis. student s t - tests and, where appropriate, regression and analysis of variance (anova) were employed to look for statistical differences between the means of two or more variables. bivariate correlations among sociodemographics, sf-36 scores, body composition, and activity level were then calculated. for each sf-36 score with significant bivariate correlation with sociodemographic variables, activity level, and obesity measures, of the 36 participants, 26 had a diagnosis of schizophrenia (61.5%) and ten had a diagnosis of schizoaffective disorder (38.5%). seventeen (47.2%) were of european, 13 (36.1%) of african, three (8.3%) of asian, and two (5.5%) of undetermined ethnic background. average age among participants was 25.1 (3.6) years ; range (1934) and duration of illness was 30 (18) months. twenty patients were receiving clozapine (n = 10) or olan - zapine (n = 10), and 16 were taking risperidone (n = 7), aripiprazole (n = 4), or ziprasidone (n = 5). twelve participants (33.1%) were of normal weight (bmi 2024.9), 13 (36.1%) were overweight (bmi 2529.9), and eleven (30.6%) were obese (bmi > 30). sixty - two percent were smokers, (8.7 8 cigarettes / day, for 48 5.2 months). sixty - seven percent were unemployed, and 60% lived independently or at home with family. physical activity level was 1707 (1186) metmin / week, and sedentary time (excluding sleep) was 2916 (1403) minutes / week. there were no significant differences in age, duration of illness, bmi, smoking, employment, and living status and no differences in physical activity and sedentary time between males and females or across ethnicities. females had higher body fat content than males (30.8% 6.9% vs 24.7% 10.6% ; t = 2.6, df = 34 ; p = 0.015). total body fat (f = 14 ; p = 0.001), lean body mass (f = 10.2 ; p = 0.001), and sedentary behavior (f = 5 ; p = 0.013) signifi - cantly increased across bmi categories (normal weight, overweight, obese). activity level (metmin / week) decreased, but the difference was not statistically significant (f = 0.3 ; p = 0.71). total body fat correlated with sedentary behavior (r = 0.62 ; p = 0.001), but not with activity level. total lean mass was not correlated with metmin / week (r = 0.049 ; p = 0.8) but was inversely correlated with sedentary time (r = 0.39 ; p = 0.03). we did not find ethnic differences in activity levels (f = 0.57 ; p = 0.64) or sedentary behavior (f = 1.8 ; p = 0.9). sf-36 scores of study subjects were compared to standardized canadian population scores and are shown in table 1. among study participants, physical functioning (f = 4.41 ; p = 0.02) and role physical (f = 6.02 ; p = 0.006) significantly worsened across bmi categories. as compared to nonobese subjects, obese subjects (bmi > 30) had worse physical functioning (t = 2.9 ; p = 0.006), role physical (t = 3.5 ; p = 0.001), and general health (t = 2.1 ; p = 0.036) scores. smokers had worse physical functioning (t = 2.1 ; p = 0.04) and more bodily pain (t = 2.8 ; p = 0.009) than nonsmokers. employed participants had significantly better role emotional functioning (t = 29 ; p = 0.044) than unemployed participants. sedentary behavior correlated with role physical (r = 0.331 ; p = 0.001) and vitality (r = 0.53 ; p = 0.001) and correlated inversely with emotional wellbeing (r = 0.403 ; p = 0.018). total body fat inversely correlated with physical function (r = 0.39 ; p = 0.027), role physical (r = 0.466 ; p = 0.008), and vitality (r = 0.49 ; p = 0.005). physical functioning was predicted by smoking status (f = 4.7 ; p = 0.016) and sedentary behavior (f = 4.4 ; p = 0.043). bodily pain was predicted by age (f = 6.1 ; p = 0.006) and total body fat percentage (f = 6.9 ; p = 0.014). total body fat (f = 14 ; p = 0.001), lean body mass (f = 10.2 ; p = 0.001), and sedentary behavior (f = 5 ; p = 0.013) signifi - cantly increased across bmi categories (normal weight, overweight, obese). activity level (metmin / week) decreased, but the difference was not statistically significant (f = 0.3 ; p = 0.71). total body fat correlated with sedentary behavior (r = 0.62 ; p = 0.001), but not with activity level. total lean mass was not correlated with metmin / week (r = 0.049 ; p = 0.8) but was inversely correlated with sedentary time (r = 0.39 ; p = 0.03). we did not find ethnic differences in activity levels (f = 0.57 ; p = 0.64) or sedentary behavior (f = 1.8 ; p = 0.9). sf-36 scores of study subjects were compared to standardized canadian population scores and are shown in table 1. among study participants, physical functioning (f = 4.41 ; p = 0.02) and role physical (f = 6.02 ; p = 0.006) significantly worsened across bmi categories. as compared to nonobese subjects, obese subjects (bmi > 30) had worse physical functioning (t = 2.9 ; p = 0.006), role physical (t = 3.5 ; p = 0.001), and general health (t = 2.1 ; p = 0.036) scores. smokers had worse physical functioning (t = 2.1 ; p = 0.04) and more bodily pain (t = 2.8 ; p = 0.009) than nonsmokers. employed participants had significantly better role emotional functioning (t = 29 ; p = 0.044) than unemployed participants. sedentary behavior correlated with role physical (r = 0.331 ; p = 0.001) and vitality (r = 0.53 ; p = 0.001) and correlated inversely with emotional wellbeing (r = 0.403 ; p = 0.018). total body fat inversely correlated with physical function (r = 0.39 ; p = 0.027), role physical (r = 0.466 ; p = 0.008), and vitality (r = 0.49 ; p = 0.005). physical functioning was predicted by smoking status (f = 4.7 ; p = 0.016) and sedentary behavior (f = 4.4 ; p = 0.043). bodily pain was predicted by age (f = 6.1 ; p = 0.006) and total body fat percentage (f = 6.9 ; p = 0.014). results from our sample indicate that early schizophrenia is associated with lower qol than observed in mentally healthy population comparisons and that this manifests primarily in lower physical functioning. it is important to note that all study participants with early schizophrenia were receiving stable doses of antipsychotic medication prior to study inclusion and were not considered actively psychotic. significant improvements in qol can be expected with initiation and maintenance of treatment with antipsychotic medication in early schizophrenia,18 and in this context ie, despite treatment the qol in our sample still remained well below population standards. given the key role the concept of qol plays in both short- and long - term outcomes of schizophrenia, we believe that the development of specific treatment interventions targeted at improving qol as early as possible in the course of schizophrenia is indicated.9 to that end, we can report that sedentary behavior, more so than physical activity, was a key determinant of low qol in early schizophrenia ; it also related more strongly than physical activity to body fat content, confirmed in larger samples as a proxy marker of cardiovascular disease risk.19,20 here we corroborate the results of vancampfort,3 who found similar relationships in patients with chronic schizophrenia. our findings suggest that a potential way to improve the physical component of qol might be targeted reduction of sedentary behavior through substitution of inactivity with habitual physical activity. employment was associated with better emotional functioning and could represent one avenue to reduce sedentary behavior ; this might well result in additional benefits, such as improvements in the mental component of qol. we would recommend that case managers and other clinicians provide more, and more frequent, activity - based interventions, such as walking groups to replace traditional groups in which participants sit for 4550 minutes. mental health facilities could establish collaborations with such diverse services as ymca / ywca, gyms, churches, and other agencies that may allow patients access to free or low - cost environments in which physical activity can be practiced, not only to improve qol but also to reduce disablement.21 we also add, albeit indirectly, to the accumulating evidence that links obesity in schizophrenia with increased cvd risk.22 our results, together with previous observations of increased body fat content5,23 and prevalent sedentary behavior2 in schizophrenia, facilitate an unfavorable cvd risk factor constellation24 that is already present early in the course of schizophrenia and can have considerable negative impact on health - related qol, as well.2 the cvd risk, emerging after relatively brief exposure to antipsychotic medication, warrants attention and suggests that more preventative action is required.25,26 smoking emerged as important determinant of qol, reducing physical functioning. because smoking is highly prevalent in patients with schizophrenia, this may represent another important treatment target, with the potential both to reduce the cvd risk inherent to smoking and to improve qol. the potential to improve qol, a key clinical outcome, may motivate patients with early schizophrenia to engage in physical activity, aimed at increasing habitual activity levels. increased habitual physical activity replacing sedentary behavior would improve body composition and lead to a more favorable cvd risk profile.27 sedentary behaviors, such as watching television, idle sitting, reading, or using a computer, can be reduced by removing environmental and access barriers to physical activity19 and by selecting subjectively enjoyable physical activities28 or providing structured rehabilitative opportunities.29 the study also highlights that the inverse relation between body weight and qol, as previously determined in chronic schizophrenia samples,4,30 is present early in the schizophrenia disease process and warrants the clinician s vigilance and rapid intervention to avert further deterioration of qol (and increase in cvd risk) in later stages of schizophrenia. ideally, a preventative framework incorporating targeted physical activity to replace sedentary behavior and perhaps smoking cessation as part of a structured lifestyle intervention would be desirable.26 there are a number of limitations associated with this preliminary study. due to funding constraints in a narsad young investigator award, the number of first - episode patients enrolled was small, limiting generalizability of results ; larger sample sizes are needed for firm conclusions, and our results may lead to conceptualization of larger follow - up protocols. the investigators were dependent on clinician referrals and agreement by patients to participate ; thus, various unmeasured sampling biases might have affected the sample. it is known that negative and depressive symptoms can increase sedentary behavior ; it is possible, therefore, that among our study participants, such symptoms may have interfered with activities, worsening sedentary behavior, and with qol. all our patients were clinically stable at time of enrollment, without significant medication changes during the preceding 2 months, however, and a majority lived successfully in the community. although the ipaq has been validated against accelerometry in patients with schizophrenia,16 we acknowledge the limitations of using a questionnaire to assess physical activity and sedentary behavior, as it may not cover short bursts of activity (less than 10 minutes). there are a number of limitations associated with this preliminary study. due to funding constraints in a narsad young investigator award, the number of first - episode patients enrolled was small, limiting generalizability of results ; larger sample sizes are needed for firm conclusions, and our results may lead to conceptualization of larger follow - up protocols. the investigators were dependent on clinician referrals and agreement by patients to participate ; thus, various unmeasured sampling biases might have affected the sample. with the provided funding, specific outreach to female patients was not feasible. also, we did not formally assess symptoms of psychopathology. it is known that negative and depressive symptoms can increase sedentary behavior ; it is possible, therefore, that among our study participants, such symptoms may have interfered with activities, worsening sedentary behavior, and with qol. all our patients were clinically stable at time of enrollment, without significant medication changes during the preceding 2 months, however, and a majority lived successfully in the community. although the ipaq has been validated against accelerometry in patients with schizophrenia,16 we acknowledge the limitations of using a questionnaire to assess physical activity and sedentary behavior, as it may not cover short bursts of activity (less than 10 minutes). as a consequence | objective : to examine adiposity and sedentary behavior in relation to health - related quality of life (qol) in patients with early schizophrenia.methods:a cross - sectional study was used to assess adiposity by dual - energy x - ray absorptiometry scans, habitual physical activity and idle sitting time by the short form international physical activity questionnaire, and health - related qol by the rand medical outcomes study sf-36. qol scores were compared with age - adjusted canadian normative population data.results:there were 36 participants with early schizophrenia, average age 25.1 (3.6). twenty - nine (72.5%) were males. mean illness duration was 30 (18) months, and mean body mass index was 28.3 (5). females had higher body fat content than males (30.8 6.9 vs 24.7 10.6 ; t = 2.6, df = 34 ; p = 0.015). total body fat (f = 14 ; p = 0.001), lean body mass (f = 10.2 ; p = 0.001), and sedentary behavior (f = 5 ; p = 0.013) significantly increased across body mass index categories. total body fat was correlated with sedentary behavior (r = 0.62 ; p = 0.001), and total lean body mass was negatively correlated with sedentary behavior (r = 0.39 ; p = 0.03). based on sf-36 scores, participants had significantly lower physical functioning (p = 0.0034), role physical (p = 0.0003), general health (p < 0.0001), vitality (p = 0.03), and physical component scores (p = 0.003) than canadian population comparisons. habitual sedentary behavior, more than activity or adiposity levels, was associated with health - related qol in early schizophrenia.conclusion:health-related qol is lower in early schizophrenia and is predominantly experienced in the physical domain. qol in early schizophrenia relates to sedentary behavior more than to activity and adiposity levels. |
the surgical technique of the midurethral sling (mus) procedure is much simpler than previous anti - incontinence surgeries, and the procedure can be performed under local anesthesia. the success rate of the procedure is reported to have reached 90% [1 - 4 ]. interlocked with the increase in social interest in urinary incontinence and in social activities of aged women, these advantages of the mus procedure have resulted in an explosive increase in operations for female urinary incontinence. most previous reports regarding mus outcomes generally tended to investigate only patients ' subjective satisfaction, but recent reports have strived to systematically observe improvement in quality of life (qol) by means of objective methods. we intended to investigate the impact of the mus procedure on not only cure and satisfaction with the surgery but also on the objective qol of incontinent patients. in addition, we intended to define clinical and urodynamic factors that affect the qol of female patients with urinary incontinence. between june 2006 and june 2007, 118 consecutive female patients with a complaint of urinary incontinence underwent the mus procedure by a single operator in our institute. because the qol of patients who had failed previous anti - incontinence surgery can differ from that of patients who had not undergone an operation previously, the former group was excluded. of the 118 patients, 93 patients (meansd age, 54.510.2 years) received follow - up tests at 1 month and 1 year after surgery and responded to the i - qol questionnaire. the remaining 25 patients who did not respond for follow - up tests were excluded. preoperative evaluations included a medical history, i - qol questionnaire, obstetric history, and physical examination including q - tip test, stress test, 3-days ' voiding diary, 1-hour pad test, and multi - channel urodynamic investigation. the analysis of urodynamic parameters included peak urinary flow, maximum cystometric capacity, post - void residue (pvr), and valsalva leak - point pressure (vlpp). each of the procedures [tension - free vaginal tape (tvt), intravaginal slingplasty (ivs), and transobturator sling (tot) ] was performed by a single experienced surgeon using the standard technique. anesthesia was selected from among a combination of light sedation with local anesthesia, spinal anesthesia, or general anesthesia after consultation with the patients and according to their general health status. all patients were asked to visit the clinic at 1 month and 1 year after surgery. at those times, they were evaluated with a careful symptom review, stress test, i - qol questionnaire, uroflowmetry, and pvr measurement and for postoperative complications. the total score was the sum of all questions (range, 0 - 110). a low total score meant that the patient was deeply troubled ; on the other hand, higher scores meant that the patient was less burdened. the i - qol questionnaire consisted of three subscales : avoidance and limiting behavior (al), psychosocial impacts (pi), and social embarrassment (se). the subscale formula was as follows : subscale score (range : 0 - 100)=[(the sum of the items - lowest possible score)100/[(highest possible score - lowest possible score) ] cure of urinary incontinence was defined as the absence of any episodes of involuntary urine leakage during stressful activities and a stress cough test. the cough test was performed with the patient in a standing position with a full bladder. improvement was defined subjectively as a significant reduction of urine leakage, such that it did not require further treatment. patients were classified as " satisfied, " " so - so, " and " dissatisfied, " for the assessment of satisfaction after surgery. urgency was defined as a sudden compelling desire to pass urine that was difficult to defer. mixed urinary incontinence (mui) was defined as complaint of an involuntary leakage of urine associated with urgency and also with symptoms of stress urinary incontinence (sui). we analyzed clinical and urodynamic factors to determine which preoperative and intraoperative factors influenced cure and satisfaction by mus for urinary incontinence. univariate analysis was performed by using fisher 's exact and chi - square tests. to determine predictive factors affecting cure and satisfaction, both " so - so " and " dissatisfied " were sorted as " not satisfied " for the analysis of satisfaction. student 's t - test was used to determine which clinical symptoms and urodynamic factors affected the qol of the incontinent patients. a 5% level of significance was used for all statistical testing, and all statistical tests were two - sided. analysis was performed by using the statistical software spss (14.0ko for windows, release 14.0.2). between june 2006 and june 2007, 118 consecutive female patients with a complaint of urinary incontinence underwent the mus procedure by a single operator in our institute. because the qol of patients who had failed previous anti - incontinence surgery can differ from that of patients who had not undergone an operation previously, the former group was excluded. of the 118 patients, 93 patients (meansd age, 54.510.2 years) received follow - up tests at 1 month and 1 year after surgery and responded to the i - qol questionnaire. the remaining 25 patients who did not respond for follow - up tests were excluded. preoperative evaluations included a medical history, i - qol questionnaire, obstetric history, and physical examination including q - tip test, stress test, 3-days ' voiding diary, 1-hour pad test, and multi - channel urodynamic investigation. the analysis of urodynamic parameters included peak urinary flow, maximum cystometric capacity, post - void residue (pvr), and valsalva leak - point pressure (vlpp). each of the procedures [tension - free vaginal tape (tvt), intravaginal slingplasty (ivs), and transobturator sling (tot) ] was performed by a single experienced surgeon using the standard technique. anesthesia was selected from among a combination of light sedation with local anesthesia, spinal anesthesia, or general anesthesia after consultation with the patients and according to their general health status. all patients were asked to visit the clinic at 1 month and 1 year after surgery. at those times, they were evaluated with a careful symptom review, stress test, i - qol questionnaire, uroflowmetry, and pvr measurement and for postoperative complications. the total score was the sum of all questions (range, 0 - 110). a low total score meant that the patient was deeply troubled ; on the other hand, higher scores meant that the patient was less burdened. the i - qol questionnaire consisted of three subscales : avoidance and limiting behavior (al), psychosocial impacts (pi), and social embarrassment (se). the subscale formula was as follows : subscale score (range : 0 - 100)=[(the sum of the items - lowest possible score)100/[(highest possible score - lowest possible score) ] cure of urinary incontinence was defined as the absence of any episodes of involuntary urine leakage during stressful activities and a stress cough test. the cough test was performed with the patient in a standing position with a full bladder. improvement was defined subjectively as a significant reduction of urine leakage, such that it did not require further treatment. patients were classified as " satisfied, " " so - so, " and " dissatisfied, " for the assessment of satisfaction after surgery. urgency was defined as a sudden compelling desire to pass urine that was difficult to defer. mixed urinary incontinence (mui) was defined as complaint of an involuntary leakage of urine associated with urgency and also with symptoms of stress urinary incontinence (sui). we analyzed clinical and urodynamic factors to determine which preoperative and intraoperative factors influenced cure and satisfaction by mus for urinary incontinence. univariate analysis was performed by using fisher 's exact and chi - square tests. to determine predictive factors affecting cure and satisfaction, both " so - so " and " dissatisfied " were sorted as " not satisfied " for the analysis of satisfaction. student 's t - test was used to determine which clinical symptoms and urodynamic factors affected the qol of the incontinent patients. a 5% level of significance was used for all statistical testing, and all statistical tests were two - sided. analysis was performed by using the statistical software spss (14.0ko for windows, release 14.0.2). on the basis of the stamey grading system, the number of female patients with grade i incontinence was 74 (79.6%), the number with grade ii was 14 (15.1%), and the number with grade iii was 5 (5.4%). clinical and urodynamic data and results from the preoperative tests are shown in table 1 and table 2. in the preoperative assessment of i - qol, the total i - qol score and subscale points (al, pi, and se points) were 61.121.0, 45.923.4, 48.426.2, and 34.926.5 points, respectively. at 1 month after surgery, the i - qol scores were higher than preoperatively (89.017.9, 75.116.7, 79.020.9, and 72.124.1 points, respectively ; p0.05) (table 3). preoperative average i - qol scores of " cured ", " improved ", and " failed " patients were 62.721.7, 56.717.2, and 66.222.1 points, respectively. i - qol scores at 1 year after surgery were 107.74.3, 94.77.3, and 54.67.3 points, respectively. i - qol scores of the cured and improved patients increased at 1 year after surgery (p0.05). patients in whom the operation had failed did not show any improvement of qol after the procedure (p>0.05) (fig. patients who had urgency or large amounts of urine leakage (more than 10 g in a 1 hour pad test) before surgery showed lower total i - qol scores than did patients without urgency or with small amounts of leakage (p<0.05). at 1 year after surgery, total i - qol and the increase in the i - qol score of the patients who had large amounts of urine leakage preoperatively were higher than in the patients with small amounts of leakage (p<0.01) (table 4). medical reports on the success rates or treatment effects of various treatments have been precisely performed with objective methods. reports about qol and patient satisfaction after treatment, however, generally tend to rely on only simple subjective measures. recently, many medical reports have strived to objectively observe the qol associated with disease and its change after treatment. accordingly, several questionnaires have been developed and used in the field of female urology, including urinary incontinence. many questionnaires for assessment of qol in urinary incontinence are being used internationally after proper official linguistic validation ; typical questionnaires include the i - qol and the king 's health questionnaire. the i - qol questionnaire, which was used in the present study, was recommended most preferentially at the 2nd international consultation on incontinence for assessing the qol of patients with urinary incontinence. papanicolaou assessed the negative impact of urinary incontinence on qol in female european patients through use of the i - qol questionnaire. they reported that the i - qol scores of patients with " low grade " or " middle or high grade " incontinence were 84.5 and 64.5 points, respectively, and that decreases in the i - qol score of incontinent patients were significantly dependent on symptom grade. in several studies of the mus procedure, the i - qol questionnaire has been used to assess the qol of incontinent patients and changes in qol after the procedure. campeau reported an increment of 38.7 points in the i - qol after the tvt procedure (from 57.8 to 96.5 points), and kim reported an increment of 32.9 points in the i - qol after the ivs procedure (from 61.5 to 94.4 points). also, kocjancic reported an increment of 40 points in the i - qol after the tot procedure (50.6 to 90.6 points). scores in our study at 1 year after the mus procedure showed an increment of more than 30 points in the i - qol from the preoperative state (61.1 to 98.4 points). our study included all patients who underwent the tvt, ivs, and tot procedures. because there are differences in approach and in the types of mesh used in each type of mus procedure, however, objections to the design of our study may arise. however, many articles have observed that there are no differences in cure or satisfaction between each type of mus. the stamey grades of the patients in this study tended toward grade i and ii. because the success rate of the mus procedure in patients with stamey grade iii incontinence is lower than that in patients with grade i and ii, grade iii patients received the remeex system procedure if indicated. the significant rise in i - qol at 1 month and 1 year after surgery can be shown as gradual improvement in qol as time elapsed after the procedure. at 1 month after surgery, however, surgical wound pain, temporary vaginal bleeding or discomfort caused by vaginal discharge may affect qol. because patients are prohibited from sexual intercourse for 1 month after surgery, question no. 22 on the i - qol about sexual intercourse may cause confusion. therefore, in order to observe the changes in the qol by time more accurately, the first postoperative i - qol assessment should be measured at least 3 months after surgery. in this study, patients who had urgency or large amounts of urine leakage before surgery showed lower total i - qol scores than did patients without urgency or with small amounts or urine leakage. however, this difference in i - qol scores was no longer evident after the procedure. higher i - qol and increment of i - qol scores were observed at 1 year after surgery in patients with large amounts of urine leakage. it may be that patients with large amounts of urine leakage were severely bothered by their symptoms and experienced a higher satisfaction with the mus procedure, resulting in a larger increase in 1-year i - qol. our finding of no difference in the average increase in i - qol between the cured and improved groups implies that simply improving symptoms through mus considerably enhances the qol of incontinent patients. therefore, for the improvement of qol, the mus procedure should be actively applied in incontinent patients, even in patients with urgency or large amounts of urine leakage. the i - qol assessment revealed a significant impairment of qol in incontinent women compared with that in continent women without voiding symptoms, but the mus procedure effectively improved these women 's qol. although urinary urgency and large amounts of urine leakage significantly reduced the preoperative qol in patients with urinary incontinence, the mus procedure effectively improved the qol regardless of these factors. even the qol of patients with only symptom improvements and who were still incontinent showed significant recovery ; therefore, the mus procedure should be actively applied to improve qol in incontinent patients, even in women with urgency or large amounts of urine leakage. | purposethis study was designed to objectively assess the impediment of incontinence to quality of life (qol) in females and its improvement by the midurethral sling (mus) procedure.materials and methodsfrom june 2006 to june 2007, 93 female patients underwent the mus procedure at our institute because of urinary incontinence. the incontinence quality of life (i - qol) questionnaire was administered to measure the qol of the incontinent patients before and 1 and 12 months after the mus procedure. preoperative data and urodynamic factors were analyzed retrospectively by i - qol scores to identify factors that may affect the qol of incontinent patients.resultsthe average preoperative i - qol score of the 93 patients was 61.121.0 points. at 1 year after surgery, the average i - qol score was found to have improved to 98.420.7 points. there were no significant differences between stress and mixed urinary incontinence in terms of cure and satisfaction (p>0.05). i - qol scores of the cured and improved patients increased at 1 year after surgery (p0.05). although urinary urgency and large urine leak amounts significantly reduced preoperative qol in incontinent patients, the mus procedure effectively improved the qol regardless of these factors.conclusionspreoperative i - qol assessment revealed a significant impairment of qol in incontinent women, but the mus procedure effectively improved these women 's qol. |
grant support was received from : isciii and feder funds pi13/00047 ; eutox, cp12/03262, cp14/00133, pi15/00298, pi14/00386, pi15/01460, pi16/01900, pi16/02057 ; diabetescancerconnect pie13/00051 ; sociedad espaola de nefrologia ; friat ; and isciii - retic redinren rd016/009. m.d.s.- n. ; joan rodes to b.f.- f ; ahnd programa intensificacin actividad investigadora (isciii / agencia lan - entralgo / cm) to a.o. | abstractexponential technologies double in power or processing speed every year, whereas their cost halves. deception and disruption are two key stages in the development of exponential technologies. deception occurs when, after initial introduction, technologies are dismissed as irrelevant, while they continue to progress, perhaps not as fast or with so many immediate practical applications as initially thought. twenty years after the first publications, clinical proteomics is still not available in most hospitals and some clinicians have felt deception at unfulfilled promises. however, there are indications that clinical proteomics may be entering the disruptive phase, where, once refined, technologies disrupt established industries or procedures. in this regard, recent manuscripts in ckj illustrate how proteomics is entering the clinical realm, with applications ranging from the identification of amyloid proteins in the pathology lab, to a new generation of urinary biomarkers for chronic kidney disease (ckd) assessment and outcome prediction. indeed, one such panel of urinary peptidomics biomarkers, ckd273, recently received a food and drug administration letter of support, the first ever in the ckd field. in addition, a must - read resource providing information on kidney disease - related proteomics and systems biology databases and how to access and use them in clinical decision - making was also recently published in ckj. |
the causes of subluxation of the lens include trauma, marfan 's syndrome, weill - marchesani syndrome, homocystinuria, idiopathic, and hereditary cases. with the development of newer techniques and devices, complications in these cases these devices include capsular tension ring (ctr), modified ctr with single or double fixation point [13 ], capsular tension segment (cts), and recently introduced capsular anchor device. additionally, the use of iris hooks has further improved the stabilization of capsular bag during the cataract surgery. manual small incision cataract surgery (msics) and phacoemulsification are the two widely practiced surgical procedures for cataract extraction. the various techniques of msics include wire loop, phaco - sandwich, and phacosection technique. the novel innovation of anterior chamber maintainer (acm) by blumenthal and moisseiev permits a high - pressure and high - flow system, providing a physiological environment throughout the surgery requiring minimal intraocular instrumentation. the procedure, with an initial learning curve, is highly effective, applicable to all grades of cataracts, has minimum intraocular instrumentation resulting in an early rehabilitation of the patient. the msics has become popular in india and has been found to be effective and economical [9, 10 ] requiring less capital investment although phacoemulsification gives better unaided visual acuity. therefore, this study was carried out with the aim of comparing the feasibility and complications of cataract surgery with endocapsular supporting devices and intraocular lens implantation in subluxated cataract between phacoemulsification and msics. this was a prospective, interventional, consecutive case series performed at the guru nanak eye centre, new delhi. sixty eyes with subluxated cataract, who presented between january 2007 and march 2011, were enrolled consecutively and randomly distributed in two equal groups with thirty patients each. in group a, phacoemulsification and in group b, manual small incision cataract surgery was done with implantation of posterior chamber intraocular lens (iol) and use of endocapsular supporting device as required. all patients with subluxated crystalline lens with visually significant cataract were evaluated for cataract surgery. the assessment included evaluation of visual acuity, refractive error, and intraocular pressure (iop). the biomicroscopy was performed for grading of nuclear sclerosis, degree of subluxation with zonular loss or any weakness, vitreous in anterior chamber, and injury to any other ocular structure. the imaging included ultrasound biomicroscopy for anterior chamber angle and zonular status and b - scan for posterior segment evaluation. the exclusion criteria included age less than 18 yrs, intraocular pressure more than 21 mm of hg, scleral thinning, more than 210 degree subluxation, pseudoexfoliation, active uveitis, corneal opacity / scarring, and history of open globe injury and retinal detachment. the degree of subluxation was divided into three groups (mild 180 degree). the different endocapsular supporting devices were used according to the extent of zonulysis based on the decentration / tilt of the lens preoperatively as well as its mobility intraoperatively. a capsular tension ring (ctr) was used for mild degree of zonulysis, a single - point fixation capsule device (cionni modified ctr / single cionni) was used for moderate degree, and for cases with severe degree, a two - point fixation capsule device was used. in case of inadvertent capsular injury, the endocapsular supporting device was not used and transscleral suture fixation of pmma iol was carried out. in all other cases, foldable, all the surgeries were performed under local anaesthesia using mixture of 3 ml lignocaine 2% and 3 ml bupivacaine 0.5% by a single surgeon (r.g.), competent in both the msics and phacoemulsification. depending on the site of zonulysis, the wound was constructed either superiorly or temporally. in group a, a clear corneal incision of 3.0 mm was made and anterior vitrectomy was done to remove any vitreous in the anterior chamber. anterior capsulorhexis of adequate size (55.5 mm) was carried out using forceps and high - molecular weight viscoelastic device. disposable nylon hooks to stabilize the capsular bag were placed in patients with > 180 degree zonulysis. phacoaspiration was then performed and posterior chamber iol implantation was done. in group b, msics was performed using 6.06.5 mm sclerocorneal tunnel using modified blumenthal technique. the anterior chamber maintainer (acm), a hollow steel tube with a 0.9 mm outer diameter and 0.65 mm inner diameter, was fixed away from the site of zonulysis. the nucleus was prolapsed out of the bag and then out of the tunnel using assisted delivery, if required. cortical clean up was carried out, then iol was implanted and using figure of infinity the section was sutured. any additional procedure required in two groups the procedures like anterior vitrectomy, pupilloplasty, and iridodialysis repair were labelled as minor and procedures like lensectomy with transscleral suture fixation of iol (sfiol) was labelled as major. the patients were followed up for a period of three months, and following parameters were recorded : visual acuity, refractive error, iol centration / tilt and complications if any were noted. the method of guyton and coauthors and the formula of kozaki and coauthors were used to calculate iol decentration and tilt. the main outcome measures were the feasibility and success of performing the cataract surgery with use of endocapsular supporting devices and iol implantation in two groups. postoperative complications, best corrected visual acuity (bcva), and iol decentration / tilt were also compared. the statistical analysis was carried out using mann whitney test (for parametric data) and mantel haenszel and fisher exact test (for nonparametric data). the mean age at surgery in group a (phacoemulsification) was 41.80 12.80 years and group b (msics) was 39.86 12.75 years. the majority of the patients in both the group were male, 24 (80%) in group a and 20 (66.7%) in group b. for the statistical analysis the decimal acuity was used. the mean visual acuity in group a was 0.14 0.10 and in group b was 0.13 0.10, with kruskal - wallis h value of 0.01 and p value of 0.91. the majority of the patients had a moderate degree of subluxation, 18 (60%) in group a and 17 (56.67%) in group b. between the two groups, the p value of mild and moderate type of subluxation was calculated using the mantel haenszel test, and for severe subluxation, fisher exact test was used due to few number of cases. the cause of subluxation was trauma (56.7% in group a and 70% in group b) followed by idiopathic (36.7% in group a and 20% in group b). the congenital causes included the marfans syndrome in two patients in group a and three patients in group b. the groups were statistically comparable. table 1 also shows the distribution of the patients according to the grade of nucleus in the two groups. the majority of the patients, 19 (63.33%) in group a and 20 (67.67%) in group b had grade of 2 + or less. the mean value was 2.2 0.80 in group a and 2.13 0.81 in group b (kruskal - wallis h value of 0.10 and p value of 0.74). table 2 shows the postoperative comparison of two groups. in group a, the implantation of the endocapsular supporting device and in the bag iol was successful in 27 (90.0%) patients, while in group b, it was successful in 23 (76.67%) patients. this difference was statistically insignificant (= 1.89, p value = 0.16). the comparison of groups for the implantation of an intended endocapsular devices showed that in 25 (83.33%) patients in group a and 20 (66.67%) patients in group b it was successful. this difference was statistically insignificant (= 2.19, p value = 0.14). in group a, 3 cases required transscleral suture fixation of iol and 2 cases required two - point fixation ctr in place of single fixation device (due to increased dehiscence during chopping). in group b, 7 cases required transscleral suture fixation of iol and 3 cases required two point fixation ctr in place of single fixation device. one case in group b had increase in dehiscence from 100 to 160 degree intraoperatively and intended single fixation capsule device was implanted. the comparison of groups in relation to requirement of additional procedure showed that 9 (30%) and 13 (43.33%) cases required it in groups a and b, respectively. though the number of these procedure were more in group b, but it was not significant (= 1.15, p value = 0.28). similarly, more intraoperative complications were noted in group b, 36.67% versus 20% but this was insignificant (= 2.02, p value = 0.15). the mean bcva achieved in groups a and b was 0.66 0.23 and 0.68 0.28, respectively (2 = 0.11, p value = 0.73). the mean postoperative spherical and cylindrical error, in group a, was 0.11 0.25 d and 0.58 0.43 d, respectively, and group b was 0.15 0.33 d and 0.95 0.48 d, respectively. the iol decentration of > 1 mm and tilt of > 15 degree was considered significant. the iol decentration developed in one patient in group b. in this case, dehiscence increased to 210 degree from 160 degree intraoperatively and implantation of two - point fixation ctr was carried out. the iol tilt was noticed in one patient in group a, where 200 degree of subluxation was present and implantation of two - point fixation device was done. the success of cataract surgery in subluxated cataract depends upon the ability to implant an endocapsular supporting device and in - the - bag iol. historically, surgical removal of the subluxated lens has been undertaken with great caution because of numerous reports of complications and poor visual outcomes. the intracapsular cataract surgery is avoided in such patients due to risk of vitreous loss, retinal detachment, and persistent inflammation as well as anterior chamber iol-(aciol-) related complications. retention of capsular bag is preferred, unless the lens is dislocated in the posterior vitreous, where pars plana lensectomy is indicated. when the posterior capsule ruptures or there is lack of zonular support, an iol can be placed in the anterior chamber between the cornea and iris, as in iris - fixated and closed or open - loop aciol, or it can be implanted in the posterior chamber within the ciliary sulcus posterior to the iris, as in sutured iris - fixated and scleral fixated posterior chamber iol. because of their anatomic advantage, sfiol provides better visual acuity and binocularity and avoids the complications of aciols, which are seen more with rigid closed loop iols than with open - loop and iris - claw iols. during cataract surgery in patients with subluxated lens, further damage to compromised zonules is achieved by use of dispersive viscoelastics, bimanual method of capsulorhexis using forceps and cystitome, cortical cleavage hydrodissection, endocapsular supporting devices, osher 's slow - motion phacoemulsification technique, tangential stripping motion, and use of foldable acrylic iol [16, 17 ]. in a randomized controlled trial in india, msics has been shown to be as effective and safe as phacoemulsification with a small difference in uncorrected visual acuity and astigmatism. moreover, it also provides closed stable chamber with a well - expanded and stable capsular bag for greater control, as well as minimizing further vitreous loss and therefore, risk of retinal detachment, glaucoma, and other complications. our study compared phacoemulsification and msics (blumenthal technique) in subluxated cataracts in a randomized controlled case series. in 90.0% cases of phacoemulsification and 76.67% cases of sics, the procedure was performed successfully without the need of major additional procedure and implantation of endocapsular supporting device and posterior chamber iol although, this difference was not significant (p = 0.14). most common was inadvertent increase in zonular dehiscence (4 cases). in three cases, it occurred during chopping, out of which two required lensectomy with sfiol and one case was managed with the insertion of two - point fixation cionni ring. in one case, during implantation of single - point fixation ctr, zonular dehiscence increased during rotation into capsular bag. so the device was removed as it failed to provide stability and lensectomy with sfiol was done. in another patient, one patient had small capsulorhexis, which during postoperative follow up lead to development of capsular phimosis. intraoperative complications in msics were more frequent than phacoemulsification group and occurred in 11 (36.67%) cases, although this difference was insignificant (p = 0.15). the most frequent was increased zonular dehiscence during nuclear prolapse and escaped capsulorhexis with four cases each. in three cases where zonular dehiscence increased, nuclear sclerosis of grade 3 was present, and hence prolapsing a bigger nucleus out of bag was difficult which resulted in increase dehiscence. in one case, increase in zonulysis occurred in region of acm, and this was managed with a placement of a two - point fixation cionni ring. the other noted complication was escaped capsulorhexis, probably related to attempt at a larger capsulorhexis which is a prerequisite to prolapse the nucleus out of bag in msics. the capsular injury was noted in one case during the insertion of a single - point fixation cionni ring. in two cases, in one such case, nucleus failed to rotate due to the presence of lens coloboma (figure 1) and lensectomy was required. the inability to rotate nucleus was also encountered in fellow eye of the same patient during phacoemulsification but was overcome by dividing nucleus into pieces., who recommend the use of endocapsular device in such a case. a second case, in which nuclear prolapse was impossible, had nuclear sclerosis of grade 4 and zonulysis of 190 degree. after the difficult insertion of a two - point fixation cionni ring, the nucleus edge got stuck in the eyelet and had to be managed by intracapsular extraction and placement of sfiol. therefore, the implantation of the double point fixation device in cases with the grade - four nucleus could be difficult and may hinder the nuclear prolapse out of bag. the use of ahmed capsular tension segment in such cases can provide effective lens stabilization before nucleus management. the statistical analysis of intraoperative complications (table 3) in relation to grade of nucleus in each group, showed that in group b (msics) they were significantly related to 3 grade of nucleus (p = 0.009), but the intergroup difference was insignificant. we also compared the implantation of intended capsular device based on grade of nucleus and degree of zonulysis (tables 4 and 5). within each group, this was found to be significantly related to the grade of nucleus (p = 0.04 and 0.004 for groups a and b, resp.) and failure to implant an intended capsular device was seen more often in nucleus grade 3 in both the groups, although difference between the two groups was insignificant. the implantation of intended capsular device was not found significantly related to the degree of zonulysis, but in 3 out of 4 cases in sics with zonulysis 180 degree, there was failure to implant an intended capsular device. the most common long - term complication reported with the use of endocapsular supporting device is posterior capsule opacification (pco). as follow up in our study was only 3 months, we did not compare the pco rates in our series. we noted difficulty in performing the aspiration of cortical matter in cases where endocapsular supporting device was used. the visually significant postoperative complications noted in our series were cystoid macular edema (cme) and vitritis in each group. cme seen in msics group was probably caused by the increased manipulation of the iris as this case required the replacement of already placed single cionni with double cionni ring. the visually insignificant complications were low - grade anterior uveitis (one case in each group), and transient vitreous haemorrhage was noted in two patients in msics group in which transscleral suture fixation of the posterior chamber iol was done. the limitation of our study is short - term followup. few studies have reported the long - term results of endocapsular devices with rate of iol dislocation ranging from 5.4 to 8.5% [21, 22 ]. with sfiol, complications like suture rupture can occur in 6% of eyes at mean of 4.9 years and up to 24% can have iol dislocation after 710 years. in a histologic study, iol stability was the result of intact scleral sutures and not to fibrous encapsulation nor correct placement of the haptic in the ciliary sulcus. as a result, iol dislocation is likely to occur if sutures are inadvertently removed or if suture fatigue occurs. iol decentration was seen in one case in msics where sfiol implantation was done due to escaped capsulorhexis (figure 2). the capsular phimosis, probably caused by small size capsulorhexis and retained lens matter, was present in one case of phacoemulsification group, which led to the iol tilt. we did not relate astigmatism between the groups, because incision site varied in each patient according to the area of zonulysis. postoperatively, both groups achieved good visual outcome, considering who definition of visual impairment as vision worse than 20/60 (equivalent to 0.33), there were 28 patients (93.34%) in phacoemulsification and 27 patients (90%) in msics who were benefitted with the surgery. capsular bag retention in subluxated lenses is possible in 90% cases of phacoemulsification and 76.67% cases of msics. both techniques achieved excellent visual outcome. the most common intraoperative complication noted in phacoemulsification was increased zonulysis and that in msics was increased zonulysis and escaped capsulorhexis. msics was performed with difficulty in cases of severe subluxation (> 180), larger grade of nucleus (3), and lens coloboma. this was due to a larger - sized capsular opening required for prolapse the nucleus out of the bag. with greater zonulysis, there is more difficulty in creating a large capsular opening. during a follow - up period of 3 months, both the techniques were comparable in terms of bcva, complications, and iol decentration / tilt. | purpose. to compare the feasibility of cataract surgery with implantation of endocapsular supporting devices and intraocular lens (iol) in subluxated cataract in phacoemulsification and manual small incision cataract surgery (msics). design. prospective randomized intervention case series consisting of 60 eyes with visually significant subluxated cataract. method. the patients were randomly distributed between the two groups equally. the main outcome measure was implantation of in - the - bag iol, requirement of additional procedure and complications, if any. results. capsular bag retention in subluxated lenses is possible in 90% cases in phacoemulsification versus 76.67% cases in msics (p = 0.16). both groups, achieved similar best corrected visual acuity (p = 0.73), although additional procedures, intraoperative, and postoperative complications were more common in msics. conclusions. achieving intact capsulorhexis and nuclear rotation in msics may be difficult in cases with large nucleus size and severe subluxation, but subluxated cataracts can be effectively managed by both phacoemuslification and msics. |
in the guidelines endorsed by the european association of cardiovascular imaging (eacvi) and the american society of echocardiography (ase), it is recommended that echocardiographic reports should contain information on the presence and grade of left ventricular (lv) diastolic dysfunction (ddf) when the technical quality is adequate and the findings are not equivocal. an algorithm structured as a two - level decision tree is proposed in which early diastolic myocardial tissue velocities and left atrial (la) volume indexed by body surface area (lavi) provide information on the presence or absence of ddf at the first branch point. subsequently, at a second branch point, more traditional diastolic doppler variables are used to grade patients with ddf into mild (grade i), intermediate (grade ii), or severe (grade iii). however, the algorithm, as presented in the eacvi / ase document, is not unequivocal as no guidance is given on how to handle discordant measurements. the effect of these ambiguities can be seen in less than optimal inter - reader agreement, with kappa values of 0.710.76 even when interpreters were given the same pre - measured variables. additionally, reports indicate that large differences in ddf definitions exist between studies. as the concordance of measures of diastolic function has been shown to be poor, even small differences in the specific algorithm used can be expected to yield large differences in subject classification and prevalence. to our knowledge, no systematic review has been performed to study how the definitions of ddf varied between studies claiming adherence to the eacvi / ase recommendations, nor has the effect of such variations on the reported prevalence been described previously. we aimed to explore how the eacvi / ase diagnostic scheme has been interpreted in the scientific community by means of a systematic literature review and subsequent analysis of the consequences of using different interpretations of definitions on the prevalence of ddf in a community - based sample. studies citing the eacvi / ase consensus document recommendations for the evaluation of left ventricular diastolic function by echocardiography published in the european journal of echocardiography in 2009 were identified through the thomson reuters web of science citation index on 3 december 2014 (n = 498). articles with titles and/or abstracts containing relevant key phrases (diastolic function, diastolic dysfunction, ddf, diastolic left ventricular dysfunction, diastolic lv dysfunction, diastolic heart failure, the titles and abstracts of these studies were screened, and clinical studies on adult human populations employing echocardiography written in the english language were retained (n = 197). finally, full - text versions of these articles were reviewed and all studies that (i) classified subjects by the presence or absence of ddf, (ii) specified which variables had been used for the classification, and (iii) cited the eacvi / ase document as the source of classification were included in the study (n = 60). included studies were subsequently analysed and coded independently by two researchers (j.s. and p.h.). algorithm for classification of ddf specified yes or no.grading of ddf into grade i, ii, or iii or similar present yes or no.if grading was present, was the classification of ddf and grading carried out by a one - level classification tree (criteria were presented for each grade and ddf was defined as fulfilment of the criteria for any one of these grades) or a two - level classification tree (criteria for ddf were defined and, if fulfilled, subsequent grading took place with additional variables)?if the variables used for classification had been specified, these variables were recorded. if a two - level classification tree had been used, the variables used for ddf classification and subsequent grading were recorded separately. if multiple parameters had been used within one level of the classification tree, the logical operator used was recorded ; in the context of the classification algorithms described, the words and and or were interpreted as the logical operators and (all listed criteria had to be fulfilled) and or (at least one of the listed criteria had to be fulfilled), respectively. yes or no. if grading was present, was the classification of ddf and grading carried out by a one - level classification tree (criteria were presented for each grade and ddf was defined as fulfilment of the criteria for any one of these grades) or a two - level classification tree (criteria for ddf were defined and, if fulfilled, subsequent grading took place with additional variables) ? the participants were recruited from the control group of the vstmanland myocardial infarction study (vamis). in the vamis study, subjects hospitalized for acute myocardial infarction were included from november 2005 to may 2011. for each included patient, a control subject was recruited from the general population. from the swedish population register in which all swedish citizens are registered, a subject of the same sex with the nearest date of birth and living in the same municipality as the vamis patient was identified and invited to participate. all subjects underwent clinical examination, electrocardiography, echocardiographic examination, and blood sampling. from the control group of the vamis study (n = 855), we excluded subjects with a left ventricular ejection fraction (lvef) 55%) or mildly to severely depressed. for the assessment of la volume, the single - plane modified simpson 's rule was used in the apical four - chamber view in the frame immediately preceding mitral valve opening. the peak early (e) and late (a) transmitral diastolic flow velocities, the e / a ratio, and the deceleration time of the early filling velocity were obtained at the peak of the mitral leaflets. the peak velocity of the early diastolic wave (e) was measured using pulsed - wave tissue doppler with the sample volume close to the mitral valve annulus in the apical four - chamber view in the septal (esep) and lateral (elat) walls. the e / e ratio was calculated on the basis of the transmitral e wave and the average of elat and esep (eavg). continuous data were expressed as mean standard deviation (sd) and categorical data as counts and percentages. skewed continuous data (i.e. nt - probnp) stata version 12.1 (statacorp lp, college station, tx, usa) was used for all statistical analyses. area - proportional ellipse - based euler diagrams were created using the open - source software eulerape v3. studies citing the eacvi / ase consensus document recommendations for the evaluation of left ventricular diastolic function by echocardiography published in the european journal of echocardiography in 2009 were identified through the thomson reuters web of science citation index on 3 december 2014 (n = 498). articles with titles and/or abstracts containing relevant key phrases (diastolic function, diastolic dysfunction, ddf, diastolic left ventricular dysfunction, diastolic lv dysfunction, diastolic heart failure, the titles and abstracts of these studies were screened, and clinical studies on adult human populations employing echocardiography written in the english language were retained (n = 197). finally, full - text versions of these articles were reviewed and all studies that (i) classified subjects by the presence or absence of ddf, (ii) specified which variables had been used for the classification, and (iii) cited the eacvi / ase document as the source of classification were included in the study (n = 60). included studies were subsequently analysed and coded independently by two researchers (j.s. and p.h.). algorithm for classification of ddf specified yes or no.grading of ddf into grade i, ii, or iii or similar present yes or no.if grading was present, was the classification of ddf and grading carried out by a one - level classification tree (criteria were presented for each grade and ddf was defined as fulfilment of the criteria for any one of these grades) or a two - level classification tree (criteria for ddf were defined and, if fulfilled, subsequent grading took place with additional variables)?if the variables used for classification had been specified, these variables were recorded. if a two - level classification tree had been used, the variables used for ddf classification and subsequent grading were recorded separately. if multiple parameters had been used within one level of the classification tree, the logical operator used was recorded ; in the context of the classification algorithms described, the words and and or were interpreted as the logical operators and (all listed criteria had to be fulfilled) and or (at least one of the listed criteria had to be fulfilled), respectively. if grading was present, was the classification of ddf and grading carried out by a one - level classification tree (criteria were presented for each grade and ddf was defined as fulfilment of the criteria for any one of these grades) or a two - level classification tree (criteria for ddf were defined and, if fulfilled, subsequent grading took place with additional variables) ? the participants were recruited from the control group of the vstmanland myocardial infarction study (vamis). in the vamis study, subjects hospitalized for acute myocardial infarction were included from november 2005 to may 2011. for each included patient, a control subject was recruited from the general population. from the swedish population register in which all swedish citizens are registered, a subject of the same sex with the nearest date of birth and living in the same municipality as the vamis patient was identified and invited to participate. all subjects underwent clinical examination, electrocardiography, echocardiographic examination, and blood sampling. from the control group of the vamis study (n = 855), we excluded subjects with a left ventricular ejection fraction (lvef) 55%) or mildly to severely depressed. for the assessment of la volume, the single - plane modified simpson 's rule was used in the apical four - chamber view in the frame immediately preceding mitral valve opening. the peak early (e) and late (a) transmitral diastolic flow velocities, the e / a ratio, and the deceleration time of the early filling velocity were obtained at the peak of the mitral leaflets. the peak velocity of the early diastolic wave (e) was measured using pulsed - wave tissue doppler with the sample volume close to the mitral valve annulus in the apical four - chamber view in the septal (esep) and lateral (elat) walls. the e / e ratio was calculated on the basis of the transmitral e wave and the average of elat and esep (eavg). continuous data were expressed as mean standard deviation (sd) and categorical data as counts and percentages. skewed continuous data (i.e. nt - probnp) stata version 12.1 (statacorp lp, college station, tx, usa) was used for all statistical analyses. area - proportional ellipse - based euler diagrams were created using the open - source software eulerape v3. in all included studies (n = 60), a classification of ddf, with or without grading, was presented, and the eacvi / ase recommendations were cited as the source of this classification. in 13 of these, the variables used for ddf classification were presented but no classification algorithm was specified. in the remaining 47 articles, a classification algorithm was described : 13 studies used a one - level classification tree, 18 studies used a two - level classification tree, and 16 studies only defined the criteria for ddf without any grading. in studies using a one - level classification tree, e and lavi were used in 1 study out of 13, whereas in studies utilizing a two - level classification tree, e was used in 16 and la size measurements in 7 of the 18 studies (table 1). a summary of how the different variables were combined, ignoring the logical operators used, is displayed in table 2. the most common combination, studies in which e (septal and/or lateral or averaged) and a measurement of la size were the only parameters used to define ddf, was seen in 17 of the 47 studies (14 of these used a two - level classification tree and in 3 studies no grading was performed). a summary of the detailed ddf definitions used in these 17 studies, including the logical operators, is shown in table 3. in eight studies, a singular parameter was used (esep 34). in two studies, the logical operator and was used to combine two or more parameters, whereas the remaining seven studies used the logical operator or. table 1variables used for ddf classification and grading grouped by different interpretations of the eacvi / ase 2009 classification algorithm (n = 60)variableall (n = 60)algorithm interpretation specified (n = 47)algorithm interpretation not specified (n = 13)classification and grading of ddf by a one - level classification tree (n = 13)classification and grading of ddf by a two - level classification tree (n = 18)classification of ddf only, no grading (n = 16)classification and grading of ddf by unspecified algorithm (n = 13)e / a44 (73%)13 (100%)16 (89%)3 (19%)12 (92%)deceleration time37 (62%)9 (69%)14 (78%)2 (13%)12 (92%)ivrt7 (12%)3 (23%)1 (6%)1 (6%)2 (15%)any e28 (47%)1 (8%)16 (89%)3 (19%)8 (62%) esep15 (25%)0 (0%)11 (61%)1 (6%)3 (23%) elat14 (23%)1 (8%)7 (39%)2 (13%)4 (31%) eavg6 (10%)0 (0%)2 (11%)1 (6%)3 (23%) e (location not specified)2 (3%)0 (0%)2 (11%)0 (0%)0 (0%)any e / e40 (67%)7 (54%)12 (67%)12 (75%)9 (69%) e / esep5 (8%)1 (8%)1 (6%)2 (13%)1 (8%) e / elat10 (17%)5 (38%)2 (11%)1 (6%)2 (15%) e / eavg21 (35%)0 (0%)6 (33%)10 (63%)5 (38%) e / e (location not specified)5 (8%)1 (8%)3 (17%)0 (0%)1 (8%)pulmonary flow indices8 (13%)6 (46%)0 (0%)0 (0%)2 (15%)left atrial size19 (32%)1 (8%)7 (39%)6 (38%)5 (38%) lavi16 (27%)1 (8%)6 (33%)6 (38%)3 (23%) lad or laa3 (5%)0 (0%)1 (6%)0 (0%)2 (15%)valsalva reversal of e / a3 (5%)2 (15%)0 (0%)0 (0%)1 (8%)s1s60 denotes references available in supplementary material online.ddf, diastolic dysfunction ; e, early diastolic inflow velocity ; a, late diastolic inflow velocity ; e, early diastolic myocardial tissue velocity ; esep, e of the septal wall ; elat, e of the lateral wall ; eavg, averaged e ; lavi, left atrial volume index ; lad, left atrial diameter ; laa, left atrial area. table 2combinations of variables used for ddf classification and grading grouped by different interpretations of the eacvi / ase 2009 classification algorithm in studies in which the algorithm interpretation was specified (n = 47)classification and grading of ddf by a one - level classification tree(n = 13)classification and grading of ddf by a two - level classification tree(n = 18)classification of ddf only, no grading(n = 16)for the definition of ddf and grading : for the definition of ddf : for the definition of ddf : e / a, e / e, and dt (n = 4) e (n = 8) e / e (n = 6) e / a and pv (n = 3) e and la size (n = 6) la size and e / e (n = 3) e / a, e / e, vsr, dt, and ivrt (n = 1) e / e (n = 2) dt and ivrt (n = 1) e / a, pv, e / e, and dt (n = 1) e, la size, and e / e (n = 1) e (n = 1) e / a, pv, dt, and ivrt (n = 1) e, e / a, and dt (n = 1) e, e / e, and e / a (n = 1) e / a, pv, dt, ivrt, and e/a (n = 1) la size (n = 1) e / a, vsr, and dt (n = 1)for the subsequent grading of ddf : la size, e / e, and e / a (n = 1) e, la size, e / a, and e / e (n = 1) e / a and dt (n = 5) e / e, e / a, and dt (n = 1) e / a, e / e, and dt (n = 4) e and la size (n = 1) dt (n = 2) e / a (n = 2) e / a, e / e, dt, and ivrt (n = 1) e, e / a, e / e, and dt (n = 1) e, la size, e / a, and e / e (n = 1) variables used not specified (n = 2)s1s60 denotes references available in supplementary material online.ddf, diastolic dysfunction ; e, septal, lateral, average, or unspecified early myocardial tissue velocity ; e, early transmitral flow velocity ; a, late / atrial transmitral flow velocity ; la size, left atrial volume, diameter, or area ; dt, deceleration time ; ivrt, isovolumetric relaxation time ; vsr, valsalva reversal of e / a ; pv, pulmonary venous flow indices. table 3detailed definitions of ddf, including used logical operators, in studies in which ddf classification was based on e and/or left atrial size only (n = 17)esep 34 (n = 4)esep 34 (n = 1)esep 34 (n = 1)lavi > 34 (n = 1)eavg 34 (n = 1)esep 125 (pg / ml)204 (29%)0 (0%)132 (40%) 34)84 (8287)67 (5975)94 (9096)(esep 34)77 (7380)50 (4159)88 (8592)(elat 34)76 (7379)54 (4563)86 (8290)(esep 34)73 (7076)53 (4562)84 (8088)(eavg 34)20 (1723)5 (210)32 (2737)(esep 34)16 (1319)2 (05)26 (2131)(eavg 34)15 (1218)2 (05)24 (2029)(elat 34)14 (1217)2 (05)22 (1827)(esep 34)12 (1015)1 (04)20 (1625)values are percentages (95% confidence intervals).e, early diastolic myocardial tissue velocity ; esep, e of the septal wall ; elat, e of the lateral wall ; eavg, average of elat and esep ; lavi, left atrial volume index. figure 1euler diagram showing overlapping of la dilatation (lavi > 34 ml / m) with low myocardial tissue velocities in the septal (esep 34 ml / m) with low myocardial tissue velocities in the septal (esep 34). in two studies, the logical operator and was used to combine two or more parameters, whereas the remaining seven studies used the logical operator or. table 1variables used for ddf classification and grading grouped by different interpretations of the eacvi / ase 2009 classification algorithm (n = 60)variableall (n = 60)algorithm interpretation specified (n = 47)algorithm interpretation not specified (n = 13)classification and grading of ddf by a one - level classification tree (n = 13)classification and grading of ddf by a two - level classification tree (n = 18)classification of ddf only, no grading (n = 16)classification and grading of ddf by unspecified algorithm (n = 13)e / a44 (73%)13 (100%)16 (89%)3 (19%)12 (92%)deceleration time37 (62%)9 (69%)14 (78%)2 (13%)12 (92%)ivrt7 (12%)3 (23%)1 (6%)1 (6%)2 (15%)any e28 (47%)1 (8%)16 (89%)3 (19%)8 (62%) esep15 (25%)0 (0%)11 (61%)1 (6%)3 (23%) elat14 (23%)1 (8%)7 (39%)2 (13%)4 (31%) eavg6 (10%)0 (0%)2 (11%)1 (6%)3 (23%) e (location not specified)2 (3%)0 (0%)2 (11%)0 (0%)0 (0%)any e / e40 (67%)7 (54%)12 (67%)12 (75%)9 (69%) e / esep5 (8%)1 (8%)1 (6%)2 (13%)1 (8%) e / elat10 (17%)5 (38%)2 (11%)1 (6%)2 (15%) e / eavg21 (35%)0 (0%)6 (33%)10 (63%)5 (38%) e / e (location not specified)5 (8%)1 (8%)3 (17%)0 (0%)1 (8%)pulmonary flow indices8 (13%)6 (46%)0 (0%)0 (0%)2 (15%)left atrial size19 (32%)1 (8%)7 (39%)6 (38%)5 (38%) lavi16 (27%)1 (8%)6 (33%)6 (38%)3 (23%) lad or laa3 (5%)0 (0%)1 (6%)0 (0%)2 (15%)valsalva reversal of e / a3 (5%)2 (15%)0 (0%)0 (0%)1 (8%)s1s60 denotes references available in supplementary material online.ddf, diastolic dysfunction ; e, early diastolic inflow velocity ; a, late diastolic inflow velocity ; e, early diastolic myocardial tissue velocity ; esep, e of the septal wall ; elat, e of the lateral wall ; eavg, averaged e ; lavi, left atrial volume index ; lad, left atrial diameter ; laa, left atrial area. table 2combinations of variables used for ddf classification and grading grouped by different interpretations of the eacvi / ase 2009 classification algorithm in studies in which the algorithm interpretation was specified (n = 47)classification and grading of ddf by a one - level classification tree(n = 13)classification and grading of ddf by a two - level classification tree(n = 18)classification of ddf only, no grading(n = 16)for the definition of ddf and grading : for the definition of ddf : for the definition of ddf : e / a, e / e, and dt (n = 4) e (n = 8) e / e (n = 6) e / a and pv (n = 3) e and la size (n = 6) la size and e / e (n = 3) e / a, e / e, vsr, dt, and ivrt (n = 1) e / e (n = 2) dt and ivrt (n = 1) e / a, pv, e / e, and dt (n = 1) e, la size, and e / e (n = 1) e (n = 1) e / a, pv, dt, and ivrt (n = 1) e, e / a, and dt (n = 1) e, e / e, and e / a (n = 1) e / a, pv, dt, ivrt, and e/a (n = 1) la size (n = 1) e / a, vsr, and dt (n = 1)for the subsequent grading of ddf : la size, e / e, and e / a (n = 1) e, la size, e / a, and e / e (n = 1) e / a and dt (n = 5) e / e, e / a, and dt (n = 1) e / a, e / e, and dt (n = 4) e and la size (n = 1) dt (n = 2) e / a (n = 2) e / a, e / e, dt, and ivrt (n = 1) e, e / a, e / e, and dt (n = 1) e, la size, e / a, and e / e (n = 1) variables used not specified (n = 2)s1s60 denotes references available in supplementary material online.ddf, diastolic dysfunction ; e, septal, lateral, average, or unspecified early myocardial tissue velocity ; e, early transmitral flow velocity ; a, late / atrial transmitral flow velocity ; la size, left atrial volume, diameter, or area ; dt, deceleration time ; ivrt, isovolumetric relaxation time ; vsr, valsalva reversal of e / a ; pv, pulmonary venous flow indices. table 3detailed definitions of ddf, including used logical operators, in studies in which ddf classification was based on e and/or left atrial size only (n = 17)esep 34 (n = 4)esep 34 (n = 1)esep 34 (n = 1)lavi > 34 (n = 1)eavg 34 (n = 1)esep 125 (pg / ml)204 (29%)0 (0%)132 (40%) 34)84 (8287)67 (5975)94 (9096)(esep 34)77 (7380)50 (4159)88 (8592)(elat 34)76 (7379)54 (4563)86 (8290)(esep 34)73 (7076)53 (4562)84 (8088)(eavg 34)20 (1723)5 (210)32 (2737)(esep 34)16 (1319)2 (05)26 (2131)(eavg 34)15 (1218)2 (05)24 (2029)(elat 34)14 (1217)2 (05)22 (1827)(esep 34)12 (1015)1 (04)20 (1625)values are percentages (95% confidence intervals).e, early diastolic myocardial tissue velocity ; esep, e of the septal wall ; elat, e of the lateral wall ; eavg, average of elat and esep ; lavi, left atrial volume index. figure 1euler diagram showing overlapping of la dilatation (lavi > 34 ml / m) with low myocardial tissue velocities in the septal (esep 34 ml / m) with low myocardial tissue velocities in the septal (esep < 8 cm / s) and lateral (elat < 10 cm / s) walls in all participants (a ; n = 714), the low - risk subgroup (b ; n = 129), and the high - risk subgroup (c ; n = 344), respectively. the central overlapping of all three ellipsoids corresponds to the use of the logical operator and, whereas the total area of all three ellipsoids corresponds to the use of or. in this study, we have demonstrated that the eacvi / ase recommendations for the evaluation of lv diastolic function by echocardiography with regard to ddf definition have been interpreted differently across different studies. furthermore, the data show that even among the substantial minority of studies that utilized a two - level classification tree with e and/or lavi at the first branch point an interpretation that we believe most closely resembles the eacvi / ase standpoint there were numerous variants of the actual definition of ddf. finally, the data show that these differences in interpretation have a huge impact on the obtained prevalence in a community - based sample. early attempts to classify diastolic function relied mainly on mitral inflow parameters. over time, numerous additional parameters have been introduced. each new parameter has had its inherent shortcomings, and no single parameter could describe the complexities of diastolic function on its own. the proposed solution has been to use a multiparametric approach by taking into account several parameters simultaneously. however, relatively sparse data exist on how these parameters should be weighted, and although the current guidelines present a list of parameters with suggested cut - offs, the integration of these is left to individual judgement. we observed substantial differences with respect to the variables and logical operators used, and the overall structure of the classification tree between the studies reviewed. among studies, a one - level decision tree was utilized, in which the criteria for each grade were presented and ddf was defined as the fulfilment of the criteria for any one of these grades. secondly, a two - level decision tree was utilized in which criteria for ddf was defined, and in a subsequent step, grading of ddf with the aid of additional parameters was performed.. a substantial minority of the studies reviewed used only e and/or lavi to define ddf. we believe that this approach most closely resembles the intention of the eacvi / ase recommendations. however, even among these studies, there was considerable heterogeneity with regard to the specific definition used (table 3). to demonstrate the effects on the reported prevalence obtained by this heterogeneity, we applied several possible interpretations of the first branch point of the two - level strategy on a community - based sample (table 5). several of these interpretations were encountered in the studies reviewed. using the eacvi / ase - endorsed fixed cut - offs, the prevalence of ddf calculated for the entire study group varied between 12 and 84% depending on the diagnostic algorithm used and between 1 and 67% in the low - risk subgroup. because of the exclusion of subjects with exertional dyspnoea, diabetes, hypertension, ischaemic heart disease, obesity, lv hypertrophy, and elevated nt - probnp from the low - risk subgroup, the prevalence of clinically relevant ddf could reasonably be expected to be very low. if we make the assumption that the true prevalence of ddf was well below 10% in this low - risk subgroup, it was evident that strategies that did not utilize lavi and strategies that used the logical operator or severely overestimated the prevalence of ddf (3667%). consequently, the specificity of such strategies will be poor. only algorithms utilizing lavi in combination with the logical operator and resulted in what might be considered a reasonable prevalence of ddf in the low - risk subgroup. however, owing to the use of the logical operator and, such ddf definitions are incompatible with the frequently stated fact that grade i ddf can be observed with normal la size. furthermore, in these algorithms, ddf classification will depend almost entirely on lavi, whereas e will have only a minor impact. the large influence of lavi stems from the fact that almost all of the subjects with increased atrial size had elat or esep below the cut - offs, whereas most subjects with low elat or esep had normal atrial size, as can be seen in figure 1a. the large difference in the prevalence of high lavi and low e, respectively, as can be observed in figure 1, is explained by the closeness of the proposed cut - off for lavi of 34 ml / m2 to the upper normal limit (mean + 2sd) of about 37 ml / m in healthy subjects, whereas the proposed cut - offs of 8 cm / s for esep and 10 cm / s for elat are close to the mean of healthy middle - aged subjects reported in several studies. thus, the prevalence of abnormal lavi of 5%, esep of 50%, and elat of 51% found in our low - risk subgroup is very much in line with what can be expected. the eacvi / ase recommendations for the evaluation of lv diastolic function provide a comprehensive overview of the full range of echocardiographic methods for evaluating diagnostic function and also provide a validated algorithm for the prediction of elevated filling pressures. the ambiguities of the classification scheme result in a less than optimal interobserver agreement, as identified by others, and large interstudy differences with regard to the method of classification, as demonstrated in this study. the latter can potentially make interstudy comparison difficult and hazardous because subject classification can vary substantially between studies. our observation is not unique, as the large variance in ddf definition across studies has also been observed by others. furthermore, the sensitivity of the eacvi / ase algorithm for identifying subjects with heart failure with preserved ejection fraction has been questioned previously, and our data indicate that some interpretations of the algorithm most likely lack specificity. we believe that if the eacvi / ase two - level classification scheme is to be used, it has to be revised and clarified. with the improvement of specificity in mind, one potential solution could be to keep the proposed cut - offs for lavi, esep, and elat and stress the use of and as the logical operator, accepting the fact that the resulting ddf classification will be driven mainly by la dilatation. such a strategy resulted in a reasonable ddf prevalence of 1% in our low - risk subgroup. another potential solution could be to adjust the esep, elat, and lavi cut - offs so that they more closely resemble the age - specific reference limits in healthy populations. age - specific limits, which are more restrictive than the cut - offs endorsed by the eacvi / ase, might make the use of or as the logical operator possible without introducing large proportions of false positives. the use of age - specific reference limits, in the context of ddf classification, has been advocated by others. however, how the potential alterations outlined in the discussion above would affect the diagnostic accuracy for identifying ddf can not be discerned from our data owing to the lack of a reference method. first, a selection bias with regard to reviewed studies is possible because only articles referring to the eacvi / ase recommendations as published in the european journal of echocardiography and not the journal of the american society of echocardiography were included. in addition, inclusion was limited by the presence of defined key phrases in the title / abstract of the screened studies. the full inclusion of all studies citing the eacvi / ase recommendations might have altered the relative frequency of different interpretations, but it would not have changed the main finding that a multitude of different interpretations exist in the published literature. secondly, the coding of reviewed studies was sometimes difficult because of vague classification definitions. this was addressed by independent coding by two researchers, where any disagreement was resolved by consensus. however, it is possible that in some cases we interpreted the ddf definitions in a different way from what was intended by the authors. thirdly, because no gold standard for ddf was available, the diagnostic accuracy of different ddf classification scheme interpretations could not be established, and the discussion of specificity was based on the presumed successful exclusion of subjects with ddf from our low - risk subgroup. finally, la volumes were calculated by the monoplane rather than the biplane simpson s rule, as recommended by the guidelines. because the cut - off of 34 ml / m is based on biplane measurements, our approach might have resulted in a slight over- or underestimation of the prevalence of atrial dilatation. first, a selection bias with regard to reviewed studies is possible because only articles referring to the eacvi / ase recommendations as published in the european journal of echocardiography and not the journal of the american society of echocardiography were included. in addition, inclusion was limited by the presence of defined key phrases in the title / abstract of the screened studies. the full inclusion of all studies citing the eacvi / ase recommendations might have altered the relative frequency of different interpretations, but it would not have changed the main finding that a multitude of different interpretations exist in the published literature. secondly, the coding of reviewed studies was sometimes difficult because of vague classification definitions. this was addressed by independent coding by two researchers, where any disagreement was resolved by consensus. however, it is possible that in some cases we interpreted the ddf definitions in a different way from what was intended by the authors. thirdly, because no gold standard for ddf was available, the diagnostic accuracy of different ddf classification scheme interpretations could not be established, and the discussion of specificity was based on the presumed successful exclusion of subjects with ddf from our low - risk subgroup. finally, la volumes were calculated by the monoplane rather than the biplane simpson s rule, as recommended by the guidelines. because the cut - off of 34 ml / m is based on biplane measurements, our approach might have resulted in a slight over- or underestimation of the prevalence of atrial dilatation. in this study, we have demonstrated that the eacvi / ase recommendations for the evaluation of lv diastolic function by echocardiography have been interpreted differently across studies with regard to the classification of ddf. furthermore, the findings show that these differences are important and can have a huge impact on subject classification and the obtained prevalence in a community - based sample. further research that is focused on the development and validation of multiparametric algorithms for the classification of diastolic function this study was supported by grants from sparbanksstiftelsen nya, the county of vstmanland, selanders stiftelse, and the swedish medical association. funding to pay the open access publication charges for this article was provided by centre for clinical research, uppsala university, vstmanland county hospital, vsters, sweden. | aimsthe aim of this article is to examine how the european association of cardiovascular imaging (eacvi) and the american society of echocardiography (ase) recommendations on the classification of diastolic dysfunction (ddf) are interpreted in the scientific community and to explore how variations in the ddf definition affect the reported prevalence.methods and resultsa systematic review of studies citing the eacvi / ase consensus document recommendations for the evaluation of left ventricular diastolic function by echocardiography was performed. the definition of ddf used in each study was recorded. subsequently, several possible interpretations of the eacvi / ase classification scheme were used to obtain ddf prevalence in a community - based sample (n = 714). in the systematic review, 60 studies were included. in 13 studies, no specification of ddf definition was presented, a one - level classification tree was used in 13, a two - level classification tree in 18, and in the remaining 16 studies, a ddf definition was presented but no grading of ddf was performed. in 17 studies, the ddf definition relied solely on early diastolic tissue velocity and/or left atrial size. in eight of these studies, a single parameter was used, in two studies the logical operator and was used to combine two or more parameters, and the remaining seven studies used the logical operator or. the resulting prevalence of ddf in the community - based sample varied from 12 to 84%, depending on the ddf definition used.conclusiona substantial heterogeneity of definitions of ddf was evident among the studies reviewed, and the different definitions had a substantial impact on the reported prevalence of ddf. |
surgery for otosclerosis involves an exposed inner ear and this puts the patient at risk of developing tinnitus, vertigo, sensorineural hearing loss, and even complete deafness. there is a risk of sensorineural hearing loss given that the stapedial tendon, which protects the inner ear from noise damage, is routinely cut in stapes surgery. the harmful noise may be continuous (e.g., concerts), receptive impulsive (e.g., industrial noise), or pure impulsive (e.g., explosion, gunshot noise). the damage mechanisms differ depending on the type of the noise and are of dual origin : mechanical and metabolic. mechanical damage develops when movement of the basilar membrane is excessive, inducing detachment of hairs from the tectorial membrane. metabolic disorders have multiple origins : ionic, excitotoxic, and production of cochlear free radicals. otoacoustic emission (oae) testing is a suitable tool for objective non - invasive assessment of inner ear function, particularly that of the organ of corti. this study aimed to evaluate the effects of noise after dividing the stapedial tendon in guinea pigs using oae testing, auditory brainstem response (abr) measurement, and histological examination. histopathological demonstration of apoptotic changes in the inner and outer hair cells of the cochlea is undoubtedly the most objective way to determine cochlear damage. the apoptotic cells can be easily seen under a light microscope following tdt - mediated dutp - biotin nick - end labeling (tunel) staining. to the best of our knowledge, this is the first study in the english literature that demonstrates the protective effect of the stapedial tendon against acoustic trauma. the stapedial tendon in each right ear was cut and the left ears served as the control in each animal. tympanometric examinations were performed to exclude middle ear pathology, which can affect the oae results. one week after surgery, a 4-khz octave band noise stimulus at an intensity of 110 db spl was presented by the audiometer (interacoustics ac 40) in a silent room for 6 h. the animal cage was put exactly half - way between 2 loudspeakers, positioned at 40 cm from each loudspeaker. the noise level variation was less than 3 db within the space available to the animal. measurement of oaes, tympanometric examination, and abr measurements were repeated after noise exposure. tympanometric examinations were performed using a low - frequency (226 hz) probe tone (az-26 interacoustics). the experimental protocol was approved by the hacettepe university animal care and use committee (protocol no. 2005/15 - 2). general anesthesia with ketamine (50 mg / kg) and xylazine (5 mg / kg) was administered intramuscularly. a parallel incision to the posterior of the bony ear canal 12 mm from the tympanic membrane was performed to the right ear of the guinea pigs with an otomicroscope. the tympanomeatal flap was elevated and the stapedial tendon was located posteroinferiorly and divided. after the completion of the procedure, all guinea pigs were euthanized. for recording of the distortion product otoacoustic emissions (dpoaes), a biological system (audx scout sport) was used. the acoustic probe was hand - held at the opening of the external ear channel with a slight pressure. the stimulus level of both frequencies was 70 db spl, and the noise to peak level of cubic dpoaes 2f1-f2 was measured. the background noise level in the exposure room was below 50 db spl and room temperature was controlled at approximately 25c. a total of 24 measurements at 1, 1.5, 2, 3, 4, and 6 khz were performed in each ear of each animal before and after noise exposure. animals were anesthetized with ketamine and xylazine. hearing thresholds of the guinea pigs were assessed using abr. the sound stimulus consisted of a 15-ms tone burst, with a rise - fall time of 1 ms at 2, 4, 8, and 16 khz. thresholds were defined as the lowest intensity required to produce a reproducible abr wave form. the temporal bones of the animals (n=20) were removed and processed for histological examination. the specimens were fixed with 4% formaldehyde and then decalcified using 0.1 mol / l ethylenediamine tetra - acetic acid (edta). then the cochlea was removed. a microtome was then used to obtain 1315 slices of 1 m in 650700 m per block. then, the percentages of apoptotic cells were determined in the experimental and control groups using light microscopy. general anesthesia with ketamine (50 mg / kg) and xylazine (5 mg / kg) was administered intramuscularly. prophylactic antibiotics were administered before surgery and immediately after surgery. a parallel incision to the posterior of the bony ear canal 12 mm from the tympanic membrane was performed to the right ear of the guinea pigs with an otomicroscope. the tympanomeatal flap was elevated and the stapedial tendon was located posteroinferiorly and divided. after the completion of the procedure, all guinea pigs were euthanized. for recording of the distortion product otoacoustic emissions (dpoaes), a biological system (audx scout sport) was used. the acoustic probe was hand - held at the opening of the external ear channel with a slight pressure. the stimulus level of both frequencies was 70 db spl, and the noise to peak level of cubic dpoaes 2f1-f2 was measured. the background noise level in the exposure room was below 50 db spl and room temperature was controlled at approximately 25c. a total of 24 measurements at 1, 1.5, 2, 3, 4, and 6 khz were performed in each ear of each animal before and after noise exposure. the sound stimulus consisted of a 15-ms tone burst, with a rise - fall time of 1 ms at 2, 4, 8, and 16 khz. thresholds were defined as the lowest intensity required to produce a reproducible abr wave form. the temporal bones of the animals (n=20) were removed and processed for histological examination. the specimens were fixed with 4% formaldehyde and then decalcified using 0.1 mol / l ethylenediamine tetra - acetic acid (edta). then the cochlea was removed. a microtome was then used to obtain 1315 slices of 1 m in 650700 m per block. then, the percentages of apoptotic cells were determined in the experimental and control groups using light microscopy. high inter - individual differences were found due to age differences between animals. distinct change in dpoaes was found at 1 khz (p=0.033), 1.5 khz (p=0.031), 2 khz (p=0.428), 3 khz (p=0.025), 4 khz (p=0.010), and 6 khz (p=0.003). the only frequency at which there was no statistically significant difference after noise exposure was 2 khz. the mean values of dpoaes in the experimental and control groups before and after noise exposure are shown in figure 1. in the control group there was no difference in tympanometric examination before and after surgery. at the start of the experiment, mean abr thresholds ranged from 23.5 to 31 db spl across frequencies for the right ears and from 23 to 31 db spl for the left ears. there was no statistically significant difference between right and left ear measurements before noise exposure. thresholds measured 10 days later were higher in right ears, and ranged from 40 to 56 db spl. the noise exposure produced significant abr threshold elevations for right ears at 2, 4, 8, and 16 khz (p=0.07, p=0.01, p=0.001, and p=0.001, respectively). thresholds measured 10 days after noise exposure ranged from 24 to 30.5 db spl for left ears, and this difference was not significant (p=0.8, p=0.5, p=0.9, p=0.1, respectively). tunel - positive cells were nearly absent in the inner and outer hair cells of the cochleas of the control group ; however, the prevalence of tunel - positive cells was high in the experimental group (table 1, figure 2). in this study, we examined the effect of acoustic trauma on the cochleas of guinea pigs using dpoaes, abr thresholds, and histopathological examination, and we attempted to demonstrate the protective effect of the stapedial tendon. we have demonstrated increased damage in the ear with divided stapedial tendons. to our knowledge, there are no other studies in the literature that have investigated the extent of acoustic damage following division of the stapedial tendon. various animal models have been used for experimental middle ear surgery, including primates, rats, dogs, and cats. the cat has been favored in studies of the histological and audiological effects of stapedectomy. due to ethical and financial considerations, the guinea pig model is established at the preferred model in many areas in otologic research. one study described the guinea pig model as an efficient and low - cost alternative to the standard feline model for stapedectomy. the effect of noise on hearing has traditionally been studied using auditory brain stem response (abr) on guinea pigs. in these studies, noise - induced abr threshold shifts were observed and it was reported that dexamethasone, geranylgeranylacetone, and tocopherol had protective effects [911 ]. in our study, dpoaes and abr thresholds were found to be more appropriate, as these tests reflect the condition of the outer hair cell more easily and accurately. preyer and gummer have shown that the non - linearity of the mechanoelectrical transducer function of the outer hair cells, as seen in guinea pigs, is essential for the non - linear movement of the basilar membrane, a source of dpoae generation. in another study, dpoaes results of this study suggest that the ability of the cochlea to generate dpoaes is associated with the condition of the outer hair cells. industrial noise, even though it does not cause visible damage to the outer hair cells, or even loss of inner hair cells, can result in microdamage to stereocilia, leading to loss of hearing function, and this is reflected by diminished dpoaes. in another study, the effects of different types of realistic occupational noise (as well as impulse noise) on loss of dpoaes in guinea pigs were tested. the study showed a link between changes in dpoae amplitudes and the type of occupational noise exposure. used 65-db spl broadband noise continuously for 3 or 11 days to evoke changes in dpoaes in guinea pigs. clark and pickles showed that exposure to high - intensity pure tones over a period of 530 min damaged outer hair cells according to intensity levels and exposure time. a similar study with guinea pigs showed that exposure to realistic noise for 2 h resulted in changed dpoaes. histopathological demonstration of damaged inner and outer hair cells is the most objective way to show cochlear damage. it was noteworthy to demonstrate significantly more tunel - positive cells in the organs of corti of the experimental animals when compared to controls. this result indicates that the damage due to acoustic trauma was significantly greater in ears with divided stapedial tendons. the beneficial impact of stapedial surgery on improving hearing in otosclerotic patients is well proven ; the immediate success rate is between 80% and 90% or even higher, and the air - bone gap can usually be reduced or closed. it has been argued that the high - frequency sensorineural hearing loss seen in postoperative stapedectomy patients is simply related to presbycusis and is not different from aged - matched controls. conversely, some have suggested that the increase in high - frequency thresholds is caused by trauma experienced during the surgery itself, or expansion of otosclerotic focus into the cochlea. one known cause of hearing loss following stapes surgery is inner ear barotrauma. in a study investigating the effects of barotrauma after stapedectomy in guinea pigs, cochlear effects were determined using electrocochleographic thresholds and cochlear hair cell counts. it is suggested that stapedectomy does not appear to predispose the guinea pig model of diving - related barotraumas to cochlear squeal. there is no study that proves convincingly that noise is a cause of sensorineural hearing loss after stapes surgery. however, it is known that one of the most important mechanisms in protecting the ears from noise damage is the stapedial tendon, and this is cut during the surgery. we found that noise significantly affected abr thresholds at all frequencies and all dpoae measurements except 2 khz in guinea pigs that were exposed to a high degree of noise over a long time. this noise altered all outer hair cells ; although the change in dpoae results at 2 khz was not statistically significant, it still changed. in light of these findings, it can be assumed that noise can cause damage to the inner ear after stapes surgery. in future studies, these findings may be further supported by the examination of defects in the outer hair cells using electron microcopy. in the clinic | backgroundthe effect of division of the stapedial tendon on susceptibility to noise - induced inner ear damage has not been previously studied. this study aimed to evaluate the effects of noise exposure following division of the stapedial tendon in guinea pigs.material/methodsten adult albino guinea pigs were used. the stapedial tendon of each right ear was cut. the stapedial tendon in each left ear was left intact and these ears served as a control group. dpoaes and abr tests were performed before and 10 days after noise exposure. the animals were exposed to a 110-db noise stimulus for 6 h in a silent room a week after surgery. cochleas of the animals were removed, and inner and outer hair cells were examined under a light microscope.resultswe found that noise exposure adversely affected dpoae measurements at all frequencies except 2 khz in experimental ears. noise exposure also produced significantly elevated abr thresholds in experimental ears at 2, 4, 8, and 16 khz. on histopathological examination, we found a significantly greater prevalence of apoptotic cells in the experimental ears.conclusionsbased on these findings, we can conclude that after division of the stapedial tendon, noise exposure may cause damage to the inner ear. this is the first study in the english literature that demonstrates the potential protective effect of the stapedial tendon against acoustic damage. |
angola and namibia share common boundaries along three provinces of angola (cunene, namibe, and caundo cubango) and four provinces of namibia (kunene, omusati, oshana, and ohangwena) [figure 1 ]. communities along the angolan namibian border immunization is one of the high - impact public health interventions that have significantly reduced morbidity, mortality, and disability among children. in other to strengthen the performance of immunization system and other health - related issues along their borders, a memorandum of understanding was signed on march 18, 2011. although cases of overdose and or intramuscular instead intradermal injection have been well documented, however, no published case of a child receiving two separate doses of bacillus calmette guerin (bcg) vaccine within the first 4 days of life as in the present report. against this context, we present a case of double bcg vaccination of a child living in calai town, cuando cubango province of angola. the said child was born on the april 25, 2013 and was bcg administered at birth in line with the angolan national immunization schedule. four days later, the mother on health reasons crossed into rundu district of namibia, where the same child was revaccinated with bcg april 30, 2013 with date of birth recorded as april 28, 2013 [figure 2 ]. the health worker of calai health centre (where the child was first vaccinated for bcg) and the provincial team made a follow - up visit when they noticed that the child missed the subsequent vaccination visits at 2 and 4 months of age at which time the child is supposed to receive the second and third doses of pentavalent, poliomyelitis, and hepatitis b vaccines. the child was traced and verified with the information on the child 's vaccination card and the nominal immunization register which contain mother name, and her address. furthermore, the vaccination site was checked for scarification, and two bcg scars were noticed on the left arm [figure 3 ]. on further probing, the mother presented two child immunization cards (angolan and namibian) that indicated the child was vaccinated for bcg in both countries within an interval of 3 days [figure 2 ]. the child immunization cards of angola and namibia the bacillus calmette guerin scars of the two vaccinations received cross - border immunization activities are conducted in different parts of africa to improve herd immunity of people living in the border areas and ultimately sustain the gains recorded in polio eradication initiative, measles, and neonatal tetanus elimination. furthermore, these border areas were reported to be sanctuaries for diseases earmarked for eradication (polio) and or elimination such as measles and neonatal tetanus. the time interval between bcg vaccination and scar formation has been reported to be influenced by the type of strain that was used in developing the vaccine, as it determines the virulence and antibody production. it has been reported that the time interval between bcg vaccination and scar formation is commonly within 6 weeks, although extreme cases up to 3 or more months have been documented. hence, the duration between the two bcg vaccinations received for the said child was inadequate for scar formation and therefore, the health worker who administered the second dose could only avoid such instances with a detailed history. however, it is possible that clients / patients from angolan border communities claim to be nationals of namibia to avoid paying fees for service rendered as people along the borders of both countries speak same local kuangale language. although immunization is free in both countries, but once the child has a namibian child vaccination card (called child passport in namibia), it will open access to other maternal and child health care services since non - namibians are charge higher than citizens of namibia. the differences in policy on whether all services are free irrespective of nationality as is obtained in angola and not so in namibia could be the underlying reason for the mother to subject her child to a second dose of bcg. having the namibian child immunization card could facilitate access to other health care services, which otherwise must be paid for. the cost of health care services has been reported as one of the major obstacles affecting the access and utilization maternal and child services. another plausible reason could be poor community knowledge that vaccination and the treatment for tuberculosis, leprosy and aids are all offered free in both countries. the fact that local community leaders (traditional, religious) are generally not involved in the regional / provincial meetings where decisions are made rub all the opportunity to interact with representatives of these border communities that would have provided better insights of the community concerns and how to tackle them. there is, therefore, the need for systematic engagement of local leaders to enhance their proactive participation in planning and community mobilization activities. while vaccines are highly effective in reducing the burden of vaccine - preventable diseases, however, they have also been reported to cause adverse events following immunization (aefi). the occurrence of this case of dual bcg vaccination also raises concern on the magnitude of such cases and the sensitivity of the surveillance for aefi. hence, the two countries need to strengthen the surveillance aefi through mobilization of communities, training of focal health workers, supervision, and monitoring by the district and provincial teams, to identify such cases and implement necessary remedial actions. finally, the current incident underscored the need for consensus by experts, government, and partners on guidelines on how to deal with such cases and policy framework on cross - border immunization activities to guide field workers that work in similar circumstances in both countries and other parts of the world. | without a doubt, the synchronization of public health intervention on health issues along the international border will enhance the control of epidemic - prone disease and other health - related behavior. however, the lack of holistic planning and the involvement of the members of border communities could result in undesired health related events. this report advanced reasons that could have resulted in a child receiving two separate doses of bacillus calmette guerin vaccine within the first 4 days of life in two different health facilities. finally, this report highlighted the need for proactive community participation and the need for consensus by experts on guidelines on how to deal with such cases particularly in the unfortunate event of adverse reactions. |
pediatric physiological interactions differ from those of the adult population ; there is therefore an inherent danger when utilizing products in children that have only been tested in adults. policymakers and healthcare professionals recognize the need to increase the number of pediatric randomized controlled trials (rcts) and have responded by creating policies and incentives for industries and organizations to engage in conducting trials in children. despite governmental incentives, there is still a dearth of pediatric trials, primarily due to the difficulty in recruitment of participants. an electronic search of rcts published between 19852004 found that only 701 (13%) of all rcts were pediatric, compared to 3,328 published adult trials.1 literature reviews identify recruitment as the single most challenging obstacle in conducting pediatric trials.2,3 the drink study (decreasing the rates of illness in kids), was a clinical trial examining the role of probiotics in infectious diseases. consenting parents or caregivers were asked to have their participating child consume a strawberry flavored dairy drink for 90 days. due to seasonal variation on illness, this paper describes a paradigm shift in recruitment design, developed from experience with grassroots political campaigns. importantly, as community research is now being highlighted by the nih, new techniques for recruiting need to be explored. this shift allowed the project to successfully enroll 638 participants in four months, while utilizing less than 70% of the original six - figure recruitment budget. additionally this approach, which reallocated funds from a mass media campaign into grassroots techniques, contributed to a retention rate of 92% throughout the 90-day trial. this technique was largely developed from the experience of the project coordinator who worked on developing coalitions in the 2004 presidential campaign, as well as a statewide gubernatorial race in 2005. the original recruitment budget and protocol for this trial utilized costly mass media, primarily radio and newspaper advertisements.4,5 this method would generally yield several hundred callers to a screening line daily. however, mass media does not target specific populations and generally a large percentage of callers are ineligible.6 screening ineligible callers takes study personnel away from other necessary project tasks and more productive means of recruitment. in conjunction with the media campaign, like most clinical trials drink intended to emphasize the utilization of a flyer campaign at various physician and health care locations for recruitment. again, this approach is not targeted and results in poor time management for research personnel. the fundamentals of all grassroots campaigns rely on effective messages being delivered by effective messengers ; the 2004 presidential campaign further refined and improved upon these message - messenger techniques. in 2004, compiled polling data from the previous twenty years revealed that persuadable swing voters had decreased from 20% to 7% of the voting population. the focus for campaigns became one of voter turn out rather than voter conversion. to accomplish this, both democrats and republicans had to 1) identify their voters, and 2) mobilize them to the polls. thus, the 2004 elections were about mobilizing specific voters on specific issues through the use of coalition and community leaders. slogans and spokespersons for these specific voter groups like veterans for kerry, w stands for women, catholics for kerry and ypw (young professionals for w) emerged. on the ground, grassroots representatives were commissioned among different demographics, coalitions and communities focused on mobilizing the within reach fruit voters to the polls ; these voters identified voters among the networks and associations of the grassroots leaders and representatives. under this grassroots campaign technique, most successful campaign staffs were not comprised of political analysts or persons with in - depth understanding of political science ; but rather, persons who could identify, talk with, and were trusted among specific voter groups. like a grassroots campaign, drink s recruitment strategy relied on tailored messaging. believing that families would be more receptive to participating in the study and motivated to call the recruitment line if 1) they received flyers and information about the study from a known contact or 2) received study information designed to inspire trust and comfort, the goal was to have known persons in the community, daycare providers, physicians from our practice based research network (pbrn) and community leaders from religious groups and other community institutions deliver hand tailored and institutional review board (irb)-approved information directly to families. information with tailored images outlining clear concise study details yields more eligible callers to recruitment lines, rather than flyers received in the mail or posted on the wall. this direct method reduced the risk of a high proportion of ineligible families being solicited for the study, and allowed for more personnel time in the field. there were a total of only 872 calls to the screening line, of which 741 candidates were screened and 681 met eligibility criteria (91%). the fundamentals of all grassroots campaigns rely on effective messages being delivered by effective messengers ; the 2004 presidential campaign further refined and improved upon these message - messenger techniques. in 2004, compiled polling data from the previous twenty years revealed that persuadable swing voters had decreased from 20% to 7% of the voting population. the focus for campaigns became one of voter turn out rather than voter conversion. to accomplish this, both democrats and republicans had to 1) identify their voters, and 2) mobilize them to the polls. thus, the 2004 elections were about mobilizing specific voters on specific issues through the use of coalition and community leaders. slogans and spokespersons for these specific voter groups like veterans for kerry, w stands for women, catholics for kerry and ypw (young professionals for w) emerged. on the ground, grassroots representatives were commissioned among different demographics, coalitions and communities focused on mobilizing the within reach fruit voters to the polls ; these voters identified voters among the networks and associations of the grassroots leaders and representatives. under this grassroots campaign technique, most successful campaign staffs were not comprised of political analysts or persons with in - depth understanding of political science ; but rather, persons who could identify, talk with, and were trusted among specific voter groups. like a grassroots campaign, drink s recruitment strategy relied on tailored messaging. believing that families would be more receptive to participating in the study and motivated to call the recruitment line if 1) they received flyers and information about the study from a known contact or 2) received study information designed to inspire trust and comfort, the goal was to have known persons in the community, daycare providers, physicians from our practice based research network (pbrn) and community leaders from religious groups and other community institutions deliver hand tailored and institutional review board (irb)-approved information directly to families. information with tailored images outlining clear concise study details yields more eligible callers to recruitment lines, rather than flyers received in the mail or posted on the wall. this direct method reduced the risk of a high proportion of ineligible families being solicited for the study, and allowed for more personnel time in the field. there were a total of only 872 calls to the screening line, of which 741 candidates were screened and 681 met eligibility criteria (91%). traditionally clinical trials primarily identify study candidates by recruiting patients already receiving care through the institution conducting the trial. an alternate traditional method places study personnel in sites like clinics, ambulatory care, and emergency rooms, to enroll potential participants in person. typically data is collected at one site, requiring participants to travel to the research study. trials often are budgeted to reimburse the travel costs of the participant, but despite these provisions recruiting from a university primarily results in a biased participant population.7 additionally, data collected in a laboratory or that of a medical center, often varies from data collected in the community. less than 10% of drink population identified themselves as learning about the study through a physician or healthcare center, but eighty - four percent of participants indicated that they learned about the study through their daycare / school or other grassroots methods (ie, from another participant, community centers, church, website or listserv). we provided cars and cell phones to research personnel, allowing more face time in the community. we believe this approach is a paradigm shift in recruitment because most sponsors and institutions are willing to spend several thousand dollars on printing and media, but have more reservations when investing in community time of personnel. by providing the staff with tools such as cell phones and cars, they have much greater access to the community and can spend more time in the community. the benefit of this approach is multifold, as research collected in the field over a laboratory increased the external validity of the study and by providing research assistants (ras) with cell phones ; participants could more easily get in contact with the ras, limiting missed follow - ups. thus, more study personnel time in the community lead to greater trust, increasing recruitment and retention. however, the nih has strict rules on expenses like travel and food for offices, often making this approach difficult when using government funds. however, we have successfully worked with nih project officers, explained the grassroots approach and have subsequently been granted permission to spend funds for these types of expenses. drink study personnel promoted the study by presenting at various meetings and gatherings and by meeting with community leaders while enrolling participants in their home or community. to identify points of contacts within the communities, the research team networked with churches, synagogues and community groups scheduling opportunities to go out and present to these different groups increased face time among potential recruitment sites thus, getting different communities comfortable with the project while continuing to network for contacts within the schools and daycare centers. less than 10% of drink population identified themselves as learning about the study through a physician or healthcare center, but eighty - four percent of participants indicated that they learned about the study through their daycare / school or other grassroots methods (ie, from another participant, community centers, church, website or listserv). we provided cars and cell phones to research personnel, allowing more face time in the community. we believe this approach is a paradigm shift in recruitment because most sponsors and institutions are willing to spend several thousand dollars on printing and media, but have more reservations when investing in community time of personnel. by providing the staff with tools such as cell phones and cars, they have much greater the benefit of this approach is multifold, as research collected in the field over a laboratory increased the external validity of the study and by providing research assistants (ras) with cell phones ; participants could more easily get in contact with the ras, limiting missed follow - ups. thus, more study personnel time in the community lead to greater trust, increasing recruitment and retention. however, the nih has strict rules on expenses like travel and food for offices, often making this approach difficult when using government funds. however, we have successfully worked with nih project officers, explained the grassroots approach and have subsequently been granted permission to spend funds for these types of expenses. drink study personnel promoted the study by presenting at various meetings and gatherings and by meeting with community leaders while enrolling participants in their home or community. to identify points of contacts within the communities, the research team networked with churches, synagogues and community groups scheduling opportunities to go out and present to these different groups increased face time among potential recruitment sites thus, getting different communities comfortable with the project while continuing to network for contacts within the schools and daycare centers. based on extensive personal research the pi (daniel merenstein) has assessed the typical clinical trial will cost $ 1050,000 per patient. a substantial part of these costs is recruitment and retention of subjects. the original protocol for drink created by the pi and sponsor set aside 10% of the entire budget for recruitment, of which 80% was allocated for the utilization of a mass media campaign. the original budget was reconfigured (table 1) to emphasize grassroots techniques, establishing relationships within the community. this reconfiguration began with providing staff with more appropriate tools and functional support to increase their availability and access to the community ; over - time pay, project cell phones, and two additional cars were included. these additional items cost 57% less than the total original budget and 37% less than the media. drink saved 30% of the total recruitment budget, only utilizing 16% toward media instead of the original 80% by engaging in a focused and specifically targeted media campaign. the original budget was reconfigured (table 1) to emphasize grassroots techniques, establishing relationships within the community. this reconfiguration began with providing staff with more appropriate tools and functional support to increase their availability and access to the community ; over - time pay, project cell phones, and two additional cars were included. these additional items cost 57% less than the total original budget and 37% less than the media. drink saved 30% of the total recruitment budget, only utilizing 16% toward media instead of the original 80% by engaging in a focused and specifically targeted media campaign. recruiting minorities and overcoming issues of mistrust for research is a common costly challenge for all clinical trials.8 utilizing mass media and practice based research networks (pbrn) for recruitment present challenges to overcoming these obstacles. there is a strong body of evidence suggesting a general mistrust of the medical / scientific community among minorities and healthcare providers do not always have the time to explain clinical trials to patients, and help them overcome this mistrust.9 minorities are more likely to enroll in studies when approached by a trusted known healthcare professional than a faceless flyer.10 overcoming barriers for families to enroll their children due to concern and mistrust was equally challenging for drink as in other pediatric clinical trials. like a grassroots campaign, drink recruitment messages were delivered by a trusted messenger and relationships were established throughout several different communities. drink staff members were not hired for their in - depth understanding of probiotics or clinical trials but rather for their personal experience and professional work histories that pointed to excellent interpersonal skills and an interest in community health. in addition to networking in schools and daycares, drink staff established relationships with communities like head start and religious organizations. the experiences of drink team members indicated competence in effectively communicating with communities of potential research populations, as well as ease and ability to move within these communities with comfort. often staff members were culturally matched with the communities they were recruiting, eg, 30% of the staff members were native spanish speakers. if staff members had intimate knowledge of certain communities based on prior experience, they were assigned these areas. again, this technique was developed from lessons from the 2004 campaign trail, which implored different community leaders to deliver political campaign messages within their respective communities and associates ; for example, the evangelical christian leaders speaking out at mega - churches and gatherings of christian communities throughout the country. traditional trials are designed top - down with the principal investigator and sponsor making decisions on how to allocate the budget for recruitment. alternatively, drink sought input from the community as well as the staff members who were most intimately connected with different communities. those recruiting in the field are the representatives of the study, and much of the recruitment success or failure is determined by how confidently they represent the project among different groups. evidence in other pediatric trials confirms the critical contribution of staff in the field to recruitment success. smith and colleagues, recruiting families with asthmatic children between 212 years old from the emergency department to partake in a two - week study, report that their high rate of enrollment was primarily due to two dedicated enrollers. the enrollers interacted with the families and emergency department staff with ease and competence, yielding successful enrollment of 527 families in sixteen months.11 the backgrounds of the drink ras were essential in the recruitment success for this project. as table 2 indicates, the grassroots approach was a success ; families represented diverse demographics, social economic status, race and ethnicity. additionally 19% of the study population identified themselves as hispanic or latino, and conducted interviews in spanish. efforts and resources were aimed at developing a trusted brand identity for the study within the community. every participant received a t - shirt with the drink logo, as well as magnets to keep study papers together and easily accessible. all research personnel used a canvas bag with the logo when they went on recruitment site visits or home visits. effective campaign messages must be authentic and representative of the messenger. although socially conservative evangelical christians may be interested in the same issues and essentially the same message as socially conservative catholics, it would not benefit a campaign to use a message with catholic imagery for a group of evangelicals ; in fact utilizing similar imagery among both groups would most likely result in deterring voters to the polls, rather than motivating them. similarly for drink, the flyer distributed by physicians offices needed a professional look and feel, congruent to the environment of a physician office, while flyers distributed by daycares and schools needed to represent the nurturing environment caring for primary - aged children. the phrase clinical trial or word probiotic could appear intimidating to parents ; recruitment information had to be presented in a manner that would set caregivers concerns at ease and with an appearance that would avoid the impression that they were unwillingly being solicited, while at the same time adhering to strict but important irb regulations. potential participants needed to trust the source of the flyer and avoid questioning the legitimacy or integrity of those conducting the project ; it was believed that developing specific flyers for specific groups would help promote confidence in those conducting the trial, and motivate potential participants to call screening lines. working closely with the irb and identifying points of contact from the community, a grassroots website was developed and versions of the flyer were tailored specific to the organization or recruitment site type. the website color scheme reflected our logo and although different versions of flyers utilized different and specific colors, every flyer carried the logo in the lower right corner. like a grassroots campaign, drink recruitment messages were delivered by a trusted messenger and relationships were established throughout several different communities. drink staff members were not hired for their in - depth understanding of probiotics or clinical trials but rather for their personal experience and professional work histories that pointed to excellent interpersonal skills and an interest in community health. in addition to networking in schools and daycares, drink staff established relationships with communities like head start and religious organizations. the experiences of drink team members indicated competence in effectively communicating with communities of potential research populations, as well as ease and ability to move within these communities with comfort. often staff members were culturally matched with the communities they were recruiting, eg, 30% of the staff members were native spanish speakers. if staff members had intimate knowledge of certain communities based on prior experience, they were assigned these areas. again, this technique was developed from lessons from the 2004 campaign trail, which implored different community leaders to deliver political campaign messages within their respective communities and associates ; for example, the evangelical christian leaders speaking out at mega - churches and gatherings of christian communities throughout the country. traditional trials are designed top - down with the principal investigator and sponsor making decisions on how to allocate the budget for recruitment. alternatively, drink sought input from the community as well as the staff members who were most intimately connected with different communities. those recruiting in the field are the representatives of the study, and much of the recruitment success or failure is determined by how confidently they represent the project among different groups. evidence in other pediatric trials confirms the critical contribution of staff in the field to recruitment success. smith and colleagues, recruiting families with asthmatic children between 212 years old from the emergency department to partake in a two - week study, report that their high rate of enrollment was primarily due to two dedicated enrollers. the enrollers interacted with the families and emergency department staff with ease and competence, yielding successful enrollment of 527 families in sixteen months.11 the backgrounds of the drink ras were essential in the recruitment success for this project. as table 2 indicates, the grassroots approach was a success ; families represented diverse demographics, social economic status, race and ethnicity. additionally 19% of the study population identified themselves as hispanic or latino, and conducted interviews in spanish. efforts and resources were aimed at developing a trusted brand identity for the study within the community. every participant received a t - shirt with the drink logo, as well as magnets to keep study papers together and easily accessible. all research personnel used a canvas bag with the logo when they went on recruitment site visits or home visits. effective campaign messages must be authentic and representative of the messenger. although socially conservative evangelical christians may be interested in the same issues and essentially the same message as socially conservative catholics, it would not benefit a campaign to use a message with catholic imagery for a group of evangelicals ; in fact utilizing similar imagery among both groups would most likely result in deterring voters to the polls, rather than motivating them. similarly for drink, the flyer distributed by physicians offices needed a professional look and feel, congruent to the environment of a physician office, while flyers distributed by daycares and schools needed to represent the nurturing environment caring for primary - aged children. the phrase clinical trial or word probiotic could appear intimidating to parents ; recruitment information had to be presented in a manner that would set caregivers concerns at ease and with an appearance that would avoid the impression that they were unwillingly being solicited, while at the same time adhering to strict but important irb regulations. potential participants needed to trust the source of the flyer and avoid questioning the legitimacy or integrity of those conducting the project ; it was believed that developing specific flyers for specific groups would help promote confidence in those conducting the trial, and motivate potential participants to call screening lines. working closely with the irb and identifying points of contact from the community, a grassroots website was developed and versions of the flyer were tailored specific to the organization or recruitment site type. the website color scheme reflected our logo and although different versions of flyers utilized different and specific colors, every flyer carried the logo in the lower right corner. the internet is looked upon by many as a new and exciting means of recruitment. often studies will be advertised at sites that investigators feel may attract the desired population. however even with this targeted population, web advertising may lead to many individuals contacting recruitment lines that are far beyond the catchment area. clinical trial websites often utilize a contact form which allows interested candidates to submit their contact information to learn more about the study. although this is a useful tool, many individuals are hesitant to supply personal contact information over the internet. we used the internet in drink but as a tool, rather than a substitute, to provide more face time in the community for research personnel and to increase trust while branding the study identity. we developed a website, www.thedrinkstudy.com, which contained pictures and short biographies of the drink staff, allowing screened participants to view the pictures of staff who would be meeting them in - person to obtain written consent and gather baseline data. additionally, the website contained the general data collection forms for the project, allowing interested families to review in advance the nature of the study and eligibility and criteria for participation. most importantly, a website link is a fast and inexpensive way for recruitment information to be circulated to several different networks. this allowed participating parents the ability to notify their friends and family effortlessly by forwarding in an email the study s website address or post the site link on networks and listservs frequented by parents. we used the internet in drink but as a tool, rather than a substitute, to provide more face time in the community for research personnel and to increase trust while branding the study identity. we developed a website, www.thedrinkstudy.com, which contained pictures and short biographies of the drink staff, allowing screened participants to view the pictures of staff who would be meeting them in - person to obtain written consent and gather baseline data. additionally, the website contained the general data collection forms for the project, allowing interested families to review in advance the nature of the study and eligibility and criteria for participation. most importantly, a website link is a fast and inexpensive way for recruitment information to be circulated to several different networks. this allowed participating parents the ability to notify their friends and family effortlessly by forwarding in an email the study s website address or post the site link on networks and listservs frequented by parents. although our recruitment goals were met efficiently in the short time allowed and at a reduced budget, this approach requires time and resources to establish rapport with communities and develop trust. most importantly, this approach is only successful with staff members who are competent at networking in the community. safety concerns also must be considered when placing staff in the field, particularly when entering foreign and possibly high violence neighborhoods. sponsors must be willing to allow researchers the freedom to use funds in the community. fortunately, we had a sponsor that trusted university guidelines and the research team but current nih guidelines make much of this approach nearly impossible to accomplish. there is an additional risk of potential participants feeling coerced to participate, just as historically belonging to certain groups required a vote for a certain candidate or participation in group activities. in our study, this was avoided as community members or leaders were not involved beyond their sharing of enrollment flyers and general information regarding the purpose and nature of the study. only the team of ten research assistants could conduct screening and enrolling, and as in all irb - approved studies, participant information was kept confidential. it was never reported back to head - start, daycare centers, churches or pediatric offices which families had enrolled. our study was limited to healthy children, which made recruiting in the community ideal. however, the pediatric epidemics under increased investigation such as asthma, obesity, type ii diabetes have environmental and social correlations allowing for targeted community recruitment. as recent public policies and literature suggest, there is a shortage of pediatric trials being conducted. many pediatric trials fail to recruit an adequate sample size for clinical significance and conducting clinical trials is costly. through the utilization of a grassroots approach and targeting potential populations, projects can meet demanding recruitment goals more efficiently and effectively, while retaining their study population and simultaneously reducing recruitment costs. they have to be willing to invest in personnel and communities, adequately for time and experience working within communities. in addition, research departments and sponsors must allow for funds to support human resources with the tools necessary to work in the field, such as cell phones, cars and fuel for transportation. we believe this investment ultimately saves money, produces studies with greater validity and respectfully involves communities. many of daycares centers, schools and the coalition of head start centers have been eager to work with us on future projects. | background : literature reviews have identified recruitment as the single most challenging obstacle in conducting pediatric trials. this paper describes a paradigm shift in recruitment design, developed from experience with grassroots campaigns through the drink study (decreasing the rates of illness in kids). the objective of this study was to explain a new method for recruiting in clinical trials based on lessons learned from grassroots political campaigning.methods and findings : the study described is a randomized controlled trial of 638 36 year olds from the washington, dc area. the design involved a comparison between new recruiting approaches modeled after grassroots campaigns and traditional techniques. traditional techniques for the purpose of this paper are defined by the use of physician referral, mass media such as radio and television advertisements, along with posters in public places like the subway. grassroots approaches alternatively developed and utilized community contacts and employed targeted small market media community. the main outcome measures were the percentage of budget used and the number of eligible participants recruited.conclusions:the results showed that the grassroots recruitment approach saved 30% of the budget, recruited 638 kids in 4 months and retained over 90% for the 90 day trial. new techniques need further exploration as community studies are stressed. |
the safety of thyroid operations may be affected by anatomical variations of the recurrent laryngeal nerve (rln), which increases the risk of nerve injury. bilateral variations of both right and left rlns in the same patient increase the risk of vocal cord palsy and may have catastrophic outcomes. extralaryngeal terminal bifurcation of rln is a common variation, which is macroscopically identified by surgical dissection along the cervical course of the nerve. terminal branches may have similar proportions, and must be fully exposed with care to preserve their integrity and avoid vocal cord dysfunction. anatomical features and bifurcation point locations are quite variable along the cervical course of rln. these features were previously discussed in total thyroidectomy cases. besides variations of extralaryngeal terminal bifurcations, variable locations of division points create many combinations that can complicate dissection of the nerve, and thus, increase the importance of this variation. bilateral occurrence of terminal bifurcation makes dissection even more challenging, and increases the risk of nerve injury by two - fold. therefore, complete knowledge of anatomical locations of bifurcation points aids in identifying and preserving both branches, decreases complication rates and increases the safety of surgery. in this study, we attempted to identify and expose bilateral extralaryngeal terminal bifurcations of both rlns from a series of total thyroidectomy cases, in order to establish anatomical features of bifurcated nerves based on bifurcation point locations along the cervical course of the nerve to elucidate the surgical importance of this variation. this prospective study evaluated surgical anatomy of rln in 146 patients with surgical thyroid diseases, who were treated with total thyroidectomy between may 2009 and october 2013. informed consent was obtained from every patient prior to the surgery. to provide a standard dissection technique, the procedure was performed by the same surgeon. a possible presence of extralaryngeal terminal bifurcation of rln was macroscopically determined, and the bifurcation point location along the cervical course of the nerve was identified according to adjacent structures. lateral sides (unilateral or bilateral) of the terminal division were also established during the procedure. after freeing and medially mobilising lateral lobes in the both sides of thyroid gland by the classical surgical approach, the inferior thyroid arteries (ita) were identified, and isolated, a loop of silk suture was placed around the arteries for traction. with the usual lateral approach, rlns were identified below the artery, and fully isolated at both sides. the nerve was carefully exposed in the tracheoesophageal groove up to its laryngeal entry point. if macroscopically and clearly delineated terminal bifurcation of the nerve was identified along its cervical course, the anatomical features of these branches in bilateral cases were macroscopically studied to compare the anatomical differences between both sides. the branches of similar dimensions were observed during the surgery, and entered into the larynx separately at different points. the classification was based on the bifurcation point location according to relatively constant landmarks, that is the neuro - vascular (rln and ita) crossing point and the laryngeal entry point of the nerve. the location of bifurcation point are classified in four segments of rln along its cervical course, as previously reported : arterial segment : bifurcation occurs at or closely adjacent to neurovascular crossing of rln and ita;post - arterial segment : bifurcation occurs after (distal to) neurovascular crossing;pre - laryngeal segment : bifurcation occurs in the most distal (15 mm to laryngeal entry) segment of the rln ; andpre - arterial segment : bifurcation occurs before (proximal to) neurovascular crossing. arterial segment : bifurcation occurs at or closely adjacent to neurovascular crossing of rln and ita ; post - arterial segment : bifurcation occurs after (distal to) neurovascular crossing ; pre - laryngeal segment : bifurcation occurs in the most distal (15 mm to laryngeal entry) segment of the rln ; and pre - arterial segment : bifurcation occurs before (proximal to) neurovascular crossing. patients with extralaryngeal terminal bifurcation of both right and left rlns were included in the study. bifurcation point locations along the cervical course of rlns in bilateral cases were classified according to the four segments. anatomical features of bifurcated nerves were compared to establish similarities or differences between two sides. after freeing and medially mobilising lateral lobes in the both sides of thyroid gland by the classical surgical approach, the inferior thyroid arteries (ita) were identified, and isolated, a loop of silk suture was placed around the arteries for traction. with the usual lateral approach the nerve was carefully exposed in the tracheoesophageal groove up to its laryngeal entry point. if macroscopically and clearly delineated terminal bifurcation of the nerve was identified along its cervical course, the anatomical features of these branches in bilateral cases were macroscopically studied to compare the anatomical differences between both sides. the branches of similar dimensions were observed during the surgery, and entered into the larynx separately at different points. the classification was based on the bifurcation point location according to relatively constant landmarks, that is the neuro - vascular (rln and ita) crossing point and the laryngeal entry point of the nerve. the location of bifurcation point are classified in four segments of rln along its cervical course, as previously reported : arterial segment : bifurcation occurs at or closely adjacent to neurovascular crossing of rln and ita;post - arterial segment : bifurcation occurs after (distal to) neurovascular crossing;pre - laryngeal segment : bifurcation occurs in the most distal (15 mm to laryngeal entry) segment of the rln ; andpre - arterial segment : bifurcation occurs before (proximal to) neurovascular crossing. arterial segment : bifurcation occurs at or closely adjacent to neurovascular crossing of rln and ita ; post - arterial segment : bifurcation occurs after (distal to) neurovascular crossing ; pre - laryngeal segment : bifurcation occurs in the most distal (15 mm to laryngeal entry) segment of the rln ; and pre - arterial segment : bifurcation occurs before (proximal to) neurovascular crossing. patients with extralaryngeal terminal bifurcation of both right and left rlns were included in the study. bifurcation point locations along the cervical course of rlns in bilateral cases were classified according to the four segments. anatomical features of bifurcated nerves were compared to establish similarities or differences between two sides. extralaryngeal terminal bifurcations of rlns were observed in 70 (48%) of 146 patients undergone with total thyroidectomy and 90 (31%) of 292 rlns. we observed bilateral bifurcation in 20 (13.7%) of 146 patients and 20 (28.6%) of 70 patients with bifurcated nerves [table 1 ]. total thyroidectomy cases with rln bifurcation bifurcation point locations along the cervical course of rln were another important result of the study. forty rlns in 20 patients with bilateral bifurcation were classified according to the anatomical location of division points. pre - laryngeal and pre - arterial locations of rln bifurcation were seldom encountered [table 2 ]. the location of bifurcation points on 40 rlns in 20 patients with bilateral bifurcation according to anatomical segments of the rln, 5 (25%) patients with bilateral bifurcation of rln presented similar locations of bifurcation point on both sides. locations of bifurcation were different in 15 (75%) patients with bilateral bifurcation of rln. the locations of bilateral bifurcations were found either in the same anatomical segments [figure 1 ] or in different anatomical segments [figure 2 ]. bifurcation points () are located in the same (pre - arterial) segments along cervical course of the nerve. (+) neuro - vascular (rln and ita) crossing extralaryngeal terminal bifurcation of both recurrent laryngeal nerves (rln). bifurcation points () are located in different (right arterial and left pre - arterial) segments along cervical course of the nerve. in this study, rates of extralaryngeal terminal bifurcation (31%) in the exposed rlns and those of bifurcation (48%) in patients undergoing total thyroidectomy indicate the common occurrence of this anatomical variation. however, some studies showed quite different results : kandil., reported bifurcation in 34% and 43% of dissected rlns, beneragama., in 36%, paradeep., in 30.7%, cernea., in 64.5% and casella., in 18.5% of rlns. the awareness of possible variations of terminal bifurcation of rln is of importance, from a surgical point of view, because a well exposing bifurcation and larger branches of the nerve separately prevents the possibility of nerve injury during thyroid surgery. inadvertent division of a branch may lead to significant vocal cord palsy, despite the surgeon believing that the nerve may be preserved. rln is particularly prone to be injured in cases of extralaryngeal bifurcation located near ita or the suspensory ligament of berry, which may develop in approximately one - third of patients. based on these findings together with the results from our study, it is suggested that bilateral bifurcation occurrence in the same patient may further increase the risk of nerve injury. our study aimed to increase surgeons awareness of possible variations of bilateral bifurcation of rln based on anatomical features of both left and right sides. extralaryngeal terminal bifurcation of rln may occur unilaterally at one side or bilaterally at both sides. unilateral bifurcation of rln was observed in 71.4% of our 70 patients with bifurcated nerves. bilateral variation was identified in 13.7% of our patients undergoing thyroidectomy cases in 28.6% of patients with bifurcated nerves. casella., reported the rate of bilateral bifurcation at 3.7%, serpell., at 8.9% and beneragama., at 18% of the patients who underwent total thyroidectomy, these rates are comparable with the rate found in our present study. kandil., reported 27% and 33.3% of bilateral bifurcation in patients undergone with total thyroidectomy, which was also in agreement with the prevalence of bilateral bifurcation that we observed in 28.6% of patients with bifurcated nerves. identification of the anatomical location of the bifurcation point along the cervical course of the nerve is crucial to securely and properly expose all branches of the nerve, and to protect nerve integrity without inadvertent injury to neural structures. in our study nerve bifurcation occurred in arterial and post - arterial segments in 70% of our patients with bilateral variations. the division point was rarely observed in close proximity to the laryngeal entry of the nerve (pre - laryngeal). this anatomical discordance between both sides can make the preservation of the motor function considerably difficult. anatomical courses of rlns were different when comparing the right with left nerves in one patient. extralaryngeal bifurcation of rln is a variation that carries great importance for the preservation of nerve integrity and function. we believe that familiarity with bifurcation of rln and bifurcation point locations along the cervical course of the nerve are considerably helpful for surgeon to avoid nerve injury. during thyroidectomy, surgeons attempting to locate rln must consider that approximately half of the patients have a bifurcated nerve. when identifying a bifurcated nerve in one side, surgeons must consider that there is approximately a 30% possibility for the presence of a bifurcated nerve in the contralateral side. because of a relatively high rate of bilateral bifurcation of rln, surgeons must be extremely cautious of exposing the nerve bilaterally to preserve the motor function of its extralaryngeal branches. the variability of bifurcation point locations along the cervical course of the nerve requires complete knowledge regarding the surgical anatomy of extralaryngeal bifurcation of rln, therefore, extensive experience on rln identification and exposure is necessary. in case of unilateral or bilateral bifurcation, the location of motor fibres in nerve branches has an extreme importance for the preservation of the motor function. incorrect identification of a relatively larger posterior branch as the main trunk of the nerve and inadvertent division of motor fibres (anterior branch) may lead to laryngeal muscle palsy, although surgeons believe that the nerve may be preserved. this anatomical variation can not be predicted preoperatively and might be associated with higher rate of nerve injury. anatomical variations such as extralaryngeal bifurcation of the nerve are good examples of high - risk situations. variability in the surgical anatomy of nerve bifurcation is a significant threat to the safety of thyroid surgery. bilateral terminal bifurcation of rln is a frequent occurrence in patients who have bifurcated rln prior to its laryngeal entry. location points may differ in left and right sides in the same patient in the majority of bilateral cases. great anatomical variability in the bifurcated nerve may increase the risk of the injury to the motor branch. therefore, a visual identification of all terminal branches of rln may be extremely helpful for the preservation of the motor function of bifurcated nerves. | background : anatomical variations of the recurrent laryngeal nerve (rln) such as extralaryngeal terminal bifurcation is an important risk for its motor function.aims:the objective is to study surgical anatomy of bilateral bifurcation of the rlns in order to decrease risk of vocal cord palsy in patients with bifurcated nerves.materials and methods : surgical anatomy including terminal bifurcation was established in 292 rlns of 146 patients. we included patients with bilateral bifurcation of rln in this study. based on two anatomical landmarks (nerve - artery crossing and laryngeal entry), the cervical course of rln was classified in four segments : pre - arterial, arterial, post - arterial and pre - laryngeal. according to these segments, bifurcation point locations along the cervical course of rlns were compared between both sides in bilateral cases.results:rlns were exposed throughout their entire courses. seventy (48%) patients had bifurcated rlns. we identified terminal bifurcation in 90 (31%) of 292 rlns along the cervical course. bilateral bifurcation was observed in 20 (28.6%) patients with bifurcated rlns. bifurcation points were located on arterial and post - arterial segments in 37.5% and 32.5% of cases, respectively. pre - arterial and pre - laryngeal segments contained bifurcations in 15% of cases. comparison of both sides indicated that bifurcation points were similar in 5 (25%) and different in 15 (75%) patients with bilateral bifurcation. permanent nerve injury did not occur in this series.conclusion:bilateral bifurcation of both rlns was observed in approximately 30% of patients with extralaryngeal bifurcation which is a common anatomical variation. bifurcation occurred in different segments along cervical course of rln. bifurcation point locations differed between both sides in the majority of bilateral cases. increasing surgeons awareness of this variation may lead to safely exposing bifurcated nerves and prevent the injury to extralaryngeal terminal branches of rln. |
metabolic syndrome is a cluster of risk factors for some diseases such as cardiovascular disease (cvd) and type 2 diabetes. according to the adult treatment panel (atp iii) criteria, any three of the five components are necessary for the diagnosis of metabolic syndrome : elevated waist circumference, blood pressure, serum triglyceride and glucose and reduced high - density lipoprotein (hdl) cholesterol. some studies have shown the relationship between metabolic syndrome and coronary artery diseases in different ethnic groups, gender, age, and postmenopausal women. several studies in europe have reported 28 - 37% prevalence of metabolic syndrome with mental illness such as schizophrenia. many other studies indicated 43% and 46% prevalence of metabolic syndrome in the united states and canada respectively. incidentally, people with psychiatric diseases such as schizophrenia have lower life expectancy than people without mental illness and subjects with coronary artery diseases. metabolic syndrome is reported in 19 - 63%, 42.4%, 12 - 36%, and 8 - 56% patients with schizophrenia, schizoaffective psychosis, relapsing depression, and bipolar affective disorder, respectively. studies on patients with severe mental health have shown that males and females schizophrenic patients have 138% and 251% more chance to have metabolic syndrome than general populations. individuals with severe mental illnesses (smi) show a higher prevalence of metabolic syndrome than those who do not a metabolic syndrome. these people indicate an important social and/or occupational dysfunction. the elevated prevalence of metabolic syndrome in these subjects may be related to the disease itself, treatment with the antipsychotic drug, obesity, consumption of high - fat diets, low physical activity, and active smoking. studies have shown that individuals with metabolic syndrome have 4 and 3 times more risk of developing diabetes and coronary heart disease, respectively. the aim of the present study was to assess the metabolic syndrome among patients with smi in gorgan (south east of caspian sea, iran). 267 patients with smi, aged 18 - 73 years (mean age 38.93 10.79 years) who were treated with antipsychotic drugs and referred to the psychiatric unit at 5 azar education hospital of gorgan faculty of medicine, golestan university of medical sciences in gorgan, iran in 2014. smi patients were defined if they are in the following category : schizophrenia, bipolar 1 mood disorder, major depressive disorder with psychotic features, psychotic or mood disorder in association with a general medical condition and schizoaffective disorder. we excluded patients without complete information, pregnant, substance abuse, anxiety disorders, mental retarded, and axis ii disorders. a 10 ml blood samples were collected after an overnight fast of 12 h. serum fasting blood glucose, hdl - cholesterol, total cholesterol and triglyceride levels were determined with commercial kits by spectrophotometer techniques (model jenway 6105 uv / vis) in the metabolic disorders research center. the friedewald equation was used to calculate low density lipoprotein cholesterol (ldl - cholesterol) level. weight was measured, while subjects were minimally clothed without shoes, using digital scales. height was measured in standing position using tape meter while the shoulder was in a normal position. body mass index (bmi) was defined as weight in kilograms divided by height in meters squared. overweight was defined as bmi 25.0 - 29.9 kg / m and obese as bmi 30 kg / m. waist circumferences were measured at the point halfway between the lower border of ribs and the iliac crest in a horizontal plane. metabolic syndrome identified if smi patients had any three or more of the following criteria, according to the atp iii as it has shown in table 1. risk factors for the metabolic syndrome according to atp iii this study was approved by the research deputy ethics committee of the golestan university of medical sciences. a software system spss for windows (version 16.0, spss inc., chicago, illinois, usa) the evaluation of results was carried out using independent sample t - test and chi - squared test. table 2 shows the biochemical data of the subjects with and without the metabolic syndrome in patients with smi. the mean waist circumference, systolic and diastolic blood pressure, triglyceride and fasting blood glucose levels were significantly higher in the smi with metabolic syndrome, but the mean hdl - cholesterol was significantly lower (p < 0.05). tables 3 and 4 show biochemical data of smi men and women with and without metabolic syndrome. there were significant differences in the mean of waist circumference, systolic (except for women) and diastolic blood pressure, triglyceride, hdl - cholesterol and fasting blood glucose in men and women with metabolic syndrome when compared with subjects without metabolic syndrome (p < 0.05). the prevalence of metabolic syndrome in smi women (32%) was higher than men (13.77%). table 5 shows the distribution of different age groups in patients with smi with and without metabolic syndrome. the most prevalence of metabolic syndrome was in age groups 50 - 59 years old. biochemical characteristic of smi with and without metabolic syndrome biochemical characteristic of smi in men with and without metabolic syndrome biochemical characteristic of smi in women with and without metabolic syndrome distribution of different age groups in smi with and without metabolic syndrome our study showed a low prevalence of metabolic syndrome (20.60%) in patients with smi according to the atp iii criteria. several studies have assessed whether an increased prevalence of the metabolic syndrome is prevalent among patients with smi in comparison to the general population. studies showed that prevalence of metabolic syndrome was estimated to be 60% and 75% among schizophrenic and mood disorder patients, respectively. recent studies of heiskanen. indicated that prevalence of metabolic syndrome in patients with schizophrenia was 37% in comparison to the general population. many other studies have reported prevalence of metabolic syndrome changes in patients with smi in different populations such as hong kong (35%), usa (28.7 - 60%), australia (54%), canada (44.7%) and finland (37.1%). interestingly asian population show a lower prevalence of metabolic syndrome in patients with smi than western populations. studies on taiwan and thailand populations indicated that prevalence of metabolic syndrome in schizophrenia patients was 22% and 20%, respectively. our results show that the prevalence of metabolic syndrome in patients with smi in gorgan is lower than western populations and almost similar to some other asian countries., in the usa showed that elevated risk of metabolic syndrome in patients with smi to be 138% and 251% for males and females, respectively. the results of this study demonstrate that lifestyle of patients with smi in gorgan are not the same as the western countries. many studies have shown that unhealthy lifestyle and poor diet of these patients have an important role in etiology of the metabolic syndrome. smi patients are at risk for metabolic syndrome, therefore, should be screened and identify to prevent probable future type 2 diabetes mellitus and cvds. smi show 2 - 3 times the incidence of higher type 2 diabetes mellitus and cvds than the general population. a recent study in the usa indicated that all cvds risk factors such as central obesity, hypertension, dyslipidemia, and hyperglycemia were higher in smi than in the general population. study of lozano. revealed that deaths due to diabetes and cvds are estimated to increases by 2020. the prevalence of metabolic syndrome in our sample of patients with smi is lower than north american and european studies, these differences may be due to geographic differences. the lower prevalence of metabolic syndrome in these patients proposed that differences in lifestyle factors might decrease susceptibility to metabolic syndrome in this area when it is compared to other populations. the influence of lifestyle factors such as physical activity and diet were not evaluated in this study. the 20.60% prevalence of metabolic syndrome is lower than the study from australia (68%). the prevalence of metabolic syndrome in mental patients in gorgan deserves more attention from health service providers. elevated mortality is reported from psychiatric disorders such as schizoaffective, bipolar, and depressive disorders. in our study, the prevalence of metabolic syndrome was lower than some other studies in patients with bipolar disorder and psychotic depression. our results suppose that psychiatric illness should be taking into account a risk factor for metabolic syndrome, which makes it necessary to screen people with any psychiatric disorder. the variation of metabolic syndrome prevalence among smi were seen mostly in ages 50 - 59 years old in comparison to other age groups. our findings indicated that metabolic syndrome among these patients differs significantly from ages 30 up to 59 years old. in our study, the prevalence of metabolic syndrome was different between genders, which is in agreement with the findings of other studies. the onset of metabolic syndrome from an early age may increase the risk of cvds in smi. cardiovascular risk factors treatment should be considered the main care in people with mental disorders in addition to screening for cvd risk factors. our results show that the prevalence of metabolic syndrome in patients with smi in gorgan is almost similar to those observed in asian countries. it should be mention that the families of mental illness subjects in our country believe that their patients must be cared better than people without mental illness. these findings of this study suggest that mental illness patients are at risk of metabolic syndrome. according to our results, risk factors such as age and gender differences may play an important role in the presence of metabolic syndrome. in our country, women do less physical activity than men. | background : metabolic syndrome is commonly associated with cardiovascular diseases and psychiatric mental illness. hence, we aimed to assess the metabolic syndrome among severe mental illness (smi).materials and methods : the study included 267 patients who were referred to the psychiatric unit at 5th azar education hospital of golestan university of medical sciences in gorgan, iran.results:the mean waist circumference, systolic and diastolic blood pressure, triglyceride and fasting blood glucose levels were significantly higher in the smi with metabolic syndrome, but the high density lipoprotein (hdl)-cholesterol was significantly lower. the prevalence of metabolic syndrome in smi patients was 20.60%. there were significant differences in the mean of waist circumference, systolic (except for women) and diastolic blood pressure, triglyceride, hdl - cholesterol and fasting blood glucose in men and women with metabolic syndrome when compared with subjects without metabolic syndrome. the prevalence of metabolic syndrome in smi women was higher than men. the most age distribution was in range of 30 - 39 years old. the most prevalence of metabolic syndrome was in age groups 50 - 59 years old. the prevalence of metabolic syndrome was increased from 30 to 59 years old.conclusion:the prevalence of metabolic syndrome in patients with smi in gorgan is almost similar to those observed in asian countries. the prevalence of metabolic syndrome was lower than western countries. these observations may be due to cultural differences in the region. it should be mention that the families of mental illness subjects in our country believe that their patients must be cared better than people without mental illness. these findings of this study suggest that mental illness patients are at risk of metabolic syndrome. according to our results, risk factors such as age and gender differences may play an important role in the presence of metabolic syndrome. in our country, women do less physical activity than men ; therefore, the incidence of metabolic syndrome is higher among women. |
most human pharmacologic investigations of depression start with criteria for major depressive disorder (mdd) from the diagnostic and statistical manual of mental disorders. 1 the overlap of signs and symptoms in primary or secondary mental disorders generates multiple diagnoses called comorbidities. a meta - analysis showed that it was difficult to differentiate between placebo and medication response unless the hamilton depression rating scale score was > 23,2,3 but patients who were more severely ill benefited from medication more than placebo. serendipitous research findings may have value, such as the antidepressant response in patients who received iproniazid to treat mycobacterium tuberculosis, but such findings may be limited in contributing to the design of new treatments for depression.4 the cause of depression is complicated by language. it has parallels in language history that convey descriptive states, such as deprimere (latin, meaning to press down), lugubrious from lugere (latin, meaning to mourn), and melancholy from melagkhola- (greek, meaning sadness, from melas and khole [black bile]).5 previous studies have demonstrated abnormal mitochondrial function and oxidative stress in various diseases. eht hypothesized that intracellular mitochondrial metabolic dysfunction in specific brain regions represents either a cause or effect of mdd. the purpose of the present study was to review the literature for evidence to evaluate this hypothesis. the brain adapts to environmental or internal challenges with alterations in synaptic efficacy, promotion of neurogenesis, suppression of pathologic apoptosis, and neuronal molding. if mitochondrial dna (mtdna) becomes compromised, various cascades of dysfunctional metabolism may impair cellular connectivity and communication. mitochondria oxidize glucose through an oxidative phosphorylation system composed of five of the 13 subunits of mtdna. the energy released during electron transport helps shift protons across the inner mitochondrial membrane, decreasing the concentration of protons on the inside of the membrane and creating gradients of acid - base and potential energy. the energy generated by protons spontaneously diffusing across the inner membrane is used to convert adenosine diphosphate to adenosine triphosphate. reactive oxygen species are free radicals (molecules with one unpaired electron) derived from molecular oxygen. in its lowest energy state, molecular oxygen (o2) has two unpaired electrons with similar spin orientation in its outer electron orbit. if one of the unpaired electrons is excited, the electron orbit changes and the oxygen molecule becomes more reactive. mitochondrial oxidative phosphorylation involves respiratory complexes that donate electrons to oxygen, producing superoxide anions and peroxides. excessive reactive oxygen species can damage intracellular proteins and lipids, oxidize mtdna and nuclear dna, and open the mitochondrial permeability transition pore to cause mitochondrial swelling and release of apoptotic effectors. if the mitochondrial outer membrane ruptures, the apoptotic factor cytochrome c is released. reactive oxygen species may function as an intracellular signal transduction regulator,6,7 and this exemplifies the subtlety of the balance of intracellular reactive oxygen species. apoptosis is induced by either extrinsic stimulation of specific death receptors on the cell membrane or intrinsic mechanisms derived from mitochondrial stress. apoptosis is tightly regulated, and increased or decreased apoptosis may cause cancer or developmental, autoimmune, or neurodegenerative diseases.8 the location of mitochondria is determined by energy need. the endoplasmic reticulum forms a network of tubules and cisternae throughout the cytoplasm, with an inhomogeneous distribution of calcium uptake and release sites. cytoplasmic signals may converge in the endoplasmic reticulum to form spatiotemporally controlled patterns of calcium release.7 in brain cells, the endoplasmic reticulum has two release channels for calcium ions, ie, inositol 1,4,5-triphosphate receptors and ryanodine receptor type 3. mitochondria linked to the inositol 1,4,5-triphosphate receptor and ryanodine receptor channels control local calcium release and cytoplasmic calcium regulation.9 mitochondria are involved in numerous metabolic activities, including the urea cycle, lipid metabolism, porphyrin synthesis, and homeostasis of steroid hormones and cellular calcium ions. calcium ions stimulate oxidative phosphorylation, upregulate the creation of adenosine triphosphate, and affect the metabolism of other molecules. calcium ions enter the mitochondria by a well regulated outer and inner mitochondrial membrane process. glutamate synapses are tripartite, involving the presynaptic neuron, postsynaptic neuron, and glial cell. there are obstacles to demonstrating mitochondrial dysfunction that may predict a depressed, lugubrious, melancholic mental state. human research is compromised by genetic variation, environmental and pharmacologic exposures, and comorbid diagnosed or undiagnosed illnesses in controls and patient groups. a study of survival or death from sepsis highlighted the importance of mtdna haploids.10 mitochondrial dna is composed of maternally inherited circular haploid groups, named from a to z in the sequence of their discovery. if mtdna becomes compromised, cascades of dysfunctional metabolism may impair cellular connectivity and communication. there is a physiologic reserve that is affected by mtdna.10 in 150 severely ill patients with sepsis who were admitted to an intensive care unit, there was a 2.12-fold (95% confidence interval 1.024.43) increased likelihood of survival at 180 days in patients with haploid group h than patients without this haploid group.10 this suggested that genetic haploids may provide a physiologic reserve that is protective against physiologic stress. this finding is similar to the observation that the presence of certain genetic material permits a physiologic reserve in transgenic rats. genetic haploids may determine the degree of vulnerability to certain diseases, such as depression. functional imaging to study brain metabolism is based on mitochondrial respiration that requires glucose, but neurons do not store glucose. glucose is transported across the plasma membrane of a cell by a glucose transporter protein (glut) that has several isoforms in different tissues. the isoform glut1 transports glucose and other hexoses across the blood brain barrier and astrocyte plasma membrane, and the isoform glut3 transports glucose into the neuron.11 positron emission tomography uses f - fluorodeoxyglucose, an analog of glucose that emits positrons when glucose is metabolized within the cell. single - photon emission computed tomography images brain metabolism using selected radiopharmaceuticals that emit gamma rays. magnetic resonance imaging (mri) provides images of the anatomy of the brain. imaging studies from positron emission tomography, single- photon emission computed tomography, and mri scans were evaluated to determine anatomic and metabolic correlates with mdd.12 in patients with a mood disorder, data were reviewed about regional changes in brain metabolism, volume, and structure. regions demonstrating brain pathology included the medial prefrontal cortex, medial and caudolateral orbital cortex, amygdala, hippocampus, ventromedial parts of basal ganglia, and anterior cingulate cortex.12 in another study, the amygdala was the only structure in which regional blood flow and glucose metabolism correlated with the severity of depression.13 there was increased metabolism in the amygdala during wakefulness and sleep. in asymptomatic periods between depressive episodes, patients with familial depression who were not receiving antidepressant treatment showed abnormally elevated blood flow and metabolism ; in contrast, patients with familial depression who were receiving antidepressant treatment had normal amygdala metabolism.13 a study with mri scans evaluated orbital frontal cortex volume in 15 patients in remission from mdd and 20 control subjects.14 patients in remission from mdd had a 32% smaller volume of the medial orbitofrontal cortex than the control subjects.14 the ongoing volume loss may have occurred because of impaired anatomic recovery from mdd, and this suggested that mdd may cause chronic brain tissue changes. abnormal affect and cognitive impairment may occur in children and adults with cerebellar cognitive affective syndrome.15 the vermis and fastigial nucleus function as a limbic cerebellum. this concept is supported by neuropsychologic testing of adults with lesions confined to the cerebellum, children with posterior fossa syndrome, patients with developmental anomalies of the cerebellum, and patients with spinocerebellar ataxia.15 another mri study evaluated 16 patients hospitalized for the first time with a manic episode, 14 patients with prior hospitalizations for manic episodes, and 15 control subjects.16 the right and left vermal areas 13 were measured. x) was smaller in patients with bipolar disorder who experienced multiple episodes of depression.16 therefore, cerebellar vermal atrophy may be a late neurodegenerative event in patients with multiple affective episodes. a post mortem study supports the importance of coexistent damage to the cerebellum and temporal lobe. in a middle- aged man with depression, dementia, and volatile behavior, histology demonstrated partial necrosis of the temporal lobe, disorganized collection of large neurons and granular cells in the vermis nodulus, and excessive numbers of bergmann astrocytes, probably the result of a closed head injury.17 extensive clinical diagnostic screening of women (aged 2386 years) with a history of recurrent mdd without medical comorbidity and normal control subjects showed that there was no difference between the two subject groups in cortisol levels obtained the day before an oral dexamethasone (1 mg) suppression test.18 evaluations confirmed that neither control subjects nor patients in this study had current depression. there was no relationship between age and volume loss of the hippocampus, but there was a direct relationship between total lifetime depression duration, bilateral hippocampus volume loss, and smaller volume of the amygdala core nuclei. in two post mortem histologic studies of brain tissue from people who had mdd, the diagnosis of mdd was confirmed using information from different sources, but the diagnosis of mdd may be unreliable because of unknown personal and medical factors.19,20 a histologic study in humans showed morphometric evidence of neuronal and glial cell changes in patients with mdd.19 there had been functional abnormalities found on imaging the left dorsolateral prefrontal cortex and left orbitofrontal cortex. patients with a retrospective diagnosis of mdd without psychosis were compared with normal control subjects. in patients with mdd, there was diminished neuronal size and cortical thickness (most prominently in the rostral orbitofrontal region), markedly diminished glial density, and moderately diminished neuronal size, without significant loss of cortical thickness in the left caudal orbitofrontal region and dorsolateral prefrontal cortical region.19 post mortem brain tissue was evaluated from area 24b of the supracallosal anterior cingulate cortex in subjects with mdd, bipolar disorder, or schizophrenia, and in normal control subjects. subjects with mdd had decreased glial cell density (22%, p = 0.004) and neuronal size (23%, p = 0.01) in layer 6 than control subjects.20 diverse monoamine oxidase activity has been observed in purified beef brain mitochondria, and enzyme properties are influenced by substrate and ph.21 the mitochondrial membrane may restrict enzymatic activity for different substrates, and this may be affected by ph and ion concentrations.21 in a study of n - methyl - d - aspartate (nmda) receptor activation in neonatal rat cardiomyocytes, overstimulation of nmda receptors by glutamate caused excessive calcium ion influx, which compromised mitochondrial membrane polarity ; glutamate overstimulation also caused an increase in reactive oxygen species and a cascade of apoptotic factors.22 tianeptine modulated the glutamatergic effects at nmda receptors and had a protective effect on neuronal and synaptic function under stress.22 creating intracellular hypoxia can induce a stress response. acute exposure to smoke inhalation in awake rats and mice causes a hypoxic environment in the cell, an increase in reactive oxygen species, inhibition of mitochondrial respiration, and oxidative dna damage.2325 however, in transgenic mice with overexpression of neuroglobin (a neuroprotective globulin protein), exposure to toxic smoke inhalation caused less oxidative damage to dna.2325 therefore, oxidative dna damage caused by inhibition of mitochondrial respiratory complexes may contribute to neuronal dysfunction and progressive brain injury. oxidative stress may cause mitochondrial fragmentation. in two cell types (human lung adenocarcinoma cells and african green monkey kidney fibroblasts transformed with simian virus 40), laser irradiation caused oxidative stress and mitochondrial fragmentation ; however, this mitochondrial damage was prevented by dehydroascorbic acid, which scavenges reactive oxygen species.26 the mitochondrial fragmentation was caused by oxidative stress and associated translocation and imbalance of profission (dynamin - related protein 1) and profusion proteins (mitofusin 2).26 repeated daily stress causes atrophy of hippocampal ca3 pyramidal neurons in tree shrews (tupaia belangeri).27 male tree shrews are territorial ; when subordinate male tree shrews were confined with dominant ones, the subordinates lost weight and had increased urinary cortisol levels. apical dendrites in ca3 pyramidal cells had a decreased number of branch points and total dendritic length in subordinate tree shrews when compared with controls.27 in rats and tree shrews, repeated stress caused volume loss in the hippocampus and retraction of apical dendrites in ca3 pyramidal cells.28 tianeptine prevented changes in hippocampal volume and preserved cell proliferation, but stress reduced neurogenesis of hippocampus neurons.28 these studies are important because one granule neuron from the entorhinal cortex of the hippocampus may innervate an average of 12 ca3 neurons, and each ca3 neuron innervates an average of 50 other ca3 neurons and 25 inhibitory cells.28 therefore, there may be a 600-fold amplification of excitation and 300-fold amplification of inhibition.28 the neuroprotective effect of topiramate was studied in young adult male wistar rats in status epilepticus triggered by pilocarpine to resemble human temporal lobe epilepsy.29 in this study, the effect of large quantities of calcium on mitochondrial metabolism was evaluated from sections of the hippocampus (cornu ammonis [ca1 and ca3 ] and dentate gyrus) and parahippocampus. most of the calcium accumulated in the mitochondria, as shown by a transient increase in green fluorescence of calcium.29 when larger amounts of calcium were added, the mitochondrial membrane permeability transition pore opened, causing swelling and release of cytochrome c from the mitochondrial intermembrane space.29 stress models of the brain have shown that elevated cortisol levels cause histologic changes, especially in the dentate gyrus. toxicity from excess excitation may be mediated in part by entry of calcium into the cell ; the high intracellular calcium level may open the membrane permeability transition pore, depolarize the mitochondrial membrane, release cytochrome c, and cause cell death. in rats with seizures induced with pilocarpine, intense neuronal activity is associated with loss of oxidative phosphorylation complexes i and iv located in the ca3 and ca1 pyramidal areas.30 imaging with rhodamine showed a decreased mitochondrial membrane potential in both pyramidal subfields, but mitochondrial oxidative phosphorylation was unaltered in the dentate gyrus and parahippocampus.30 this research showed that specific tissues may be vulnerable to neurotoxicity. superoxide dismutase protects intracellular metabolism from oxidative stress, and there are three forms of superoxide dismutase in humans. the superoxide dismutase, glutathione peroxidase, reduces lipid hydroperoxides and scavenges peroxides. in a study of oxidative stress in prion- infected or uninfected hypothalamic neuronal gt1 - 7 cells, the infected cells showed increased lipid peroxidation and apoptosis associated with decreased glutathione - dependent and superoxide dismutase activity.31 although this study focused on transmissible spongiform encephalopathy, it provided a stress model for hypothalamic cells and suggested that oxidative stress may dysregulate the hypothalamus. in humans, the adenohypophysis secretes many proteins and is controlled by hypothalamic secretions and hypothalamic hypophyseal portal vessels. the potent selective d1-dopamine receptor agonist, a68930, may protect the hypothalamic - pituitary - adrenal axis against unpredictable acute and chronic stress.32 changes in fiber density and numbers of immunoreactive neurons with tyrosine hydroxylase and glucocorticoid receptors were measured in regions of the rat brain rich in dopamine and glucocorticoid receptors. chronic unpredictable stress caused a significant decrease in the number of immunoreactive neurons located in the striatum, medial forebrain bundle, ventral tegmental area, and substantia nigra, and reduced the activity of superoxide dismutase and catalase in the cortex, striatum, and hippocampus ; the unstressed control rats showed no changes.32 therefore, the d1-dopamine agonist a68930 may have neuroprotective effects in both acute and chronic unpredictable stress.32 mitochondrial dysfunction may also be important in mood disorder because of genetic variation or mutations in nuclear and mtdna. mitochondrial diseases, such as leber s disease (hereditary optic neuropathy), have been correlated with psychiatric illness, including the effects of psychotropic drugs on mitochondria, genetic variations or mutations, polymorphisms of nuclear encoded mtdna, and impairment of the electron transport system.33 in homoplasmy, the cell has identical copies of mtdna ; in heteroplasmy, there is more than one type of mtdna present in the cell.34 many patients with mood disorder have pathologic mtdna defects and two different sets of mtdna. when some nuclear genes are disrupted, autosomal dominant or recessive mitochondrial disease may occur.34 some mitochondrial diseases may cause secondary mtdna abnormalities such as deletions or depletion of copy numbers. 34 there may be a relationship between mood disorder and inflammatory processes manifested by increased plasma levels of proinflammatory cytokines and acute phase reactants, oxidative damage to red blood cell membranes, and decreased serum zinc levels.34 the studies reviewed provide support for the hypothesis that intracellular mitochondrial metabolic dysfunction in specific brain regions is associated with mdd, either as a cause or effect. mitochondrial dysfunction caused by oxidative stress alters intracellular metabolism and may damage mtdna. the level of resilience or physiologic reserve of mitochondria to stress may explain the variation in clinical presentation and disease severity. glut glucose transporter proteins require healthy mitochondria, and failure in one region of the cell may affect the metabolism of the entire cell. impairment of a specific tissue may provide an explanation for the varied signs and symptoms of depression and effects on other parts of the body. histologic study of the rat hippocampus demonstrated enfolding of the hippocampus and cellular connections.35 there are four sections of the cornu ammonis. layer 2 of the entorhinal cortex is connected through the perforant pathway to the dentate gyrus and ca3 ; the granular cells of the dentate gyrus are connected with ca3 ; the pyramidal cells of ca3 are connected with ca1 ; the pyramidal cells of ca1 are connected with the subiculum and deep entorhinal cortex ; and the subiculum neurons are connected with the entorhinal cortex. previous studies showed that ca3 pyramidal neurons may be compromised by the effect of physiologic stress.27,28 stress requires mitochondrial physiologic reserve, and a change in ca3 pyramidal neurons may affect the interdependent connections of the hippocampus, especially considering the amplification effects of multiple neural connections.28 in the 19th century, there was controversy about the relationship between dementia, depression, paralysis, and brain localization, and there were differences in medical opinion about histopathologic post mortem findings,36,37 as reflected by mairet who observed postmortem changes in the temporal lobe of melancholic patients and hypothesized that :...... this area might be related to primary feelings of sadness and that the nihilistic delusions were in fact secondary developments made possible by the spread of the lesion to the cortex.36 this 19th century debate about the presentation of melancholic patients highlighted the diverse presentation of this illness, which may be influenced by culture. therefore, it was reasonable to infer that depression may be influenced by genetic vulnerability to stress similar to that observed in patients with sepsis. a variety of tissues may have changes in morphology and function associated with depression that may affect the clinical presentation. chronic memory problems in depressed patients in remission may be caused by a lack of resilience of ca3 pyramidal cells. the amygdala, which includes several nuclei, may enable long - term memory but does not store memory. the basolateral complex of the amygdala may affect long - term memory through its numerous projections.14 memory is promoted by stress or excitement, and impaired memory during remission from mood disorder suggests that cellular metabolic damage may persist even though stability of mood has been achieved.18,38 areas of the brain may vary in vulnerability to chronic metabolic changes in depression. symptoms of abnormal regulation of the hypothalamus observed with depression may include altered sleep, menstrual irregularities, altered appetite and thirst, autonomic nervous system dysfunction, and irritability. the cornu ammonis of the hippocampus connects through the fornix to the mammillary body and has efferent connections to the temporal lobe, and impairment of the hippocampus may include impaired memory, especially verbal memory. abnormal regulation of the prefrontal cortex may cause bradyphrenia, and amygdala dysfunction may cause abnormal emotional control. the association between mdd, decreased glial cell density, and decreased neuronal size in the supracallosal anterior cingulate cortex suggests that patients with mdd may have impaired emotional processing and impaired emotional interface with pain.20,3941 in addition, patients with motor cerebellar signs may also have an association between the vermis of the cerebellum, affect, and abnormal cognitive function.17 although there is controversy about the biologic basis of depression, morphologic and functional abnormalities associated with mitochondrial dysfunction and oxidative stress may be associated with depression. genetic variation or mutations in nuclear dna and mtdna the pathologic morphology at various sites may be associated with impaired function and may enable prediction of diverse presentations of depression and chronic cognitive problems. | there is controversy about depression being a physical illness, in part because a reproducible, sensitive, and specific biologic marker is not available. however, there is evidence that mitochondrial dysfunction and oxidative stress may be associated with abnormal brain function and mood disorders, such as depression. this paper reviews selected human and animal studies providing evidence that intracellular mitochondrial metabolic dysfunction in specific brain regions is associated with major depressive disorder. this supports the hypothesis that chronic mitochondrial dysfunction in specific tissues may be associated with depression. evaluation of mitochondrial dysfunction in specific tissues may broaden the perspective of depression beyond theories about neurotransmitters or receptor sites, and may explain the persistent signs and symptoms of depression. |
grazing cattle are continuously exposed to infection with gastrointestinal nematodes (gin) that can severely impair the health and productivity of pasture - based livestock systems (corwin, 1997, shaw., 1998, charlier., 2014). in practice, the control of gin in cattle largely relies on the routine use of anthelmintic drugs, mainly from the macrocyclic lactone (ml) family (vercruysse and rew, 2002, geurden., 2015). as a consequence, worm populations resistant to mls have been selected, and anthelmintic resistance (ar) is now becoming a serious threat to the control of bovine nematodes in several countries (sutherland and leathwick, 2011, gasbarre, 2014, sutherland and bullen, 2015). coinciding with the development of ar, concerns regarding the prophylactic use of veterinary drugs and chemical residues in both food and environment have led to stricter regulations on the use of anthelmintics in some nations (thamsborg., 1999). in 1999, denmark became the first country to introduce prescription - only use of anthelmintics in livestock, requiring a mandatory veterinary diagnosis before treatment in both organic and conventional farms (anonymous, 1998, anonymous, 2013). since 2000, there has been an additional requirement for all prescriptions in production animals to be registered in vetstat the danish system for surveillance of the veterinary use of drugs (stege., preliminary analyses in vetstat indicate that mls accounted for 85% of all anthelmintics prescribed for danish cattle between 2010 and 2012, with ivermectin (alone or in combination) representing 72% of all ml prescribed (pea - espinoza. however, and despite the significance of ivermectin for current parasite control strategies in cattle, its field efficacy against gin has not been investigated in denmark. in the absence of quantitative molecular techniques for the detection of ml - resistance, and the high cost of the controlled efficacy test (the current gold standard method for verification of anthelmintic activity ; wood., 1995), the only readily available technique for investigating field drug efficacy is the faecal egg count reduction test (fecrt). this technique estimates the efficacy of an anthelmintic to reduce the faecal egg counts (fec) of infected animals based on measurements pre- and post - treatment, or between treated and untreated individuals. the major advantages of the fecrt are that all drugs can be tested regardless of active compounds or formulation and that it relies on fec detection methods readily available in most diagnostic laboratories. the current recommendations to conduct and analyse fecrt in cattle derive from guidelines by the world association for advancement of veterinary parasitology (waavp), which were originally developed for detection of ar in sheep nematodes (coles., 1992). however, potential limitations have been highlighted concerning the use of fecrt with bovine nematodes, mainly due to the lower faecal egg excretion of cattle, compared to sheep, and the highly aggregated distribution of fec in cattle groups (coles, 2002, coles., 2006, demeler., 2010, el - abdellati., 2010, sutherland and leathwick, 2011). these factors may limit the correct analysis of fecrt data and inference of drug efficacy in cattle using the waavp guidelines. more recently, bayesian modelling using markov chain monte carlo (mcmc) methods have been advocated as robust statistical analyses to cope with low and aggregated fec data (denwood., 2010, these mcmc - based procedures, available as open - source r packages or web - interface software, are being increasingly used to infer drug efficacy and to monitor ar in horse nematodes (denwood., 2010, fischer., 2015) and cattle helminths (neves. however, the performance of these mcmc procedures with the low mean fec and parasite aggregation levels commonly found in cattle has not yet been evaluated. in addition, sensitive and species - specific tests to detect which gin species survive treatment are critical for the surveillance of ar and are urgently required for cattle (coles, 2002, sutherland and leathwick, 2011). the objectives of the present study were : 1) to assess the efficacy of ivermectin (ivm) against gin in naturally infected danish cattle by fecrt, and 2) to evaluate the performance of different statistical approaches for estimating drug efficacy using simulated bovine fec data of similar characteristics to those observed in danish cattle. in addition, we investigated the prescription patterns of anthelmintics in the study farms in order to examine a possible relationship between previous use of avermectins and ivm efficacy in the fecrt. cattle farms (50) with a history of clinical parasitism were contacted through local veterinarians across denmark during spring 2013 and 2014. farms were selected based on the following criteria : herd size 20 first - season grazing (fsg) calves with 4 weeks of grazing (before the initial screening) and not treated with anthelmintics within 8 weeks prior to sampling. in addition, the availability of a cattle crush or barn was required for the handling of animals. a total of 19 farms (8 in 2013 and 11 in 2014) that fulfilled these criteria accepted the invitation. individual faecal samples were collected from 20 fsg calves in each farm between mid - june and early september of 2013 and 2014 for analysis of fec (initial screening). due to a low number of farms with mean fec > 150 strongyle eggs per g (epg) of faeces (as recommended by coles., 1992), farms with a mean of the six farms finally included in the study, one herd was a conventional beef farm (farm # 1), three were organic dairy farms (# 2, # 4 and # 6), one was an organic beef farm (# 5) and one was a conventional dairy farm (# 3). in denmark, organic cattle farms should by law provide access to pasture from 15 april until 1 november (anonymous, 2016), while conventional farms do not have to comply with this rule. the cattle breeds in the investigated farms were danish holstein crossbreeds (# 1 and # 5), danish holstein (# 2, # 3, and # 6) and danish jersey (# 4). all the selected farms were located in the jutland peninsula and the fecrt was conducted within one to four weeks after the initial screening. the fecrt was performed to test the efficacy of ivm based on waavp recommendations (coles., 1992). pre- and post - treatment faecal samples from treated and untreated animals were included, and a total of 120 fsg calves were enrolled in the fecrt studies. on the day of treatment (day 0), 20 fsg animals from each farm were stratified by fec (based on the initial screening) and randomly allocated to a treatment group (ivm ; n = 10) or an untreated control group (ctl ; n = 10) of similar (initial) mean fec. due to a limited number of animals available in farms # 4 and # 6 at the start of the fecrt, oral formulations of ivm are not registered for use in cattle in denmark, thus injectable ivm was used. at day 0, individual body weights (bw) were estimated in the ivm group using a girth tape for cattle (rondo combi, kruuse, denmark), and the calves in the treatment group were injected with the recommended dose of ivm (0.2 mg kg bw s.c. a comparison of bw estimations between girth tape and electronic scale in a group of 30 fsg calves (bw range = 84172 kg) was performed prior to the study and demonstrated a very high correlation between the methods (pearson 's correlation = 0.98). faecal samples were collected rectally from all animals on day 0 and 14 days post - treatment (day 14). immediately after collection, the faecal samples were vacuum packed (freshield touch, cse co, gyeonggi - do, korea) to create anaerobic conditions and transported to the laboratory in a cooling box. on all farms, animals in the ivm and ctl groups grazed together on the same pastures until day 14, when all control calves were treated with the recommended dose of injectable ivm as described above. upon arrival at the laboratory, individual fec were determined using an accredited, modified mcmaster technique with a sensitivity of 5 epg (henriksen and aagard, 1975). at day 0 and day 14, pooled larval cultures were prepared from the ivm and ctl groups by mixing 10 g of faeces from each animal of the same group into a pool, which was then cultured according to roepstorff and nansen (1998). after 14 days of incubation at 20 c, nematode l3 were recovered by baermannisation and stored at 12 c. a small number of l3 were harvested in the post - treatment larval cultures from farms # 1, # 2, # 5 and # 6 (90% in cattle should not be considered a case of drug resistance (coles., 1992). in practice, most studies evaluating anthelmintic efficacy in cattle declare ar when fecr < 95% and lower 95% ci < 90, as proposed for sheep. nevertheless, it has been suggested that this criteria is biased towards declaration of ar when there is none, particularly if the mean fecr% is between 9095% and the ci is wide (lyndal - murphy., 2014). in the present study, we included the upper 95% ci in the interpretation of the fecrt to increase the certainty of detecting true cases of ivm inefficacy. a similar interpretation for fecrt studies in cattle has been reported in recent investigations by geurden. (2016). however, the effect of including the upper 95% ci in the interpretation for estimating drug efficacy using a fecrt, and how this correlates with an actual resistant phenotype confirmed by controlled efficacy tests, warrant further investigation. it is also important to note that a reduced fecr% may not necessarily be caused by ar. a lower - than - expected in vivo efficacy, or varying drug response between animals, could be the result of under dosing (e.g. due to inaccurate estimation of bw) and/or altered drug pharmacokinetics and pharmacodynamics in different animals (e.g. due to nutrition - related variations in fat reserves that may affect the persistent efficacy of ml, erratic absorption of drugs from the site of injection and/or interactions with other co - administered drugs) (gonzlez canga., 2012, areskog., 2014, de graef., 2013). these factors can impair the correct estimation of drug efficacy and detection of ar, particularly in the dose - limiting species c. oncophora. recently, c. oncophora populations that were declared resistant to the recommended dose of injectable ivm by fecrt in two swedish cattle farms (with fecr% [upper ci ] = 78% [97% ] and 79% [98% ] in each farm ; demeler., 2009) were declared ivm - susceptible when tested in calves under controlled conditions (areskog., 2014). therefore, the presence of ivm - resistant nematodes suggested by our fecrt in three farms, as well as the ar status in the farms with inconclusive results and low initial fec, should be confirmed by controlled efficacy test. the use of anthelmintics in the farms included in our fecrt was investigated to potentially detect trends in drug use and the extent of treatments with avermectins. data was retrieved from the vetstat database and used to estimate the number of animals treated with a given anthelmintic at each prescription. however, the actual number of cattle treated at each investigated prescription is unknown and our analysis aimed only to offer a rough estimate of the anthelmintic use in these farms. furthermore, vetstat does not register whether adult cattle are lactating or not at the time of treatment, and therefore the prescription of drugs not allowed for treatment of animals in lactation (e.g. ivm, levamisole) recorded in some of the studied farms deserves further investigation. based on the data retrieved from vetstat, most of the anthelmintics prescribed in the six farms between 2002 and 2012 were avermectins, mostly topical ivm products. a similar reliance on avermectins has been preliminarily detected in the entire danish cattle population in the period 20102014, constituting 80% of all treatments of which 79% were ivm, mainly in topical formulations (pea - espinoza., unpublished data). the irregular prescription of anthelmintics observed in the study farms correlates with the prescription - only regulations in denmark, illustrated by the treatment of single animals or selected groups of animals in the herds. however, prescription patterns suggesting whole - group treatments in some farms indicate that these may be recommended by veterinarians under certain conditions (e.g. during outbreaks of dictyocaulosis), and the effect of this practice on the selection for ar needs further investigation. all treatments against gin in denmark should be based on a clinical and/or laboratory examination, and preventive / strategic anthelmintic treatments without such diagnosis are illegal. organic farms are further encouraged to apply other means of parasite control than use of anthelmintics ; however, due to limited knowledge of alternative and effective parasite control methods, most farms (whether organic or not) still rely on anthelmintic drugs. therefore, and considering the relevance of ivm and other anthelmintics for nematode control in danish cattle, the true extent of ar in bovine nematodes in denmark needs to be assessed in larger surveys. until then, producers and veterinarians should be aware of potentially ineffective treatments against gin in cattle, while reducing the reliance on anthelmintics by including other parasite control strategies with documented efficacy, such as grazing management and feeding with bioactive forages (nansen. in conclusion, reduced ivm efficacy was detected by all methods for analysis of fecrt data excluding untreated controls in three of six danish cattle farms investigated. cooperia oncophora was the main species surviving ivm treatment in three farms with confirmed reduced drug efficacy, while o. ostertagi was also identified post - treatment by qpcr in one farm with reduced ivm efficacy. nevertheless, the presence of ivm - resistant nematode strains suggested by the fecrt should preferably be confirmed by controlled efficacy test. the reduced efficacy of ivm detected in this study and the widespread use of ml drugs in danish cattle suggest that farmers and their advisors should be aware of potentially ineffective treatments and larger surveys are warranted to describe the true extent of the problem. however, further validation of the design and analysis of the fecrt in cattle are urgently needed before such surveys can be implemented in cattle farms. | the efficacy of ivermectin (ivm) against gastrointestinal nematodes in danish cattle was assessed by faecal egg count reduction test (fecrt). six cattle farms with history of clinical parasitism and avermectin use were included. on the day of treatment (day 0), 20 naturally infected calves per farm (total n = 120) were stratified by initial faecal egg counts (fec) and randomly allocated to a treatment group dosed with 0.2 mg ivm kg1 body weight s.c. (ivm ; n = 10) or an untreated control group (ctl ; n = 10). individual fec were obtained at day 0 and day 14 post - treatment and pooled faeces by group were cultured to isolate l3 for detection of ostertagia ostertagi and cooperia oncophora by qpcr. treatment efficacies were analysed using the recommended waavp method and two open - source statistical procedures based on bayesian modelling : eggcounts and bayescount. a simulation study evaluated the performance of the different procedures to correctly identify fec reduction percentages of simulated bovine fec data representing the observed real data. in the fecrt, reduced ivm efficacy was detected in three farms by all procedures using data from treated animals only, and in one farm according to the procedures including data from treated and untreated cattle. post - treatment, o. ostertagi and c. oncophora l3 were detected by qpcr in faeces of treated animals from one and three herds with declared reduced ivm efficacy, respectively. based on the simulation study, all methods showed a reduced performance when fec aggregation increased post - treatment and suggested that a treatment group of 10 animals is insufficient for the fecrt in cattle. this is the first report of reduced anthelmintic efficacy in danish cattle and warrants the implementation of larger surveys. advantages and caveats regarding the use of bayesian modelling and the relevance of including untreated cattle in the fecrt are discussed. |
since the turn of the century, interest in the dynamic behavior of the radiation belts has grown immensely (for reviews, see millan and baker, millan and thorne, and hudson.). it is well known that processes leading to radiation belt relativistic electron enhancements also lead to greater losses [e.g., turner., sometimes acceleration dominates, and at other times loss, to either the magnetopause or atmosphere (precipitation), dominates. in fact, it is exactly this interplay between the various acceleration, loss, and transport mechanisms that brings about the wide range of responses in the radiation belts. new missions, such as the van allen probes, are making extraordinarily detailed measurements of the particle and wave environment throughout the radiation belts. yet solar cycle 24 has proven to be very weak, producing many fewer large geomagnetic disturbances than previous solar cycles. in a precursor to this study, reeves. statistically investigated the change of relativistic electron flux levels at geosynchronous due to moderate and large geomagnetic storms, those with minimum disturbance storm time (d s t) index less than 50 nt [gonzalez., 1994 ].) they found that large geomagnetic storms result in flux enhancements 53% of the time, no change to the flux level 28% of the time, and flux depletions 19% of the time. for example, on 14 february 2009 a small geomagnetic storm (minimum d s t 36 nt) resulted in prolonged enhancement of relativistic electrons at geosynchronous orbit by several orders of magnitude (figure 1). concurrent with this small storm, electron precipitation was detected by a balloon array for radiationbelt relativistic electron losses (barrel) payload [millan., more recent studies have even reported dramatic radiation belt activity during nonstorm times [e.g., schiller., 2014 ; su., 2014 ], underscoring the importance of investigating electron dynamics even during geomagnetically quiet times. two weeks of data are plotted, centered on the time of storm, 14 february 2009 1500 ut, which is marked by a vertical red line. (top) d s t ; horizontal lines mark 0 nt and 20 nt as a guide. d s t dropped 69 nt to a minimum of 36 nt. (bottom) geosynchronous goes 11 > 2 mev electron flux ; dashed vertical lines mark the prestorm and poststorm periods, and horizontal lines mark the corresponding 90th percentile maximum flux. therefore, it is essential to understand the statistical response of relativistic electrons in the radiation belts to small geomagnetic storms. to date, no such study has been performed. here we extend the analysis of electron response to geomagnetic storms to include the smallest storms. the method used in the present study closely follows that of reeves. and analyzes the same period beginning in october 1989 and ending in october 2000. we begin by identifying small geomagnetic storms using 1 h resolution d s t data. the time of the storm is defined as the time of minimum d s t. we use an automated stormfinding algorithm with three relatively simple criteria. the first criterion serves to select times for which d s t is at a minimum value. the third criterion requires a preceding sharp and significant drop in d s t. the criteria are as follows : (1) d s t must be the first occurrence of the global minimum during a period extending 16 h prior and 16 h after ; (2) d s t must be between 50 nt and 20 nt, inclusive ; and (3) d s t must decrease by at least 27 nt in the preceding 12 h. these criteria together select times for which d s t shows similar characteristics as during large geomagnetic storms, namely, a sharp drop that indicates the start of the storm main phase followed by a recovery period extending several days or longer. sometimes, a sudden storm commencement is indicated by a sharp rise in d s t prior to the drop. these criteria, in particular the third, have been empirically fine tuned to ensure nonstorm fluctuations in d s t do not trigger false identification of small storms while maximizing the identification of real storms. the vast majority of potential storms with smaller drops in d s t do not display stormlike d s t signatures, while those with larger drops do. the list of identified small storms has been manually verified for several randomly selected 1 year periods. further, we modify the criteria to similarly identify large geomagnetic storms to allow for a direct comparison with reeves. to validate our method., the second criterion is changed to require d s t be less than 50 nt. the third criterion is changed to require a drop of at least 55 nt in the preceding 16 h. our automatic identification of large storms is in excellent agreement with the storms identified by reeves. figure 2 shows the d s t signature of all identified storms, small and large, for the 2 week period centered on the time of storm, with the median and upper and lower quartile levels overlaid as black lines. for both sets of storms, there are occurrences when a given storm is closely preceded or followed by another storm. to assess the effect this may have, we create subgroups of isolated storms. these are storms that are separated temporally from any other storm, whether small or large, by at least 5 days. d s t signatures for all (top) small and (bottom) large storms included in this study are plotted for two weeks centered on the time of storm. median as well as upper and lower quartile levels for d s t are plotted in black lines. to quantify the change in relativistic electron flux, we use los alamos national laboratory (lanl) geosynchronous (l 6.6) satellite 1.83.5 mev electron flux data from the energy spectrometer for particles instrument [meier., 1996 ]. one hour resolution data are weighted by each of the five satellite 's lifetime average flux values and then averaged across all satellites to create one consistent time series. we then compare the 90th percentile maximum flux value in the poststorm period (0.55.5 days after) to that of the prestorm period (3.50.5 days before). see figure 1 for graphical illustration. if the poststorm / prestorm flux ratio is greater than 2, we determine that the storm resulted in flux enhancement. if the flux change ratio is less than 0.5, the storm resulted in flux depletion. if the flux change is within a factor of 2, the storm resulted in no change to the electron flux.. showed, however, that the probability a storm will result in an enhancement or depletion is independent of l shell. relativistic electron flux at geosynchronous also varies in local time due to magnetic field asymmetries. since we use only the maximum flux to quantify change, and since both prestorm and poststorm periods are several days long, this diurnal variation has little impact on the present study. use of the 90th percentile maximum flux further minimizes the impact of brief flux measurement increases that may be caused by the field asymmetry. we determine the maximum solar wind speed during each storm, from the beginning of the prestorm period through the end of the poststorm period. we then compare the relativistic electron flux change for storms during various solar wind speed ranges, as in reeves.. we identify 342 small geomagnetic storms between october 1989 and october 2000 for which geosynchronous lanl 1.83.5 mev electron flux data are available. figure 3 shows the 90th percentile poststorm flux versus 90th percentile prestorm flux for all small and large storms. first, regardless of the size of the geomagnetic storm, a very wide range of geosynchronous relativistic electron responses are possible. second, there is no correlation between prestorm and poststorm flux levels ; in other words, any poststorm flux level can be preceded by any prestorm flux level for any size of storm. finally, though not explicitly shown, the distributions of poststorm absolute flux levels are nearly identically distributed for small and large storms. poststorm flux is plotted on the ordinate, and prestorm flux is plotted on the abscissa. each storm for which lanl 1.83.5 mev geosynchronous electron flux data are available is represented by a dot. color helps differentiate between storms that result in enhancement (red), no change (green), or depletion (blue) of relativistic electron flux. of the 342 small geomagnetic storms, 42% result in enhancement, 26% result in no change, and 32% result in depletion of relativistic electron flux. the large storms identified in this study result in flux enhancement / no change / depletion 52%/26%/22% of the time, respectively. the proportions for large storms are in excellent agreement with reeves., providing confidence in our new stormfinding technique and analysis. overall, small storms show very similar proportions of flux enhancement / no change / depletion as large storms, though small storms are slightly less likely to result in enhancement and slightly more likely to result in depletion. small and large storm relativistic electron response for small and large storms, the ranges of possible flux change are equally as wide and similarly distributed. figures 4a and 4b show histograms of flux change ratios for small and large storms. figure 4c shows cumulative distribution function (cdf) curves for small (black) and large (red) storms. these cdf curves represent the probability that a storm has a flux change ratio less than a certain value. the largest vertical difference between two cdf curves and the sizes of the corresponding subsets are used in a kolmogorovsmirnov test to determine the statistical likelihood that the difference between the distributions is random. in figure 4c, the largest difference between the curves is 13%, and the difference between the distributions is unlikely to be random (700 in red. the largest vertical difference between any two curves in each plot is marked by a thick vertical black line. in both plots, this largest vertical difference is between the subgroups occurring during the fastest and slowest solar wind conditions. the largest vertical difference for small storms is 43% (chance random < 0.004%) and for large storms is 31% (chance random < 2%). we have examined the relativistic electron response at geosynchronous orbit for 342 small geomagnetic storms between 1989 and 2000. we have demonstrated that even though d s t remains above 50 nt, small storms have important effects on radiation belt relativistic electron fluxes. as a validation of our method, we identified 234 large storms during the same period and successfully reproduced results of reeves.. contrary to what is often expected, the effects on radiation belt relativistic electron fluxes of small geomagnetic storms are comparable with those of large storms. overall, the enhancement / no change / depletion proportions are very similar for small and large storms. flux enhancements and depletions can be equally as extreme, and further, both small and large storms result in similarly distributed poststorm flux levels. to rule out the possibility that the effects of small storms are merely the lingering effects of storms that occur in quick succession, we similarly analyzed isolated storms. the enhancement / no change / depletion proportions remain nearly the same for the isolated subset of large storms. though their enhancement / depletion proportions are slightly more skewed than those for all small storms, isolated small storms exhibit the same very wide range of possible effects on radiation belt electrons. we have also shown that faster solar wind conditions increase the likelihood of a flux enhancement for all storms. as the horizontal separation of cdf curves in figure 5 shows, faster solar wind drivers are not merely more likely to result in flux enhancements, but stronger flux enhancements. that this is much more evident for small storms suggests that the effects of solar wind drivers might be more easily distinguishable during less geomagnetically disturbed times nonetheless, as other studies confirm [e.g., kilpua., 2015 ; reeves., 2011 ], all classes of solar wind drivers can produce electron flux enhancement, no change, or depletion. there remains a difference between the distributions of flux changes for small versus large storms. small storms are less likely to result in flux enhancements and more likely to result in flux depletions than large storms. though statistically significant, this difference is not large, indicating that the response to small and large storms is similar in both cases. the trend is slightly exaggerated for isolated storms, though the much smaller sample sizes limit conclusive interpretation. what is most remarkable is that the distributions of flux changes for small and large storms are so similar and equally as wide. this study demonstrates that for all geomagnetic storms, even the smallest storms, the d s t index is a poor predictor of relativistic electron dynamics in the radiation belts. d s t is often thought of as a proxy for ring current ions, though other currents may also contribute significantly to d s t [zhao., 2015 ]. in contrast, the processes that lead to relativistic electron acceleration, loss, and transport depend heavily on the electron seed and core populations as well as waves, some of which depend on source electrons (several to tens of kev electrons). one might expect that with less intense geomagnetic activity, radiation belt response would also be less intense. indeed, many phenomena do scale with d s t, for example, number and intensity of injections, generation of waves, erosion of the plasmasphere, and radial diffusion. in other words, one might expect the enhancement and depletion of radiation belt electrons to be less extreme for small storms, even if the corresponding proportions are roughly the same as for large storms. instead, we find that neither the range nor the proportions of possible radiation belt responses scale with size of storm. this emphasizes the fact that radiation belt dynamics is highly complex, and we fundamentally do not understand how radiation belt responses scale with the drivers. recent studies have begun to investigate more closely the complete narrative of electron acceleration or loss in the radiation belts [e.g., boyd., 2014 ; breneman., 2015 ; jaynes., in addition to more accurately determining the causes of acceleration or loss, new studies must also separately quantify the amount of acceleration and loss during events. since multiple processes may occur simultaneously, often with competing effects, this can be a difficult task. further, small storms occur when the magnetosphere is less disturbed, by definition, and thus provide opportunities to more clearly analyze cause and effect relationships as well as quantify acceleration and/or loss. this becomes even more significant in light of the relatively quiet geomagnetic conditions of solar cycle 24. our stormfinding algorithm applied to january 2008 through october 2015 identifies less than half as many large storms (72) than the corresponding period following the start of solar cycle 23 in may 1996 (157). given the plethora of new data sources and tools that can powerfully address this issue, now is a fantastic time to be investigating the causes of radiation belt relativistic electron acceleration and loss as well as the delicate balance between these competing mechanisms. | abstractpast studies of radiation belt relativistic electrons have favored active storm time periods, while the effects of small geomagnetic storms (d s t > 50 nt) have not been statistically characterized. in this timely study, given the current weak solar cycle, we identify 342 small storms from 1989 through 2000 and quantify the corresponding change in relativistic electron flux at geosynchronous orbit. surprisingly, small storms can be equally as effective as large storms at enhancing and depleting fluxes. slight differences exist, as small storms are 10% less likely to result in flux enhancement and 10% more likely to result in flux depletion than large storms. nevertheless, it is clear that neither acceleration nor loss mechanisms scale with storm drivers as would be expected. small geomagnetic storms play a significant role in radiation belt relativistic electron dynamics and provide opportunities to gain new insights into the complex balance of acceleration and loss processes. |
extracellular nucleotides, such as adenosine triphosphate (atp), are important signalling molecules involved in many biological processes. under basal conditions endogenous regulation of atp concentration is mediated by ectoenzymes : the family of ectonucleotidases (e - ntpdases) and ecto-5-nucelotidase (cd73 ; e.c. 3.1.3.5) located on the cell surface. four plasma membrane - bound e - ntpdaess have been cloned : ntpdase1 (cd39 ; e.c.3.6.1.5), ntpdase2, ntpdase3, and ntpdase8, each with distinct localization and biological properties. ntpdase1 hydrolyzes atp and adenosine diphosphate (adp) equally well ; ntpdase2 preferentially hydrolyzes adp ; ntpdase3 ; ntpdase8 have intermediate hydrolysis profiles. the hydrolysis of atp and adp generates adenosine monophosphate (amp), which is then hydrolysed by cd73 to adenosine. cd39 is the rate - limiting enzyme in this cascade and thus is the prime regulator of nucleotide and adenosine concentrations within the microenvironment. hypoxia upregulates both ectoenzymes cd39 through sp1-dependent pathways and cd73 through binding of hif-1. further, within the cd73 gene, promoter region is a camp response element (cre) which regulates transcription through camp - dependent cre - binding protein (creb). activation of adenosine receptors increases camp and creb suggesting that the enzymatic product of cd73 (adenosine) may transcriptionally regulate its expression (reviewed in). finally, the glucocorticoid dexamethasone increases amp hydrolysis and cd73 expression which is mitigated by protein kinase c (pkc) inhibition. like atp, adenosine is constitutively expressed at low levels with a dramatic increase during metabolic stress such as hypoxia and ischemia consequent to atp hydrolysis. adenosine is a biologically active molecule that signals through four g - protein - coupled receptors denoted a1, a2a, a2b, and a3. activation of a1 and a3 inhibits adenylyl cyclase activity through coupling to gi resulting in a decrease in intracellular cyclic amp (camp), whereas a2a and a2b subtypes are coupled to gs or go to stimulate adenylyl cyclase and lead to an increase of camp. furthermore, the a2br is also coupled to gq/11 stimulating phospholipase c (plc) reviewed in and the a3r signals via plc-2/3. adenosine can also activate phosphoinositide 3-kinase (pi3k), mitogen - activated protein kinases (mapks) and extracellular receptor signal - induced kinase (erk). additional effector mechanisms include activation of akt to inhibit apoptosis by a3r, a1r activation which promotes the influx of ca and efflux of k and the activation of the arrestin pathway by adenosine receptors (reviewed in). the adenosine receptors are ubiquitously distributed in the body and the overriding effect of adenosine receptor activation in any one cell is dependent on the repertoire of receptors expressed. two distinct subtypes are recognised : autoimmune diabetes (type 1 diabetes, t1d) typically afflicting the young and associated with destruction of -cells and nonimmune diabetes (type 2 diabetes, t2d) typically arising in those of older age, obese, and with the metabolic syndrome. although the pathogenesis of the two disorders is distinct, central to both is that of pancreatic -cell failure and hypoinsulinemia. features unique to t2d include peripheral insulin resistance and failure of the incretin effect. despite the disparate aetiologies, the sequelae hyperglycemia and its associated complications are common to both disorders. in this paper, the role of purinergic signalling via the cd39-adenosinergic axis will be discussed in the context of the pathogenesis of t1d and t2d. insulin synthesis and secretion is tightly regulated in order to maintain stable blood glucose levels. when blood glucose levels increase, insulin secretion is augmented ; hyperglycemia increases the metabolic demand of -cells causing a rise in intracellular atp concentrations and atp is released with insulin reaching concentrations of 25 m at the cell surface. a number of p2 receptors have been implicated in nucleotide - mediated regulation of insulin including p2y1, p2y6, p2y13, p2x3, and amongst others [1215 ]. ectonucleotidase expression has been defined within the mouse, rat, and human endocrine pancreas [16, 17 ]. using immunohistochemical and enzyme histochemical techniques, ntpdase1/cd39 was expressed in all blood vessels and acinar tissue ; ntpdase2 was localised in capillaries and in connective tissue surrounding islets and acini and ntpdase3 was expressed exclusively in langerhan islet cells. similarly inhibition of ectonucleotidases with arl 67156, an inhibitor of ntpdase1 and 3, augmented insulin secretion from human pancreas. intriguingly cd73 was expressed exclusively in rat islet cells but not in human or mouse (and chia. notably both cd39 and cd73 are secreted from acinar tissue together with atp directly into the fluid controlling pancreatic exocrine function (reviewed in). in t1d islet destruction secondary to the autoimmune infiltration of cd4 t cells and macrophages results in the loss of insulin secretory capacity of -cells. treatment with multiple daily injections of insulin slows but transplantation of the whole pancreas or islets is a potential cure for the disease ; however, there remains the risk of recurrent disease culminating in graft failure. the nonobese diabetic (nod) mouse is the prototypical mouse model for t1d and shares a number of clinical, serological, and immunological features with the human condition. nod mice spontaneously develop diabetes at ~25 weeks of age after progressing through a prediabetic stage correlating with increasing insulitis. t - cell - mediated diabetes can also be induced chemically using multiple low dose streptozotocin (mlds). streptozotocin is a glucosamine - nitrosourea compound that enters the pancreatic -cell through the specific glucose transporter 2 (glut2) expressed on its surface. administered in high dose (250 mg / kg) streptozotocin is cytotoxic causing islet death. however, streptozotocin administered in low dose (50 mg / kg for 5 days) results in repetitive low - grade -cell damage, which incites a local inflammatory response comprised principally of cd4 t cells that is maximal at 1214 days. the delay in the onset of hyperglycemia suggests immune - mediated damage to -cells, rather than direct toxicity predominates. further t cell depleted or deficient mice are resistant to mlds - induced diabetes. cd4 regulatory t cells are integral to the maintenance of immune homeostasis and abnormalities in number and or function results in autoimmune disease. indeed low numbers of resting regulatory t cells have been reported in nod mice and human patients with t1d. cd39 is expressed on both murine [3, 24 ] and human [25, 26 ] cd4 regulatory t cells and is essential for the full suppressive activity of these cells in mice. further, mice deficient in cd39 (cd39ko) develop an immune diathesis and spontaneous autoimmune alopecia. as anticipated, these mice are highly susceptible to mlds - induced diabetes with a rapid rate of onset of diabetes (within 10 days) and 100% incidence. insulitis and reduction in insulin staining was evident at the onset of diabetes. when reconstituted with wild - type bone marrow comprising functional regulatory t cells, the kinetics and incidence are reduced to that of wild - type mice with the development of diabetes at day 42 and overall diabetes incidence of 57% (chia. cd39ko mice also have evidence of hepatic insulin resistance, which will be discussed in detail below. cd39 colocalises to -cells without perturbing glucose homeostasis and these mice are resistant to mlds - induced diabetes : minimal insulitis was evident and diabetes occurred in only 14% of animals. this robust protection persisted even following reconstitution with bone marrow from immunodeficient cd39ko mice (chia., submitted manuscript) which may reflect enhanced cell regenerative capacity due to increased pancreatic ntpdase activity. indicate a role for adenosine signalling in cell specific regeneration. in a zebrafish model, the nonselective agonist neca did not alter protection against cell death but promoted cell regeneration by increasing the proportion of new cells that proliferate through a2a - dependent mechanisms. interestingly, neca did not significantly increase the number of cells in normal development. further in mice treated with streptozotocin at 150 mg / kg for 2 days, bgl were 30% lower in mice concurrently treated with neca and cell mass was 8 times larger. although cd73 is not expressed in mouse or human islets, it is widely expressed on leukocytes and plays an essential role in leukocyte trafficking. cd73 is expressed on cd4 regulatory t cells in mice [3, 33 ], but interestingly is not expressed by human cd4 regulatory t cells. the biological relevance of cd73 had become evident from a number of small animal models : cd73 activity attenuates hypoxia - induced vascular leakage fmlp (formyl - met - leu - phe - oh)-stimulated neutrophil adhesion to endothelial cells and neutrophil accumulation in tissues [34, 36, 37 ]. cd73ko mice have a proinflammatory phenotype with increased vcam-1 expression on endothelial cells and heightened susceptibility to vascular inflammation and neointima formation. these effects are a consequence of the loss of both enzymatic and nonenzymatic functions of cd73. contrary to these reports, we have shown that cd73ko mice are protected in a model of renal ischemia - reperfusion injury [3941 ]. similarly, cd73ko mice are resistant to mlds - induced diabetes (figure 1), presumably a consequence of impaired leukocyte trafficking. in alloxan - induced diabetes in rats, a model which produces a pattern of t1d, adenosine signalling has emerged as a regulator of glucose homeostasis through modulating insulin and glucagon release. all four adenosine receptors are expressed in whole pancreas of cd-1 mice ; in isolated islets a1, a2a, and a2b receptors are expressed at the mrna level (chia. a1 receptor expression is downregulated, a2a expression is unchanged, and a2b receptor expression is augmented (chia., submitted manuscript). basal levels of adenosine in isolated islets are in the micromolar range, which is sufficient to stimulate glucagon release and inhibit insulin release via the a1 receptor. thus the peri - islet adenosine concentration is inversely related to extracellular glucose concentrations and may act as a paracrine or autocrine signal. using the -cell line ins-1 cells in vitro, treatment with the nonspecific agonist neca or a1, a2a, and a3 agonists reduced insulin secretion in a dose dependent manner. the effect of neca was completely antagonised by a2b receptor inhibition. in two mouse models of diabetes (cyclophosphamide treated nod and mlds), a1 receptor agonism mitigated diabetes but was less efficacious than the nonspecific agonist neca. in our hands, antagonism or agonism of the a1 receptor did not influence the rate of diabetes in c57bl/6 wild - type (wt) mice (chia. mice lacking the a2a receptor (a2arko) are highly susceptible to mlds - induced diabetes with rapid onset (within 10 days) and 100% diabetes incidence. like cd39ko mice, to delineate the site - specific importance of the a2a receptor, a series of adoptive transfer experiments were performed. deletion of the a2a receptor either on the tissues or the circulating cells increased the susceptibility of these mice to the effects of mlds (chia. neca ameliorated diabetes in a2arko mice and treatment with an a2ar agonist had no effect in wild - type mice following mlds. the prevention of mlds - induced diabetes in cd-1 mice by neca was reversed by pretreatment with a selective a2b receptor inhibitor. similarly, we have identified a role for the a2b receptor particularly in the early response to mlds. the rise in blood glucose following mlds in c57bl/6 wild - type mice was quicker, reaching hyperglycemia by 810 days, although the overall rate of diabetes was unchanged (chia. the protection conferred by cd39 overexpression was mitigated by deletion of the a2a receptor or by pharmacological inhibition of the a2b receptor. complete blockade of both receptors did not further exaggerate the diabetic phenotype (chia. involvement of more than one adenosine receptor parallels the effects of adenosine in renal iri, where a2a receptor signaling predominates on circulating cd4 t cells and macrophages, while a2b receptor signaling within the renal parenchyma is also important. intriguingly, cd73ko mice coadministered with an a2b receptor inhibitor became susceptible to the effects of mlds, with an onset of diabetes at day 10 and a diabetes incidence of 66% (figure 2). insulin resistance characterises t2d, however, -cell dysfunction must coexist for hyperglycemia to occur. indeed it is progressive -cell dysfunction that underpins the progression from normoglycemia to impaired glucose tolerance to overt diabetes. mice deficient in cd39 demonstrate impaired glucose tolerance following oral glucose tolerance testing a consequence of hepatic insulin resistance rather than peripheral muscle resistance. there was an associated increased level of hepatocyte c - jun nh2-terminal kinase (c - jnk) in response to extracellular nucleotides and aberrant insulin receptor substrate (irs)2 phosphorylation in the liver of these mice. there was no abnormality in glucose handling following an intraperitoneal glucose load in mice overexpressing cd39 nor intriguingly in cd73ko mice (figure 3). in human t2d poor glycemic control was associate with proportions of cd39 + cells particularly within the cd19 + subset. further, platelet - associated cd39 enzymatic activity was increased in patients with t2d, hypertension, and coexisting t2d and hypertension. platelet - associated cd73 enzymatic activity was only increased in patients with hypertension or coexisting hypertension and t2d and not t2d alone. cd39 expression also influences the susceptibility to diabetes - induced renal disease in both mice and humans. in african americans, a common entpd1 (cd39) two - single nucleotide polymorphism haplotype all adenosine receptors are expressed at the mrna level in skeletal muscle of mice and the role of adenosine receptor blockade in reversing insulin resistance in skeletal muscle from diabetic rats has been realised for some time [57, 58 ]. in keeping with this treatment of wild - type c57bl/6 mice with neca promoted impaired glucose tolerance by inhibiting glucose disposal. although initially thought to be mediated by the a1 receptor, studies with a1rko and a2rko mice show that these receptors have a minimal effect on skeletal muscle uptake of glucose. rather it appears that activation of a2b receptor promotes peripheral insulin resistance and blockade of the receptor in diabetic kka mice enhances glucose disposal into skeletal muscle and adipose tissue as well as reducing hepatic glucose production. further, in goto - kakizaki rats, which resemble t2d, insulin levels were increased temporarily following a2b receptor inhibition, although without effecting blood glucose level. there may however be a role for a1 receptor activation through the suppression of lipolysis and free fatty acid levels (ffa) both of which are involved in the pathogenesis of t2d. indeed, mice overexpressing the a1 receptor in diet - induced insulin resistant mice have lower ffa levels and insulin resistance compared to controls. the effect of the null mutation of a1r on glucose homeostasis following a high fat diet is controversial : faulhaber - walter. demonstrated decreased glucose tolerance with increased bgl and insulin levels in a1rko mice (c57bl/6 and swiss compared to controls) as early as 5 weeks following a high fat diet., however, reported a1rko mice (c57bl/6) clear blood glucose more efficiently, however, following a high fat diet both wt and a1rko mice develop glucose intolerance. the incretin hormones glucagon - like peptides-1 (glp-1) and glucagon intestinal peptide (gip) are released from the gastrointestinal tract in response to food and promote insulin secretion in a glucose concentration - dependent manner in -cells and inhibit glucagon secretion. the incretins are rapidly metabolised by dipeptidyl peptidase-4 (dpp-4) and drugs that inhibit this enzyme are very effective in the treatment of t2d. ddp-4, also known as cd26 or adenosine deaminase (ada), enzymatically and irreversibly converts adenosine to inosine. ada activity has been found in most organs but is notably high in adipose tissue, liver, skeletal muscle and heart. an increase in ada activity has been reported in patients with t2d and a relationship with insulin resistance has been postulated. high ada activity is associated with low adenosine levels ; however a direct relationship between adenosine and the incretin effect in t2d has not yet been defined. the cd39-adenosinergic axis is involved in the pathophysiology of pancreatic dysfunction and thus drug development targeting different components of the pathway may be of relevance in the treatment of both type 1 and type 2 diabetes. there remain a number of unanswered questions including the source of cd73 enzymatic activity given the lack of expression within the pancreas ; the mechanisms behind protection observed with cd73 deletion in mlds- induced diabetes and the role of purinergic signalling in the incretin effect, which is of particular importance in the pathogenesis of t2d. | diabetes mellitus encompasses two distinct disease processes : autoimmune type 1 (t1d) and nonimmune type 2 (t2d) diabetes. despite the disparate aetiologies, the disease phenotype of hyperglycemia and the associated complications are similar. in this paper, we discuss the role of the cd39-adenosinergic axis in the pathogenesis of both t1d and t2d, with particular emphasis on the role of cd39 and cd73. |
brugada syndrome(brs) is known for its catastrophic course with heightened risk of sudden death in seemingly healthy patients. diagnosis of brs in patients with suggestive history is established either by spontaneously occurring type 1 brugada ecg pattern or by inducible type 1 brugada ecg pattern. non - type 1 brugada ecg pattern (type 2 and type 3 brugada ecg patterns), though are suggestive, are not diagnostic. drug challenge with sodium channel blockers is commonly employed to unmask type 1 brugada pattern among those without type 1 brugada ecg pattern. studies,,,,, support the importance of this type of tests for the appropriate evaluation of patients with suspicious brs and syncope of unknown etiology. however, their sensitivity and specificity are variable and is better with ajmaline compared to other agents,,,,. usage of these drugs, (either the drug or the form of drug ; example intravenous form of flecainide), are limited in many countries given their nonavailability. given its limited utility, ajmaline is not easily available in all electrophysiology laboratories. and non - availability of intravenous flecainide and procainamide in many countries has made many laboratories to employ, freely available oral flecainide, to unmask type 1 brugada pattern, and has been reported as case studies,. however a systematic analysis of such data is limited. on the other hand, many patients in community have non - type 1 brugada pattern ecg with atypical symptoms, relevance of which is not clear. unmasking of type 1 brugada pattern in these patients would help in diagnosing brs which has significant impact on prognosis and treatment options. though some studies, suggest repeating the test to improve sensitivity, given the prevalence of the condition, more so in eastern part of world, it may not be prudent to repeat the test in all patients with negative result. determining the predictors of positive challenge would improve our understanding and facilitate appropriate usage of these challenge tests. we hypothesized that certain clinical & electrophysiological characteristics of patients like aborted sudden cardiac death (scd), spontaneously occurring ventricular arrhythmia, inducible ventricular arrhythmia or family history of brs could help predict positive flecainide challenge test (fct) and thereby in identification of patients with type 1 brugada pattern which would help us in better risk stratification of these non - type 1 brugada pattern patients. we aimed to study the clinical and electrophysiological profile of patients who underwent flecainide challenge test with the objective to study and compare the clinical, genetic and electrophysiological profile of patients with positive and negative fct in patients without type 1 brugada ecg pattern. we aimed to study the clinical and electrophysiological profile of patients who underwent flecainide challenge test with the objective to study and compare the clinical, genetic and electrophysiological profile of patients with positive and negative fct in patients without type 1 brugada ecg pattern. this study is a part of prospective registry, involving all consecutive patients who underwent fct for suspected brs or to look for inducibility of ecg pattern in non - type 1 brugada pattern at sree chitra institute of medical sciences and technology, trivandrum, india between january 2008 to april 2015. patients suspected to have brugada byecg non type 1 brugada pattern (type 2 or type 3 brugada pattern).f / h / o brugada syndrome.patients for whom fct was contemplated as a part of workup to rule out brugada syndrome.h/o aborted scd.unexplained syncope / pre - syncope.documented ventricular arrhythmia. patients suspected to have brugada by ecg non type 1 brugada pattern (type 2 or type 3 brugada pattern). patients for whom fct was contemplated as a part of workup to rule out brugada syndrome. evidence of structural heart disease that explains their symptoms.spontaneous type 1 brugada pattern.contraindication to flecainide.patient who did not give their consent. iv : 2 mg / kg for 10 min as infusion, max 150 mg. oral : 400 mg stat.ecg monitoring : (apart from continuous bed side telemetry).normally placed 12 lead ecg and one space above right sided leads (v1, v2, v3r, v4 r).for iv protocol : ecgs every minute for 10 min & every 5 min thereafter till 30 min or till ecg abnormalities revert.for oral protocol : ecgs every 5 min for first 30 min, and then at 30 min interval till 6 h or till abnormalities revert.positivity : inducible type 1 brugada pattern in atleast 2 right sided leads were considered as positive fct.type 1 brugada pattern is characterized bya coved st - segment elevation 2 mm (0.2 mv) followed by a negative t wave.type 2 st - segment elevation has asaddleback appearance with a high takeoff st - segment elevation of 2 mm, a trough displaying 1 mm in depth and wide s wave was defined as > 1 mm in width on a ecg recorded with standard speed of 25 mm per sec and 10 mv / mm voltage. twelve of 29 patients had prominent or wide s wave in lead i, of which 9 patients had type 2 or type 3 brugada pattern (8 had positive fct) and 3 had normal baseline ecg (all 3 were positive for fct). on univariate analysis, this parameter failed to achieve statistical significance (p = 0.057), though there was a trend towards increased incidence among fct positive patients. a recent article by calo., had shown that prominent or wide s in lead i was useful predictor of sudden death among brugada patients. our study, though showed the trend of increased incidence of prominent s in lead i among type 2 or type 3 brugada pattern with fct positivity with a high odd s ratio of 24, it was not statistically significant on either univariate or binary logistic regression. this could be because of the study design with a conscious exclusion of all definitive brs patients who would be at higher risk of scd and also can be affected by smaller sample size. other ecg parameters like fragmentation of qrs and early repolarisation pattern were not found to be significant in our study, unlike other studies which report variable degree of significance. oral fct though considered as alternative to ajmaline provocation test, due to its nonavailability, the fct protocol is not yet standardised. bioavailability of oral flecainide in its standard dose averages 70% (range 6086%), and higher bioavailability is achieved by higher doses. thus, in consistent with other studies,, we used single dose of 400 mg flecainide tablets as the challenge dose for oral fct. in this study, we observe that maximum time to positivity was 3 h and maximum time to subsequent normalization was 6 h. we suggest, that there is no need to observe beyond 6 h. shahrzad. observed some clinical and electrocardiographic predictors of positive response to the intravenous sodium channel blockers in patients suspected of the brs. during test, a transient episode of a second - degree atrioventricular block and isolated ventricular ectopics, a qrs prolongation 30%, baseline qrs duration in v1 110 ms and a st - segment elevation 0.17 mv in v2 had a good sensitivity and specificity for a positive response. however, our study showed only an insignificant prolongation of the qtc, qrs and pr intervals after drug administration. there were neither 2nd nor 3rd degree av block in our patients. thereby providing evidences for safety of oral fct, nevertheless, we suggest monitoring for ecg changes, arrhythmias and hemodynamic parameters as with any other drug challenge test. risk stratification aimed at the identification of patients at risk for sudden death is an important goal of research teams worldwide. the inducibility of ventricular tachycardia (vt)/fibrillation (vf) during eps may forecast risk, although some studies,, failed to find an association between inducibility and recurrence of vt / vf among both asymptomatic and symptomatic patients with brs. the role of eps is still a controversial topic in patients with brs ; priori.in their prelude study (programmed electrical stimulation predictive value) showed that eps was unable to identify high - risk patients. in this study high proportion of patients underwent ep study compared to other studies, but like other studies failed to find an association with fct. however given the variable sensitivity of provocating drugs, type 1 pattern may not be unmasked on some of the occasions. studies, have shown that repeating the test improves sensitivity albeit with a warning of increased incidence of drug adverse events. with the conflicting evidence of utility of repeating fct to improve sensitivity, coupled with the potential danger of inducing malignant arrhythmia and associated mortality, we suggest that, decision of repeating the test should be based on highly suspicious clinical profile. and in this regard, among those with non - type 1 brugada pattern, a family h / o scd could serve as a clinical indicator to repeat the test on a different day in case of initial negativity. second, we did not use other sodium channel blocking drugs for challenge for comparison. also we did not intend to prove efficacy and safety comparison between intravenous and oral fct. given the variable sensitivity of provocating drugs, type 1 pattern may not be unmasked on some of the occasions. few studies, have shown increased sensitivity on repeating the test, but with potential risk of serious drug adverse events. these studies had often used intravenous form of sodium channel blocking drugs. however in our study, we did not have any serious drug adverse events. finally, though we screened for sodium channel mutation in few of patients, non - sodium channel mutations were never screened for. family history of scd. in the present study, 14 (48.2%) out 29 patients who underwent fct had positive family history of scd. of which 10 patients (7 with type 2 or type 3 brugada pattern and 3 with normal ecg) were tested negative. interestingly, all 7 who had family history of scd with baseline type 2 or type 3 brugada pattern were tested positive. in univariate and binary logistic regression analysis, family history of scd many previous studies,,, have shown that family history of scd does not predict increased event rate or inducible ventricular arrhythmia or positive drug challenge test among brs (all types included). however none of them have analyzed the data for differential risk between type 1 and non type 1 brugada pattern. our study, by its nature, addresses this major clinical issue of risk stratification among type 2 or type 3 brs patients. prognosis of inducible type 1 brugada pattern is known to be poor compared to those with negative drug challenge test. therefore sodium channel blocker challenge test can be considered in all patients with family history of scd in patients with baseline ecg showing type 2 or type 3 brugada pattern.b)h / o syncope. seventeen (58.6%) of 29 patients had presyncope (n = 7) or syncope (n = 10), of which 09 were tested fct positive (6 with type 2 or type 3 brugada pattern and other 3 had normal baseline ecg). history of syncope predicted the fct positivity with an odd s ratio of 7.5 between group 1 and group 2, though statistically insignificant. unlike other arrhythmic risk predicting studies,,,,,, our study did not find the history of syncope to be useful for predicting positive response of drug challenge test. this possibly could be because of our inclusion criteria of including all unexplained syncope and conscious exclusion of definitive cases of type 1 brugada pattern and any other structural heart disease. also likely that the etiology of syncope need not be arrhythmic in this family history of brugada. in this study, 3 (10.3%) of 29 patients had family history of brs, of which 2 had type 2 or type 3 brugada pattern (1 was fct positive) and other had normal baseline ecg (fct was negative). neither univariate nor binary logistic regression, found this to be predictor of fct positivity among non - type 1 brugada pattern. this finding extends the general notion of limited usefulness of family history of brugada syndrome in predicting further clinical events among brs patients (all put together) to subgroup of non - type 1 brugada patients also. in our study, family history of scd has the strongest prediction with odds ratio of 21 (p = 0.04). first, the disease prevalence in the family may be more than anticipated and many may be asymptomatic. third, various biophysical factors affecting ionic channels and other genetic moderators might be playing their role in determining the nature of symptoms. a study by priori., had shown that upto 90% of family members of affected pro - bands could be asymptomatic, more than half of them had negative phenotype (silent mutation carriers) or had diagnostic ecg only after provocative challenge test. these results suggest the need of aggressive approach towards family members of victims of scd, more so in those who have type 2 or type 3 brugada pattern in resting ecg. long - term follow - up of patients diagnosed with brs from the finger registry have shown that event rates in asymptomatic patients is low (0.5% per year) and bigger in patients with aborted scd (7.7% per year) or syncope (1.9% per year). our study, which included largely symptomatic patients, and hence at higher risk, portrays family h / o scd, as a strong predictor of unmasking of type 1 brugada pattern with flecainide challenge test. a)prominent or wide s in lead i. a)prominent or wide s in lead i. prominent s wave was defined as > 1 mm in depth and wide s wave was defined as > 1 mm in width on a ecg recorded with standard speed of 25 mm per sec and 10 mv / mm voltage. twelve of 29 patients had prominent or wide s wave in lead i, of which 9 patients had type 2 or type 3 brugada pattern (8 had positive fct) and 3 had normal baseline ecg (all 3 were positive for fct). on univariate analysis, this parameter failed to achieve statistical significance (p = 0.057), though there was a trend towards increased incidence among fct positive patients. a recent article by calo., had shown that prominent or wide s in lead i was useful predictor of sudden death among brugada patients. our study, though showed the trend of increased incidence of prominent s in lead i among type 2 or type 3 brugada pattern with fct positivity with a high odd s ratio of 24, it was not statistically significant on either univariate or binary logistic regression. this could be because of the study design with a conscious exclusion of all definitive brs patients who would be at higher risk of scd and also can be affected by smaller sample size. other ecg parameters like fragmentation of qrs and early repolarisation pattern were not found to be significant in our study, unlike other studies which report variable degree of significance. oral fct though considered as alternative to ajmaline provocation test, due to its nonavailability, the fct protocol is not yet standardised. bioavailability of oral flecainide in its standard dose averages 70% (range 6086%), and higher bioavailability is achieved by higher doses. thus, in consistent with other studies,, we used single dose of 400 mg flecainide tablets as the challenge dose for oral fct. in this study, we observe that maximum time to positivity was 3 h and maximum time to subsequent normalization was 6 h. we suggest, that there is no need to observe beyond 6 h. shahrzad. observed some clinical and electrocardiographic predictors of positive response to the intravenous sodium channel blockers in patients suspected of the brs. during test, a transient episode of a second - degree atrioventricular block and isolated ventricular ectopics, a qrs prolongation 30%, baseline qrs duration in v1 110 ms and a st - segment elevation 0.17 mv in v2 had a good sensitivity and specificity for a positive response. however, our study showed only an insignificant prolongation of the qtc, qrs and pr intervals after drug administration. thereby providing evidences for safety of oral fct, nevertheless, we suggest monitoring for ecg changes, arrhythmias and hemodynamic parameters as with any other drug challenge test. risk stratification aimed at the identification of patients at risk for sudden death is an important goal of research teams worldwide. the inducibility of ventricular tachycardia (vt)/fibrillation (vf) during eps may forecast risk, although some studies,, failed to find an association between inducibility and recurrence of vt / vf among both asymptomatic and symptomatic patients with brs. the role of eps is still a controversial topic in patients with brs ; priori.in their prelude study (programmed electrical stimulation predictive value) showed that eps was unable to identify high - risk patients. in this study high proportion of patients underwent ep study compared to other studies, but like other studies failed to find an association with fct. however given the variable sensitivity of provocating drugs, type 1 pattern may not be unmasked on some of the occasions. studies, have shown that repeating the test improves sensitivity albeit with a warning of increased incidence of drug adverse events. with the conflicting evidence of utility of repeating fct to improve sensitivity, coupled with the potential danger of inducing malignant arrhythmia and associated mortality, we suggest that, decision of repeating the test should be based on highly suspicious clinical profile. and in this regard, among those with non - type 1 brugada pattern, a family h / o scd could serve as a clinical indicator to repeat the test on a different day in case of initial negativity. second, we did not use other sodium channel blocking drugs for challenge for comparison. also we did not intend to prove efficacy and safety comparison between intravenous and oral fct. given the variable sensitivity of provocating drugs, type 1 pattern may not be unmasked on some of the occasions. few studies, have shown increased sensitivity on repeating the test, but with potential risk of serious drug adverse events. these studies had often used intravenous form of sodium channel blocking drugs. however in our study, we did not have any serious drug adverse events. finally, though we screened for sodium channel mutation in few of patients, non - sodium channel mutations were never screened for. we conclude that oral flecainide is useful and safe for unmasking of type 1 brugada pattern. in our study, family history of sudden cardiac death was a major predictor of flecainide test positivity among those with non - type 1 brugada pattern. | backgroundmany subjects in community have non - type 1 brugada pattern ecg with atypical symptoms, relevance of which is not clear. provocative tests to unmask type 1 brugada pattern in these patients would help in diagnosing brugada syndrome. however sensitivity and specificity of provocating drugs are variable.methodswe studied 29 patients referred to our institute with clinical presentation suggestive but not diagnostic of brugada or with non - type 1 brugada pattern ecg. flecainide challenge test (fct) was done in these patients (iv flecainide test in 4 patients and oral flecainide in 25 patients). resting 12-lead ecg with standard precordial leads and ecg with precordial leads placed 1 intercostal space above were performed after flecainide administration every 5 min for first 30 min and every 30 min thereafter until ecg became normal or upto 6 h. the positivity was defined as inducible type 1 brugada pattern in atleast 2 right sided leads.resultmedian age was 35(range = 565) years. in 16 (55%) patients the type 1 brugada pattern was unmasked. there were no episodes of major av block, atrial or ventricular tachyarrhythmia. three groups were considered for analysis : group 1(n = 9) fct positive among patients with non - type 1 brugada ecg pattern, group 2(n = 4) fct negative among the patients with non - type 1 brugada ecg pattern, and group 3(n = 7) fct positive among patients with no spontaneous brugada ecg pattern. binary logistic regression analysis found that family h / o scd was predictive of fct positivity in group 1 (odd s ratio 21, 95% confidence interval 1.04 to 698.83, p = 0.004).conclusionoral flecainide is useful and safe for unmasking of type i brugada pattern. in our study, among the many variables studied, family history of sudden cardiac death was the only predictor of flecainide test positivity among those with non - type 1 brugada pattern. |
necrotizing enterocolitis (nec), characterized by coagulation necrosis and inflammation of the intestine, is a serious condition that usually affects preterm infants, with high mortality rate (1). the disease occurs in 1 - 5% of neonatal intensive care admissions, but 5 - 10% of very low birth weight (vlbw) infants have nec (2). the mortality rate of vlbw preterms has continued to reduce over the time, due to better prenatal and neonatal care, antenatal corticosteroid therapy use, and noninvasive respiratory support in the neonatal intensive care units (nicu) (3, 4). despite advances in the care of premature infants, nec remains one of the leading causes of morbidity and mortality in this population (5). although the exact etiology of necrotizing enterocolitis (nec) remains unknown, researchers suggest that it is multifactorial. prematurity (with immature git and host defenses) is the primary risk factor (6) ; ischemia and/or reperfusion injury, exacerbated by activation of pro - inflammatory intracellular cascades may play a significant role (7). various studies have identified risk factors for the development of nec, including genetic predisposition (8), alterations in the normal bacterial colonization of the gastrointestinal tract (9), and introduction and advancement of enteric feeding (10). awareness of the risk factors for nec changes a practice to reduce the risk, including early trophic feeding with breast milk and following the established feeding guidelines. administration of probiotics in recent time has been shown to reduce the incidence of nec (11). despite advances in management of vlbw infants, aggressive and invasive treatment is needed to achieve survival of extremely preterm infants, especially in countries in which there is a low rate of antenatal corticosteroid usage (12). however, understanding of possibly harmful effects of any management alternative is crucial in reducing the morbidity rate of and disease, including nec ; a disease in which therapeutic interventions include : red blood cell transfusions, long term antibiotic therapy for nosocomial infections, mechanical ventilation, infusion of morphine to relieve pain, reduce the stress response, and of medications including methylxanthines and h2 blockers. this retrospective study was performed on all nec preterm infants (< 37 weeks gestation at birth) admitted in the nicu of our institution over a period of five years, from 2008 to 2012. gestational age was routinely determined from that last menstrual period, early ultrasound investigation or using the new ballard score, and recorded as completed weeks (13). diagnosis of nec was made based on the presence of clinical, radiological and/or histopathological evidence that fulfilled the criteria of bell s (14) as well as walsh s modification of these criteria (15). definition of nosocomial infection (ni) : ni infection is defined as an infection that occurs after 48 hours of hospitalization, resulting in a positive blood, cerebrospinal fluid (csf), or urine culture with clinical manifestations such as hospital - acquired bloodstream infections, nosocomial pneumonia, sepsis, urinary tract infection and meningitis. medical nec was defined as the presence of radiological signs of intestinal pneumatosis and when the disease is treated with antibiotics for more than two days. the infants medical records were reviewed daily for medical course information until hospital discharge or death of infant. statistical analysis was performed using spss 16.0 (spss inc, chicago, il, usa). the number of infants with each investigated factors (nosocomial infections, mv, ncpap, morphine sulfate, inotropes, rbc transfusions, h2 blockers) was compared between groups with and without nec. the means of continuous variables were compared using student s t test, and the data are presented as mean (sd). the influence of relevant confounding variables, identified by univariate analysis, was assessed using multivariate logistic regression analysis. confidence intervals presented for odds ratios are adjusted for the clustering of infants within participating nurseries. statistical level of 95% (p<0.05) was considered as significant for all performed tests. statistical analysis was performed using spss 16.0 (spss inc, chicago, il, usa). the number of infants with each investigated factors (nosocomial infections, mv, ncpap, morphine sulfate, inotropes, rbc transfusions, h2 blockers) was compared between groups with and without nec. the means of continuous variables were compared using student s t test, and the data are presented as mean (sd). the influence of relevant confounding variables, identified by univariate analysis, was assessed using multivariate logistic regression analysis. confidence intervals presented for odds ratios are adjusted for the clustering of infants within participating nurseries. statistical level of 95% (p<0.05) was considered as significant for all performed tests. during the study period, 830 preterm infants were admitted in the nicu ; 51 (6.1%) got nec. control group consisted of 71 randomly selected preterm infants that were not significantly different in bw and ga from premature infants with nec. in the group of patients with nec, based on the diagnostic criteria (12, 13), presence of the medical nec established in 30 patients (58.8%) while the surgical nec found in 21 patients (41.2%). average gestational age of preterm infants with nec was 30.2 gw (sd 3.7), average birth weight 1502.75 g (sd 781.5). postnatal age in time of appearance of nec was 18.2 days (sd 12.8) (2 - 57 days) ; 49% infants were older than 2 weeks. in one patient nec developed before starting of enteral feeding. the most common gastrointestinal symptoms in the study group were : abdominal distension in 89% cases, macroscopic or microscopic blood in the stool in 56.9% and increasing gastric residuum in 46% cases. risk factors related to the hospitalization and treatment of vlbw infants 29/51 (56.9%) of premature infants with nec had at least one or more of ni prior to nec diagnosis. in the control group of patients, there was statistically significant difference regarding infections between groups 2 (1, n = 122) = 12.328, p = 0.0004. 37/51 (72.5%) infants in nec group received morphine sulfate during the hospitalization, (average 2.7 days), in control group 14/71 (19.7%), average 0.37 days. statistically significant difference was found related to morphine sulfate treatment between groups (1, n=122) = 31.914, p=0.0001. 33/51 (64.7%) infants in nec group were treated with inotropes an average of 2 days, compared to the control group 12.7% (9/71). statistically significant difference is noted between group of nec and control group. (1, n=122) = 33.325, p=0.0001. statistically significant difference was found in number of days of mechanical ventilation in nec group (md=8, n=43) compared to control group (md=3, n=22), u=262.00, z=2.955, p=0.0031. there was statistical difference between average number of days on ncpap in nec group (md=5 n=25) and control group (md=3 n=39). u=313.50, z=2.413 ; p=0.0158. model for logistic regression factors of treatments 20/51 (39.2%) infants in nec group received h2 blockers (an average of 2.2 days), in control group 6/71 (8.5%) (an average of 0.25 days), which is statistically significant (1, n=122) = 14.967, p=0.0001. there was statistical difference in rbc transfusions in nec group (mean 1.53 n=31) and control (mean 0.55 n=21). (1, n=122) = 10.578, p=0.0011. logistic regression is done to estimate the influence of factors related to treatment of sick infants on possibility to develop nec. model consists of 7 independent variables (nosocomial infections, mv, ncpap, morphine sulfate, inotropes, rbc transfusions, h2 blockers). whole model with all predictors was statistically significant, (7, n=122) = 49.522, p<0.0001, which indicates that model can recognize infants who may develop nec later. two independent variables (nosocomial infections and h2 blockers use gave statistically significant attribution to the model. logistic regression analysis showed that there was a statistically significant association in the number of nosocomial infections prior nec diagnosis with the development of nec (p<0.05). based on the result of logistic regression analysis, it can be concluded that each additional infection increased the odds of developing nec by 3 times, and use of h2 blockers 1.5 times. incidence of nec in our study was 6.1% (51/830) consequently admitted preterm infants in nicu. the incidence generally varies from neonatal units, regions and countries, as can be determined by different definition of nec (2). 15), in literature still exists mismatch of uniform recognition and classification of suspected nec in vlbw and elbw infants (2). to determine potential risk factors and predictors for nec development (clinical, radiological and laboratory), we tried to achieve the most homogeneous study group of infants with nec and control group ; there was no statistical difference between groups in gestational age and birth weight. 1461.08 781.47 g, which is in agreement with similar studies (16, 17). investigations related to nosocomial infection and developing of nec indicate that increasing number of infections before clinical manifestation is associated with increasing risk for nec (18). although there is no exact identification of reasons for this kind of connection in the literature, it is assumed that can be in relationship with duration of parenteral nutrition. parenteral nutrition has been shown to have immunosuppressive effect with decreasing the grade of phagocytosis and neutralization coagulase negative staphylococci. results of our study showed that the number of nosocomial infections before clinical manifestation of nec was significantly higher than in control group (p=0.0004). respiratory insufficiency of preterm infants is relatively frequent, including inability to maintain normal gas exchange. that is why respiratory support routinely carried out especially in vlbw and elbw infants, using mechanical ventilation (mv) or continuous positive pressure ventilation through nasal prongs (ncpap). ncpap as non - invasive procedure used today as important alternative, has reducing the incidence of chronic lung disease, retinopathy, intraventricular bleeding and incidence of neurodevelopmental disorders. meyer. (20) concluded that vlbw and modality of respiratory support can be a risk factor for nec development, so it can serve as a predictor for development and severity of the disease. possible explanations can be in cognition that vlbw infants have higher need for respiratory support before gastrointestinal symptoms, compared to infants of the same birth weight without nec. dolgin. (21) in their study found that preterm infants with surgical nec have higher need for respiratory support results of our study showed that duration of mechanical ventilation in days prior gastrointestinal symptoms in infants with nec was statistically significantly higher compared to control group (p=0.0031). although anemia of prematurity is the usual in this group of age, caused by incomplete placental transport of fe, leak of complete fetal erythropoiesis, iatrogenic blood loss, low level of erythropoietin in plasma and increasing needs due to fast body growth, frequent red blood cell transfusions were common clinical practices in nicus. reasons for this are in the cognition of relationship between rbc, acute intestinal injury and serious gastrointestinal reaction, especially in elbw and extremely preterm infants (22, 23). however, mechanism of which rbc causes injury on gi tract of preterm infants is not researched enough. (24) offered some explanations as decreased capacity of nitric oxide storage in packed rbc, excessive intestinal immunological response and alteration of mesenteries blood flow after rbc transfusion result in intestinal hypoxia and intestinal mucosal injury. all of that suggest that rbc transfusions can cause alteration gastrointestinal microcirculation in the supply of oxygen during this vulnerable period and significantly contribute to nec developing. our study showed that preterm infants with nec had significantly more rbc transfusions before clinical signs of nec compared to control group (p=0.0005). this result is in concordance with similar studies that determined positive correlation between rbc transfusions and appearance of nec (22, 23). morphine sulfate is used to be common praxis in ventilated infants in nicus due better of synchronization with ventilator, pain relief and reduction of stress response. its use has been decreasing steadily because of its adverse effects including hypotension, bradycardia, delay in beginning of enteral feeding and reduction of gastrointestinal motility. general acceptability of morphine administration in preterm infants in era of non - invasive ventilation and high antenatal steroid use is now questionable (25). (26) first noticed that duration of morphine sulfate administration have significant influence on nec development. additional explanation is that reduction of gastrointestinal motility allows prolonged contact gi bacteria with feeding substrates and gut wall, bacterial translocation with increasing possibility for development of disease. our results showed that infants with nec had statistically significant higher number of days of morphine administration compared to control group (p<0.0001). use of inotropes (dubutamine, dopamine) is common in presence of shock or hypotension, to achieve cardiovascular stabilization. action of dobutamine is based on its action on receptors, resulting in improving of heart contractibility, vasodilatation and mild tachycardia. (27) using doppler ultrasonography on mesenteric artery, determined increased intestinal perfusion and concluded that is no influence on nec development, although pathophysiological mechanism of this connections still is not clear. use of inhibitors gastric acid secretion can cause insufficient elimination of ingested pathogens and increase risk of nosocomial infections, due to alkalization of gastric content which normally presents the main non - immune mechanism of defense against infection (28). histaminh2 receptors blockers and proton pump inhibitors (ranitidin, famotin, and cimetidin) increase the risk of infection and nec in neonatal period (28). administration of those medicaments reduces proteolytic activity of gastric secretion, allowing gastric colonization with gram negative strains, and consecutive pneumonia and gram negative sepsis. (28) use of histamine h2 blockers in nicus is empiric, especially in infants with proven gi bleeding and gi reflux. although in these cases administration can protect mucosa from extensive production of gastric acid and prevent stress ulcers, in the same time it can neutralize natural defense against overgrowth propagation (28). in study in terin (28) in infants receiving h2 blockers number of infection was 4 times higher. logistic regression of 7 independent variables (nosocomial infections, mv, ncpap, morphine sulfate, inotropes, rbc transfusions, h2 blockers) was statistically significant, (7, n=122) = 49.522, p<0.0001, which indicates that model can recognize infants who may develop nec later. two independent variables (nosocomial infections and h2 blockers use gave statistically significant attribution to the model. based on the result of logistic regression analysis, it can be concluded that each additional infection increased the odds of developing nec by 3 times, and administration of h2 blockers 1.5 times. | background : necrotizing enterocolitis is a serious condition that affects mostly preterm infants, with high mortality rate.aim:to estimate the influence of potentially contributing factors of this multifactorial disease.methods:the study group included 51 necrotizing enterocolitis infants who were less than 37 week gestation who were hospitalized in nicu during a five year period. the control group consisted of 71 patients with approximately the same gestational age and birth weight. average gestational age in the study group was 30.2 weeks (sd 3.7), average birth weight 1502 g (sd 781.5). average postnatal age in the time of the presenting nec was 18.2 days (sd 12.8).results : logistic regression estimates the influence of risk factors, which in our study related to the treatment of preterm infants on the likelihood of nec development. our regression model consisted of seven independent variables (nosocomial infections, mechanical ventilation, nasal continuous positive pressure, morphine, inotropes, blood transfusions, and h2 blockers), which were shown to have a statistically significant impact, x2 (7, n=1222) = 49.522, p<0.0001 ; two independent variables (nosocomial infection and h2 blockers use) were statistically significant. preterm infants with nosocomial infection had a three times greater chance of developing nec, and infants who received h2 blockers had a 1.5 higher risk.conclusions:underlying pathology of very low birth weight infants and their treatment in nicu contribute to nec development. identifying risk factors can be crucial for the early diagnosis and outcome of disease. awareness of risk factors should influence changes in practice to reduce the risk of nec. |
post - transcriptional rna modifications are ubiquitous in biology, with more than 100 different types being found in cellular rnas, including trna, rrna (rrna), small nuclear rna, and mrna. rrna is the most abundant noncoding rna inside the cell, bearing from 10 to 200 rna methylations and pseudouridines depending on the species. the ribosomes from escherichia coli have a total of 36 modified nucleotides resulting from the action of at least 29 known protein enzymes (22 methyltransferases and 7 pseudouridine synthases) that carry out site - specific modifications (table 1). based on a few studies investigating the functional role of individual rrna modifications, they are generally considered to stabilize local structure of the ribosome, affect its translational activity and modulate antibiotic drug resistance. while providing selective benefits for cell - survival, many of the rrna modification enzymes found in bacteria and yeast are dispensable. in higher organisms, however, deficiency in a single modified nucleoside can cause severe morphological defects and embryonic lethality. moreover, deficiency or mutations in small nucleolar rna genes, guiding rrna modifications in eukaryotes, have been implicated in congenital diseases and different types of cancer in humans. the bacterial ribosome is a complex macromolecular machine consisting of the small (30s) and large (50s) subunits, in which core rrna components 16s and 23s, respectively, are bound to a total of 54 ribosomal proteins. the modification of rrna is carried out during the ribosome biogenesis process where it is coupled to a series of steps, including rrna folding, binding of ribosomal proteins, and rna processing. these steps are tightly coordinated in space and time to produce accurate and efficient protein synthesis machinery, essential for growth and division of every living cell. the complete set of methyltransferases and pseudouridine synthases have been successfully identified in e. coli. using recombinant modification enzymes, their substrate specificity has been characterized in vitro (table 1), demonstrating that some enzymes prefer naked rrna (16s or 23s), while others modify the assembled subunits (30s, 50s) or even complete ribosomes (70s) (table 1). despite recent progress in biochemical and structural characterization of rrna modifying enzymes, how individual rrna modification steps are integrated into the entire ribosome biogenesis process is poorly understood. one of the main reasons that mechanistic studies of rrna modifications inside the cell remain limited is that accurate and efficient detection and quantification of post - transcriptional modifications are technically difficult. historically, discovery and localization of the new rna modifications have been carried out using a reverse transcriptase primer extension technique, relying on premature stops of the enzyme at the modified site of interest. the major disadvantage of this method is that it is not amenable to quantitative analysis. additionally, many known modifications (e.g., pseudouridine, 5-methylcytosine, 7-methylguanine) do not generate a reverse transcriptase stop, requiring modification - specific chemical derivatization. another approach is based on p1 nuclease digestion of rna with subsequent tlc or hplc separation of the resulting nucleotide products. taking advantage of different migration of modified and unmodified residues, radio labeling or uv detection is then used to measure their relative stoichiometry. recently, this technique was applied to analyze e. coli rrna modifications present in incompletely assembled ribosomal particles that accumulated as a result of antibiotic treatment. in this study, however, reliable quantitative characterization was obtained only for those modified nucleosides which were resolved as individual chromatographic peaks. furthermore, the method was not suitable for analysis of nonunique modifications, found at two or more positions in the 16s or 23s rna molecule. mass spectrometry has recently become practical for oligonucleotide analysis and in the last 10 years has played a primary role in the identification of new post - transcriptional modifications. a bottom - up approach, in which nucleotide - specific ribonucleases (rnase t1, a) produce rna fragments of the size amenable for maldi or lc - ms detection, has been widely used. furthermore, applicability of tandem ms analysis to rna has been demonstrated providing sequence characterization of oligonucleotide fragments. despite these successes, to distinguish pseudouridine from its structural isomer uridine, protocols based on chemical derivatization of pseudouridines with acrylonitrile and carbodiimide reagents have been reported. while these approaches have been useful to identify new pseudouridines, incomplete or nonspecific derivatization in addition, several laboratories took advantage of the tandem ms to design a method for identification and reference free quantitative profiling of pseudouridines via pseudouridine specific fragmentation products. furthermore, to our best knowledge, there are only a few published studies where ms was applied for quantitative analysis of rna. the most recent work describes a convenient workflow for identification and relative quantification of rna using stable isotope labeling with n. by combining the light (n) and heavy (n) rrna isolated from e. coli, the authors reliably identified nucleolytic fragments bearing all known base and ribose methylated residues in 16s rna. in summary, existing technologies and approaches for ms analysis of rna provide an excellent platform for development of new applications which would enable mechanistic studies of rna modifications inside living cells. our laboratory has utilized stable isotopic labeling and quantitative ms (qms) analysis of ribosomal proteins to characterize the ribosome assembly process in vitro and in vivo. in these analyses, protein identification and quantitation were based on finding unique pairs of n- and n - labeled peptides from a tryptic digest of ribosomal proteins. a robust and powerful least - squares fourier transform convolution (ls - ftc) method was introduced to fit observed isotope distributions from each of the nitrogen species, providing relative quantitation with exceptional precision. the present study expands this qms framework to establish a tool for a global monitoring of rna modifications in bacterial ribosomes. modifications are profiled via their signature nucleolytic fragments using n and n masses for their identification. in conjunction with new metabolic labeling strategies introduced for analysis of rna methylations and pseudouridines, 80% of all modified nucleotides in the rrna of e. coli were identified and quantified. the method was applied to measure relative levels of rrna modifications present in the distribution of preribosomal particles in an e. coli cell lysate. the data reveal groups of rna residues that are modified at different points during assembly, providing new insights into the global picture of ribosome biogenesis in bacteria. in general, the proposed method should be applicable for quantitative analysis of rrna and other rna modifications in a wide variety of systems, to better understand rna modification processes, their functions, and their implication in diseases. 16s rna has 10 methylated (m) nucleosides and 1 pseudouridine (). 23s rna has 14 methylated nucleosides (m), 1 dihydrouridine (h), 1 hydroxycytidine (ho), and 9 pseudouridines (). rna methylations are denoted according to the previously used nomenclature : mn, base methylations ; nm, 2-o - ribose methylation. modification observed by qms : (+ + +), systematically ; (+ +), occasionally ; (+), but quantitation is difficult (figure s1e) ; (), never been observed in this work (table s2). e. coli mre-600 cultures were grown at 37 c in m9 minimal medium supplemented with trace amounts of vitamins and minerals. cells were chilled by adding ice directly to the culture, then harvested by centrifugation. cell lysis was carried out using a mini - beadbeater (biospec products) in nondissociating buffer a (10 mm mgcl2, 100 mm nh4cl, 20 mm tris - hcl (ph = 7.8), 0.5 mm edta, 6 mm 2-mercaptoethanol), and cell debris was removed by two consecutive rounds of centrifugation. ribosomal particles were separated by loading cell lysate on top of a 1040% (w / w) sucrose gradient, prepared in buffer a, and centrifugation of the gradient in a sw-32 rotor (beckman coulter) at 26 000 rpm for 18 h at 4 c. after gradient fractionation using a260 detection, rrna from each individual fraction was extracted using trizol reagent (invitrogen) and isopropanol precipitated in the presence of 20 g / ml of glycogen. rna pellets were redissolved in depc treated water and additionally purified by dialysis or by rapid spin filtration using amicon ultra 30k mwco columns (millipore). rna samples were stored frozen in water at 20 c or lyophilized. in a similar manner, n - labeled ribosomes were prepared for use as an external standard in ms experiments, by cell growth in m9 minimal medium with 0.5 g / l n - ammonium sulfate as the nitrogen source. cell lysis was carried out in buffer b (1 mm mgcl2, 100 mm nh4cl, 20 mm tris - hcl (ph = 7.8), 0.5 mm edta, 6 mm 2-mercaptoethanol) with a low mg concentration to dissociate ribosomal subunits and prevent their reassociation during ultracentrifugation. sucrose fractions containing separated 30s and 50s particles were analyzed using 1.2% agarose gel electrophoresis. fractions containing pure 16s or 23s rna were combined and processed as described above. for metabolic labeling of rrna methyl groups, cells were grown in the presence of 50 mg / l of cd3-methionine (cambridge isotope laboratories). for metabolic labeling of pseudouridines, cells were grown in the presence of 25 mg / l 5,6-d - uracil (cambridge isotope laboratories). methionine (meta) and pyrimidine (pyrc) auxotrophs obtained from the keio knockout collection were initially used to confirm rrna labeling. a set of titration experiments using the pyrc strain was performed to determine the minimum required concentration of 5,6-d - uracil in the m9 medium. at 25 mg / l, uptake of 5,6-d - uracil was shown to inhibit de novo pyrimidine biosynthesis in the wild - type cells harvested at 0.50.8 a600, and ms peaks corresponding to unlabeled pyrimidines were not detected. for lc - ms analysis, 10 pmol of rrna sample was mixed with n - labeled 16s or 23s rna isolated from 70s ribosomes, in an 1:1 molar ratio, based on a260 measurements. the rrna mixture was suspended in 25 mm ammonium acetate (ph = 5.8) and then denatured at 90 c for 3 min, followed by rapid cooling on ice. the rna digestion reaction was carried out using one of three commercially available ribonucleases : rnase a (30 units), t1 (50 units), or u2 (1 unit) for 1 h at 55 c in a 5 l volume. complete digestion with rnase t1 (cleaves at g) or rnase a (cleaves at u and c) resulted in rrna fragments with 3-phosphate termini and no missed cleavages. rnase u2 digestion was carried out using a limiting concentration of enzyme, resulting in partial cleavages of the phosphodiester backbone after a and g residues, with 02 missed cleavages and either 2-3-cyclic (7080% of all observed) phosphate or 3-linear phosphate termini. digestion products were analyzed on agilent q - tof g6520b or esi - tof g1969a mass spectrometers with an electrospray ionization source coupled to an agilent-1100/1200 chromatography system. nucleolytic fragments were separated on an xbridge c18 column (waters) using buffer a (15 mm ammonium acetate, ph = 8.8) and buffer b (15 mm ammonium acetate, ph = 8.8 and 50% acetonitrile). hplc separation consisted of the following steps : (1) isocratic elution with 1% buffer b for 5 min, (2) a linear gradient from 1% to 15% buffer b over 40 min, (3) column washing with 100% b for 25 min, and (4) column equilibration with 1% b for 30 min. data were recorded over the 4001700 m / z range using negative or positive ionization. an experimental peak list, obtained using agilent qualitative analysis software, was compared against a theoretical digest containing predicted rrna nucleolytic fragments and corresponding monoisotopic m / z values for both n- and n - labeled fragments. an in - house perl script was used to generate a series of theoretical digest files to account for different ribonuclease cleavage patterns, isotope labeling strategies, and positive or negative ionization modes. a typical theoretical digest comprised nucleolytic fragments with both linear and cyclic phosphates at the 3 terminus, up to 3 missed cleavages and charge states in the 14 range. due to the high degree of sequence conservation between the seven e. coli rrna operons, sequences of 23s and 16s from a single operon (rrna) were used. for a singly modified oligonucleotide fragment, both modified and unmodified versions were included in the theoretical digest. when multiple modifications were present, all possible combinations of modifications were included in the digest. n peak pairs was performed by matching experimentally observed and predicted n and n m / z values, using a 50 ppm mass threshold for matching. additionally, n and n peaks corresponding to the same rrna fragment were required to elute within 0.1 min of each other and to exhibit the same charge state. peaks with multiple identities were excluded, unless the presence of a modified residue is suspected. further analysis was carried out to confirm their identification, including fractional spike analysis, analysis of deletion strains for rrna modifying enzymes, and metabolic labeling (table s1). for each of the identified n n peak pairs, their isotope distributions, averaged over a 0.2 min retention time window, were extracted. extracted mini - spectra were fit to theoretical isotope distributions using in - house software which implements a previously reported ls - ftc method. resulting least - squares fits were visually inspected and filtered to eliminate data with low signal - to - noise ratio or spectral overlaps that are hard to resolve. the fit parameters for the n and n amplitudes were used for relative quantification of rrna fragment levels in the n sample with respect to the n external standard. fractional rna levels (f) were calculated as f = a14/(a14 + a15), where a14 and a15 are peak amplitudes for the sample and the standard. hierarchical clustering of the rna modification and protein levels were carried out using pearson correlation distance metric implemented in gene cluster 3.0 software, and the resulting dendrograms and heat maps were visualized using java treeview. the workflow of the qms approach used to detect and quantify rrna modification is schematically illustrated in figure 1. briefly, bacterial cells are separately grown in minimal media supplemented with n- or n - labeled ammonium sulfate as the sole source of nitrogen. using sucrose gradient ultracentrifugation (black traces, figure 1), 70s ribosomes, 50s and 30s subunits, and preribosomal particles are isolated from the cell lysate, followed by rrna extraction and purification. the 16s or 23s rrna isolated from mature n - ribosomes is added as an external standard, and the combined mixture is digested with a nucleotide - specific ribonuclease t1, u2, or a prior to lc - ms analysis. pairs of the co - eluting n- and n - labeled ribooligonucleotide fragments are identified by matching their experimentally observed masses and charge states to a theoretical digest of the rrna sequence. after the lc - ms peak profiles are extracted, the isotope distributions are fitted using ls - ftc approach, and the resulting amplitudes are used for quantification. rna level parameters are calculated for each of the uniquely identified fragments as a fraction of n isotopologue compared to the n standard. the qms workflow for analysis of rrna modifications. in a typical rrna modification inventory experiment, rrna isolated from a sample of interest (n, red) is mixed with n - labeled external standard (blue), containing mature 16s or 23s rrna. after ribonuclease cleavage, the mixture is submitted to lc - ms analysis. pairs of n and n peaks are detected, and their masses are used for assignment. experimental peaks are fitted to their theoretical isotope distributions, and obtained amplitudes are used to calculate the relative amounts of rrna modifications present in the n - sample. to illustrate the approach, a control experiment was performed where n- and n - labeled 16s rna were mixed in a 1:1 ratio, and the fragments from t1 digestion were analyzed on an esi - tof instrument after a reverse - phase chromatographic separation. n peak pairs co - eluting over 25 min of the lc gradient (figure 2a). for instance, one of the signature 16s rna fragments 1518-(m2a)(m2a)ccug-1523 was found in two different charge - state isoforms, as shown in the red and cyan boxes in figure 2a. high - resolution contour plots and raw data in the m / z dimension (figure 2b) for the triply charged species show that isotopic envelopes of the n and n peaks are well resolved and that the lc - ms traces can be precisely fit using ls - ftc (green trace, figure 2b). n peak pairs, the masses were compared to the masses of the theoretically predicted 16s rna fragments. assignment based on matching monoisotopic m / z values for the n - labeled species alone may be insufficient, especially when mass accuracy is low or when a theoretical digestion list becomes more complex. for example, when mass accuracy of the ms instrument falls below 30 ppm, the n peak shown in figure 2b can be assigned to either (m2a)(m2a)ccug or acgggu (figure 2d). however, the ambiguity of peak identification is reduced significantly when both n and n m / z values are considered. this is illustrated by figure 2c, where the fraction of the experimental peaks with multiple possible identities as a function of the mass tolerance parameter is shown. the agilent ms instruments used in this study routinely achieve mass accuracy in the 530 ppm range, which permits unambiguous identification of at least 99.7% of all 16s rna peaks observed in the control experiment (figure 2c, gray area). additional ms / ms analysis could in principle be used to resolve compositional isomers that are frequently present in the rrna digest (figure 2d, marked by). however, the limited number of modified fragments present in the rrna digest makes ms / ms identification largely unnecessary for the purpose of identification and quantification of modifications. there were a few compositional ambiguities during assignment of the modified fragments, and several different approaches were used to confirm their identification (figure s1 and table s1). in this work, a previously described ls - ftc method for analysis of proteomic data has been adapted for quantification of rrna. using sequence and isotope composition of the assigned rna fragment, the theoretical mass spectrum is fit to the experimental data points, by varying baseline, mass offset, and the width and amplitude of the peaks. since all the isotopic peaks in the n and n distributions contribute to the fit, ls - ftc delivers a high precision of quantification, and minor peak overlaps can be resolved. furthermore, the method can be readily adapted to a variety of isotope labeling strategies. for instance, n peaks were fit using the n isotope enrichment as an adjustable parameter, and the resulting value of 99.3%, determined from multiple measurements, provided excellent fits to the data shown in figure 2b. finally, amplitudes of the n and n peaks (a14 and a15) resulting from least - squares fitting were used to calculate fractional rna levels (f) for each identified 16s rna fragment, where f = a14/(a14 + a15). the values were found to cluster tightly around the expected value of 0.5 (= 0.52 0.02), demonstrating accurate and reproducible quantification (figure 2e). results of the control experiment in which n- and n - labeled and individually purified 16s rna were mixed in a 1:1 molar ratio and digested with ribonuclease t1. (a) low - resolution contour plot of the lc - ms run, showing pairs of the co - eluting n / n rrna fragments (b) high - resolution lc - ms peak profiles (box 1), ms isotope distributions (red dots), and their least - squares fits (green traces) for a representative 16s fragment (box1 in a). (c) ambiguity of peak identification as a function of the mass tolerance parameter (ppm). ms peaks were matched against the 16s theoretical digest (described in d), and the fraction of experimental peaks assignable to more than one rrna fragment was calculated. peak identification was carried out using m / z values for n - labeled fragments only (black) ; m / z for both n- and n - labeled fragments and assuming that fragments should elute within 0.1 min of each other (red) ; using n and n m / z and charge state (z) of the two species (blue). (d) excerpt of the rnase t1 theoretical digest containing predicted 16s rna fragments and their monoisotopic m / z values in the vicinity of (m2a)(m2a)ccug (gray box). digest includes rna species with charges 14, with 02 missed cleavages and either linear or cyclic (> p) phosphate at 3 terminus. m is a methyl group, > p cyclic phosphate (otherwise linear), and marks compositionally nonunique rna fragments included as a single entry. (e) histogram of rna level values calculated for all 16s rrna fragments identified in the control experiment. in e. coli rrna, the majority of modified residues are base and ribose methylations and pseudouridinylations. methylated nucleotides exhibit a distinct mass shift of 14 da that makes them readily observable using ms, however, the pseudouridine substitutions are isobaric. to enable identification of pseudouridines and confirm assignment of the methylated rrna fragments, first, we demonstrated that rrna methyl groups can be selectively deuterated by growing cells in the medium supplemented with cd3-methionine. methionine serves as the precursor for the s - adenosyl - methionine, the cosubstrate and methyl group donor used by cellular methyltransferases. as a result, every rrna fragment exhibited a characteristic + 3 da shift per methyl group in the ms spectrum (figure 3a). the supplemental cd3-methionine concentration in the minimal medium was apparently sufficient to completely inhibit biosynthesis of unlabeled methionine, and the use of an methionine auxotrophic strain was not required to observe complete metabolic labeling of rrna methyl groups. pseudouridine is a structural isomer of uridine, which significantly complicates their discrimination by ms. in the next set of experiments, we demonstrated that supplementing the minimal medium with 5,6-d - uracil results in complete labeling of rrna pyrimidines with a characteristic + 2 da mass shift for every cytidine or uridine residue. in addition, metabolic labeling with 5,6-d - uracil enables observation of pseudouridines because the pseudouridylation reaction results in the exchange of the 5-deuteron with solvent, providing a convenient 1 da mass shift that distinguishes uridine from pseudouridine (figure 3b). indeed, for a number of unique rrna fragments where pseudouridine is found in the proximity of a methylated residue, the expected 1 da mass shift per pseudouridine was observed. for example, the n mass for the metabolically labeled (m)a (976 da, figure 3b, cyan) is 2 da compared to the calculated mass for (mu)au (978 da, not observed), but + 2 da compared to the mass of unlabeled (m)a (974 da, figure 3b, red). overall, by carrying out these metabolic labeling experiments using 16s and 23s rna we confirmed identification of the methylated fragments based initially on n / n m / z measurements and, most importantly, identified a number of fragments bearing pseudouridines. the comprehensive summary on ms identification of modified rrna fragments that were systematically observed in this study is given in table 2. combining results from different rnase treatments, rna fragments 211 nucleotides long were obtained. notably, some modifications can be monitored using only one enzyme (e.g., 16s : mg(1207) and 23s : mc(1962)), while others can be analyzed independently using two or three nucleases. furthermore, due to the high density of the modified residues in certain regions of the 16s and 23s rna, a number of observed fragments bear multiple modifications. thus, in 16s rnase a product 1513-agg(mg)g(m2a)(m2a)c-1520, mg(1516) is physically linked to modified nucleotides m2a(1518) and m2a(1519). obtaining m / z values for n / n pairs enabled reliable assignment for most of rrna fragments (table 2). in addition, the 5,6-d labeling protocol helped to assign fragments with pseudouridines, and in a number of cases to distinguish between compositional isomers such as 16s : 964-au(mg)-966 and 23s : 2503-(ma)g-2505, as described in table s1. in principle, due to high sequence redundancy, identification of short modified nucleolytic fragments (23 nt) can be a challenge in long and extensively modified molecules like rrna. for instance, two isobaric fragments 16s : 527-(mg)c-528 and 23s : 1835-(mg)c-1836 exhibit different retention times. to assign these fragments, spiking the sample with either n - labeled 16s or 23s rna was sufficient to demonstrate matching of one of the two fragments but not the other (figure s1a). the possibility that observed peaks resulted from the singly methylated derivative of 16s : 966-(mg)(mc)-967 is unlikely, since only mature and presumably fully modified rrna is used for a spike. furthermore, 5,6-d - uracil labeling protocol was also useful to distinguish between isobaric fragments that instead of a pseudouridine modification carry a methyl group at the c5 position of the cytosine or uracil (table 2, 1 da 5,6-d shift for 16s : mc(1407) and 23s : mu(1939)). for instance, two nonunique 16s fragments 966-(mg)(mc)-967 and 1401-g(mcm)-1402 bear c5 or n4 cytosine methylations. the two methylations were distinguished by the loss of the 5-deuteron to accommodate c5-methyl resulting in a 1 da shift in the spectrum (figure s1b), and the observed peak at 695 da (figure s1b) was assigned to 966-(mg)(mc)-967. in fact, the expected 1401- g(mcm)-1402 fragment was not observed at all, most likely because rnase a cleavage at the 2-o - methylated sites is ineffective (table 2, cm(2498) and um(2552)). finally, genetic deletions of the individual modification enzymes were used to confirm identification of a number of small rrna fragments, including 23s : 745-(mg)-746 and compositionally nonunique 23s : 2251-(gm)g-2252 (figure s1c - d). a number of modified fragments predicted by the theoretical digestion and corresponding to about 20% of all rrna modifications were either never observed in our lc - ms data sets or their quantitative analysis was difficult (table 1). the residues include five pseudouridine modifications from 23s (table s2), 23s methylation mg(2445), and hu(2449) dihydrouridine (figure s1e). to attain complete modification coverage in the future, one may try to expand the repertoire of sequence - specific nucleases and pursue other chromatography approaches to improve separation of rrna digestion products and remove spectral interference from the co - eluting products. in summary, ms analysis of the specific rnase digestion products combined with metabolic labeling approaches enabled efficient and reliable identification of about of 80% of all modified nucleotides in e. coli rrna. among them are 23 out of 24 methylated residues and 6 out of 11 pseudouridines (tables 1 and 2). cd3-methionine (a) and 5,6-d - uracil (b) labeling results in characteristic mass shifts for methylated and pseudouridinylated rrna fragments. 23s n - labeled spike was prepared by growing cells in n - ammonium sulfate (red) ; in n - ammonium sulfate with cd3-n - methionine (blue) or in n - ammonium sulfate with 5,6-d - n - uracil (cyan) added to the m9 medium. isotope distributions were fitted using 99.3% of n isotope enrichment (red) ; 99.3% of n and 98.5% of d (cyan), as was determined empirically. using cd3-n - methionine, some amount of the methionine amino group was scrambled leading to a reduced fraction of n labeling, which was adjusted to 98.0% for n and 99.3% for d (blue). for rnase t1 and a for rnase u2, only most frequently observed fragments with 2-3 cyclic terminal phosphates (> p) are included. compositionally nonunique fragments are marked by an asterisk (see table s1 for discussion). n, cd3, and 5,6-d are the mass shifts (da) observed using metabolic labeling of rrna. sucrose gradient ultracentrifugation with a subsequent fractionation is a well - established method to separate mature ribosomes (70s) from the small (30s) and the large (50s) subunits and their precursor particles. to determine the presence of ribosomal modifications in these particles, rna modification inventory experiments were carried out using cell lysate from wild - type bacterial cells that was resolved on a sucrose density gradient. each fraction across the gradient containing variable amounts of n particles was spiked individually with mature, fully modified n - labeled 16s or 23s rna to attain 1:1 molar ratio of n ton rna. rna abundances with respect to the spike (rna level, f) were calculated for every unique rrna fragment found. to account for different amounts of the n standard added to each fraction, initial rna levels for modified fragments (rna modification level, fmod) were scaled to the level of unmodified rrna fragments present in the fraction (total rrna level, ftotal). the total rrna level (ftotal) used for normalizations was calculated by averaging rna levels (f) for 315 unmodified compositionally unique 16s or 23s fragments (figure s2). throughout the gradient, unnormalized rrna levels (f) could vary between 0.2 and 0.7 with a 0.0150.04 deviation from the average. to cover all modifications, each fraction was subjected to treatment by at least two nucleases, unless only small amounts of rrna were isolated. for some modifications, independent measurements using fragments from different rnase treatments were obtained (table 2), and their average values are reported. normalized rna levels depicted in figures 4 and 5 suggest that rrna isolated from sucrose fractions corresponding to 30s and 70s peaks contains a complete set of 16s and 23s modifications (normalized fmod 1, data for 16s in the 70s peak are not shown). major changes in the stoichiometry of the rna modifications (i.e., normalized fmod < 1) were observed at the leading edges of the 30s and 50s peaks, where according to prior work assembly intermediates are found. most likely, early 30s and 50s fractions contain a heterogeneous mixture of the cosedimenting ribosomal particles, and the data report on the average relative amounts of the modified fragments in these particles. 16s inventory profiles obtained from a single sucrose gradient (figure 4a) and from two more replicate experiments (figure s3) suggest presence of at least three groups of modifications with high (red), medium (yellow), and low (cyan) abundances in the pre-30s fractions. when these groups of modifications were mapped on to the rrna structure, they were found to correlate with domain organization of the 16s molecule (figure 4b). for the large ribosomal subunit, rna levels calculated across 50s and 70s peaks of a given sucrose gradient (figure 5) reveal two groups of rna modifications. this result is further supported by the cluster analysis carried out using data from four replicate inventory experiments (figure s4). the vast majority of 23s modifications are highly abundant in the pre-50s fractions (red group), with a small distinct group (cyan) of modifications that are depleted and reach their stoichiometric levels only in the 70s region. unlike 16s rna, these results have no obvious correlations to the secondary or tertiary structure of the large subunit (see discussion). inventory of the ribosomal proteins, obtained earlier using the same e. coli strain, describes protein composition of 30s and 50s assembly intermediates. the data suggest that in general, less mature intermediates (i.e., bound to a smaller number of the ribosomal proteins) sediment slower than more mature (i.e., heavier) particles (from left to right on the gradient), and we expect that rna modifications occurring earlier during assembly should be more abundant in the pre-30s and pre-50s fractions than modifications occurring later. to better understand when individual rna modification steps might occur on the assembly pathway, we analyzed dependencies between ribosomal proteins and rna modification profiles. to see the correlations, protein and rna modification level data obtained from two different sucrose gradients were aligned (figure 6). normalized levels calculated for a subset of ribosomal proteins which mark early (s4/l24), intermediate (s7/l5), and late (s2/l16) stages of subunits assembly were coclustered with a subset of rna data for modifications representing each of the groups shown in figures 4 and 5. this analysis reveals similarities between protein profiles and rna modifications profiles across 30s and 5070s fractions (figure 6). for the 30s subunit (figure 6a), the profile for residue mg(527) correlates to s4 abundances suggesting that mg(527) is modified early on the assembly pathway. similarly, mg(1207) profile correlates to the profile for the intermediately binding s7, and the profiles for residues mg(1516), m2a(1518), and m2a(1519) correlate to the profile for the late binding s2, indicating that these modifications are taking place at intermediate and late stages of ribosome assembly. in the 50s subunit (figure 6b), profiles for residues ma(2030), mu(1939), and cm(2498) correlate to the protein levels for the early (l24) and intermediate (l5) binders, suggesting that the majority of 23s modifications are carried out relatively early on the pathway. in contrast, (1911), m(1915), (1917) residues are modified much later, likely when protein assembly is complete, as these modifications reach their stoichiometric level further on the gradient (in the 70s peak) than the late binding l16 protein. relative abundances of the 16s modifications with respect to the external standard are shown for the fractions collected across the 30s peak. rna modification levels (fmod) were normalized to amounts of unmodified rrna fragments measured in each fraction (ftotal). (a) three distinct groups of rrna modifications are shown in red, yellow, and cyan. data for fragments reporting on mg(1516) and m2a(1518), m2a(1519) abundances (table 2) were combined. (b) schematic of the 16s secondary structure with its structural domains colored to map groups of rna modifications. inventory of 23s rna modifications in wild - type e. coli ribosomes. normalized rna modification levels (fmod / ftotal) across the 50s and 70s peaks. two distinct groups of 23s modifications are colored red and cyan. for residues that are physically linked due to digestion : mg(745), (746), mu(747) ; (1911), m(1915), (1917) ; cm(2498), hoc(2501) ; and ma(2503), (2504) (table 2), inventory data were combined. rna levels calculated using individual nucleolytic fragments corresponding to these residues were found to be within the measurement error, and their averaged values are shown. relative levels of rna modifications and ribosomal proteins for the subset of the small (a) and large (b) ribosomal subunit components are shown as a heat map. rna and protein data were obtained from two separate experiments, ribosome sedimentation traces were aligned, and data linearly interpolated to account for the difference in a number of collected fractions., values for the intermediate (s7/l5) and late (s2/l16) binding proteins were normalized to those of the primary binders (s4 or l24). sensitivity, accuracy of mass determination, and automation of ms - based methods make them attractive for studying post - transcriptional modifications that are not amenable to direct rna sequencing analysis. shifts in the mass of an oligonucleotide fragment caused by most types of rna modifications can be easily resolved by modern mass spectrometers, making ms highly suitable for identification and sequence placement of the new residues. expanding the applicability of ms for quantitative profiling of known rna modifications, we have introduced an efficient method based on stable isotope labeling and precise fitting of the isotope distributions. the method enabled quantitative characterization of rrna modifications in a scope that to our knowledge has not been previously achieved. the presence of both n- and n - labeled species assures reliable assignment of nucleolytic fragments and can be used for analysis of complex fragment mixtures resulting from rna digestion. simplicity of sample preparation is such that separation of 16s and 23s molecules preceding lc - ms run is not required, as n - labeled 16s or 23s rna is used to selectively spike one of the two rnas. furthermore, automation of essentially every step during lc - ms data analysis, including peak picking, peak identification, and fitting allowed us to acquire information over hundreds of rna peaks from a single lc - ms run. finally, accurate and reproducible quantification results were obtained using the ls - ftc algorithm, which enabled us to fit rna isotope distributions from a number of different labeling experiments and account for variations in the degree of isotope enrichment or metabolic scrambling (figure 3). pseudouridines and methylations are the most abundant ribosomal modifications in all domains of life. isotope labeling strategies described in this work do not interfere with the metabolic machinery of the cell and provide powerful means for future investigation of rna modifications. using a deuterated methionine analog, one can selectively monitor methylated nucleosides in virtually any cellular rna. using 5,6-d - uracil, efficient and uniform labeling of cellular pyrimidines was achieved permitting convenient monitoring of pseudouridines concurrently with other modifications. in comparison with previously used derivatization approaches adapted for ms - based identification of pseudouridines, metabolic labeling is more efficient as it does not involve additional separation steps which increase the amount of rrna sample required for analysis. using the described qms method, extensive data sets were collected to quantify levels of rna modifications present in preribosomal particles isolated from wild - type e. coli. inventory experiments were carried out across 30s and 50s regions of the sucrose gradient, and several replicate experiments were performed to provide independent assessment of reproducibility and to compensate for occasionally missing data points. gradual increases in rna modification levels toward the 30s and 50s peaks correspond to accumulation of the rna modifications in the fractions which contain more mature subunits. moreover, observed correlations between ribosomal proteins and rna modification profiles across the gradient suggest that rrna is modified at different time points on the assembly pathways and that assembly and rna modification might be intimately related temporally and functionally. by concatenating results from multiple sucrose gradients (figures s3 and s4), rna profiles were used to determine groups of residues modified at different stages on the assembly pathway. for the 30s subunit, three groups of early, [mg(527) ] ; intermediate, [mg(966), mc(967), mg(1207) ] ; and late, [mcm(1402), mc(1407), mu(1498), mg(1516), m2a(1518), m2a(1519) ] modifications emerged. curiously, these groups are localized in the individual structural domains of the 30s subunit : the 5 body domain, the 3 head domain, and 3 minor domain correspondingly (figure 4b). this is consistent with an overall picture of 30s subunit assembly in vivo and in vitro, which proceeds by its structural domains in a 5 to 3 direction. previously reported in vitro biochemical analyses indicate that the preferable substrate for most of the 30s modification enzymes is the assembled subunit, rather than naked 16s rna (table 1). to reconcile these analyses with the data obtained here, we suggest that rather than complete assembly of the 30s particle, binding of at least a subset of the ribosomal proteins which promote structural organization of the specific 30s domains is necessary and sufficient for rna modifications to take place. thus, in vitro, the 30s ribosome serves as a better mimic of the native substrates for the modification enzymes than 16s rna, but it is likely that the true in vivo substrates for the modification enzymes are partially assembled ribosomes with partial native structure. to some degree, our inventory data are consistent with in vivo results previously obtained by the reverse - phase chromatography analysis of rrna nucleosides. in that study, mcm(1402) was postulated as an intermediate (see figure s3 for discussion) and mu(1498), m2a(1518), m2a(1519) as late assembly modifications. surprisingly, these modifications are present at a low level in the 30s subunit which might be caused by differences in growth conditions and cell lysis between our study and the previous work. the rna level profile for the 30s pseudouridine (516) is not shown, as corresponding fragments exhibited abnormally high levels in the pre-30s region of the gradient. this suggests that other pseudouridines are present in these fractions, perhaps from different rnas, and more work needs to be done to determine their origin. furthermore, our qms method has difficulty measuring intermediate modification states of residues or nucleolytic fragments expected to undergo multiple modifications. for instance, detecting whether base and ribose methylations of mcm(1402) occur at drastically different points during assembly would be challenging. similarly, detection of mg(1516) is limited to a single rnase a fragment (table 2), where mg(1516) is linked to m2a(1518) and m2a(1519). most likely, g1516 is methylated not later than a1518 and a1519, since m2a(1518) and m2a(1519) residues were monitored independently by rnase a and t1 fragments and equivalent abundance profiles were obtained. in the future, more detailed investigation of mcm(1402) or mg(1516) could be performed using qms and bacterial strains carrying deletions of rsmh / rsmi and rsma genes. for 50s, inventory profiles were used to characterize [mg(745), (746), mu(747), ma(1618), mg(1835), mu(1939), mc(1962), ma(2030), mg(2069), gm(2251), cm(2498), hoc(2501), ma(2503) and (2504) ] as early stage modifications and [(1911), m(1915), (1917) and um(2552) ] as late. a closer examination of our qms data sets also suggests that cm(2498) and mu(1939) might have been modified at an early to intermediate stage of 50s assembly (see figure s4 for discussion), consistent with previous in vivo results suggesting that cm(2498) is methylated at an intermediate point along the assembly pathway. however, more data are needed to fully confirm this observation. unlike the 30s subunit, it is difficult to reconcile structural positioning of the modified 50s nucleotides with their modification order. for example, both mg(1835) and m(1915) are surface exposed residues, though the first is modified early during subunit assembly and the second much later. likewise, gm(2251) and um(2552) form the core of the peptidyl transferase center, but gm(2251) is abundant and um(2552) is depleted in the fractions corresponding to 50s intermediates (figure 5). this discrepancy might be a consequence of the much more intricate assembly landscape characteristic of the large subunit or regulatory roles that individual enzymes (or their products) might play in biogenesis. in fact, results from a few previously published studies emphasize that rrna modification enzymes, such as rsma (aka ksga), rlud, and rlme (aka rrmj), may act as assembly chaperones, check point, or quality control markers. these essentially unknown functions of the rna modification machinery might play key roles in shaping the whole biogenesis process. the obtained 23s modification profiles are fully consistent with results of in vitro modification assays (table 1), suggesting that most 23s enzymes recognize their targets when the rna is free in solution, with the exception of rlme (um-2552), rlud (-1911, 15, 17), and rlmh (m-1915), which act on the fully assembled 50s or even 70s particles. detailed investigations of rlud- and rlmh - dependent modifications in vivo and in vitro suggest that pseudouridylations of 1911, 1915, and 1917 occur at the stage of completion of the 50s assembly and (1915) is methylated during or after subunit joining. as in case of the 16s residue mcm(1402), qms may not resolve the rlud and rlmh steps, as the corresponding modifications are monitored using the same nucleolytic fragments (table 2), requiring more in depth analyses. figure 6 provides the first illustration of how individual modification steps are integrated into the existing framework for the 30s and 50s subunit assembly. these and previously published data strongly suggest that rna modification processes are tightly coupled to the binding of the individual or groups of proteins. we envision that future protein and rna modification qms analyses will be fruitful to decipher detailed dependencies between these steps. in particular, this method offers the opportunity to analyze the relationship of rrna modifications to the roles of the ribosome assembly factors using deletion strains or other perturbations to ribosome assembly. the data presented here demonstrate feasibility of a new qms approach for efficient and accurate monitoring of rrna modifications. relative amounts of individual rna modifications were measured in the incompletely assembled ribosomal particles, providing insights into the relative order of the individual rrna modification steps. this work lays a foundation for more in depth studies of rrna modifications in bacterial and eukaryotic systems, where the proposed metabolic labeling schemes should be applicable. future experiments will undoubtedly lead to a better understanding of the mechanisms and functions of individual modification steps in ribosome biogenesis and translation. | post - transcriptional rna modifications that are introduced during the multistep ribosome biogenesis process are essential for protein synthesis. the current lack of a comprehensive method for a fast quantitative analysis of rrna modifications significantly limits our understanding of how individual modification steps are coordinated during biogenesis inside the cell. here, an lc - ms approach has been developed and successfully applied for quantitative monitoring of 29 out of 36 modified residues in the 16s and 23s rrna from escherichia coli. an isotope labeling strategy is described for efficient identification of ribose and base methylations, and a novel metabolic labeling approach is presented to allow identification of ms - silent pseudouridine modifications. the method was used to measure relative abundances of modified residues in incomplete ribosomal subunits compared to a mature 15n - labeled rrna standard, and a number of modifications in both 16s and 23s rrna were present in substoichiometric amounts in the preribosomal particles. the rna modification levels correlate well with previously obtained profiles for the ribosomal proteins, suggesting that rna is modified in a schedule comparable to the association of the ribosomal proteins. importantly, this study establishes an efficient workflow for a global monitoring of ribosomal modifications that will contribute to a better understanding of mechanisms of rna modifications and their impact on intracellular processes in the future. |
biometrics refers to a real - time identification system that is used in the identification of a person using a specific physical or behavioral characteristic which is compared with a library of characteristics of many other people. this is done using a biometric scanning device (tongue - print scan) which captures the user 's biometric data such as the tongue - print scan and converts it into a digital information that the computer interprets and verifies. tongue print is the information carried on the exposed portion of the tongue that is the shape and texture put together. the geometric shape of the tongue is usually constant, and the physiological surface texture does not vary a lot. tongue is an organ that can be easily exposed for inspection but at the same time well protected from environmental influences and therefore very difficult to manipulate or forge unlike other identification systems. the uniqueness of the tongue print is that no two tongues are the same, and studies have found that the tongue of identical twins also does not resemble each other. therefore, the use of tongue prints as a biometric authentication system is gaining a lot of momentum. in the past 10 years, research has been targeted towards developing a tongue print recognition system, and the first of its kind was proposed by liu. in 2007. recently, tongue recognition systems based on 2d dual - tree complex wavelet transform have been proposed by bade. tongue scanners are under research and being tested. in india, this system of identification is still in the grassroot level and needs more quantum of research and planning to implement the same. creation of a database is pivotal for identification, but there is no national database available currently in india. furthermore, there is no scanning device yet been created for capturing the tongue print. visual inspection and digital photography lingual impression is the impression of the dorsal surface of the tongue along with the lateral borders. this will be useful in determining the shape and the surface characteristics of the tongue and can serve as a permanent record through the cast. a small - scale study was carried out in our institution with the aim of determining the most common tongue features, its predominant shape and variations in males and females. further, the usefulness of alginate impressions and dental cast as a permanent record of the lingual impression was also evaluated. the study participants were selected on a random basis from patients attending the outpatient department of oral medicine and radiology, thai moogambigai dental college. patients with habit of smoking and any systemic illness were excluded from the study. after obtaining informed consent, clinical examination of the patient 's tongue was performed. before the examination, the patients were asked to rinse the mouth gently with water to remove any surface debris or food particles. subsequent to clinical examination, photographs (front and side view) were taken from a predetermined distance using a digital camera (7 megapixels). alginate impression of the dorsal surface of the tongue was made, and a positive replica was prepared using type ii dental stone [figure 1a and b ]. the photographs and the cast were analyzed and compared for morphological features such as shape and characteristics of fissures by two independent observers. the reference points included the region of the tongue in contact with the commissure of the lips (when protruded outside the mouth) and the tip of the tongue [figure 2a and b ]. (a and b) stone casts of tongue prints (a and b) reference points for determining the shape of the tongue a total of 20 patients participated in the study, of which there were 12 males and 8 females. when considering the direction and the number of fissures, it was observed that the presence of vertical fissure was more common in females and multiple vertical fissures were more common in males (33.3%) [figure 3 ]. it was observed that fissures were shallow in males (50%) and deep fissures were common in females (62.5%) [figure 4 ]. on analyzing the shape of the tongue, it was found that the most common shape was u shape in both males and females (83.3% and 75%, respectively) followed by v shape which was more common in females than males (25% and 16.7%, respectively) [figure 5 ]. fisher 's exact test resulted in a p = 0.993, which was not statistically significant [table 3 ]. analysis using photographs and stone casts revealed 90% matching validating alginate as a reliable aid in obtaining lingual impressions. gender - wise distribution of the location of the fissures gender - wise distribution of location of fissure gender - wise distribution of depth of fissure gender - wise distribution of the depth of the fissures gender - wise distribution of the shape of the tongue gender - wise distribution of the shape of the tongue innovative and efficient identification systems are an urgent need to combat this social issue. identification of human beings based on characteristic physiological parameters is the central dogma of biometric authentication and information security advantages of tongue prints over other biometric systems are genetic independence (no two tongue are similar), physical protection (well encased in the oral cavity) and its stability over time. research on tongue prints is at a preliminary stage. a study by diwakar and maharshi reported tongue as a reliable member of biometrics family. application of tongue biometrics system in public - use system such as banking system has been proved by naaz. in 2011. the study also proposed the use of alginate impression in obtaining lingual impression as an efficient technique. implications of tongue prints and its use as a forensic tool remain unexplored in the field of dentistry. to the best of our knowledge, the presence of fissures was the most common morphological characteristic seen in the dorsum of the tongue. the fissures were predominantly located in the central region of the tongue as observed by stefanescu. the predominant shape of the tongue in both males and females was u shape. v - shaped tongue with a sharp tip was also observed in a substantial sample of females. these observations corroborated with the findings of other studies where the authors reported increased length and width of the tongue in males compared to females. all the participants included in the study were free of any pathology affecting tongue, habits and systemic illness. this study would serve as a pilot survey for the use of tongue prints in biometrics and forensic investigations. this study represents a preliminary analysis of tongue features and its variations with respect to gender. a simple methodology to obtain lingual impression this simple procedure of obtaining lingual impressions can be adopted by dentists as a chairside technique. large - scale studies should be conducted to determine the common presentation of tongue features among males and females. dentists can play an important role by collecting images of the tongue and prepare a cast routinely for the patients along with their other dental records. this would serve as a database and a guide for identification purposes. to conclude, tongue print being a unique record and one that can not be forged is a better biometric authentication tool than others, and since it is personalized and constant, it can be used for forensic identification purposes too. | background and objectives : biometric authentication is an important process for the identification and verification of individuals for security purposes. there are many biometric systems that are currently in use and also being researched. tongue print is a new biometric authentication tool that is unique and can not be easily forged because no two tongue prints are similar. the present study aims to evaluate the common morphological features of the tongue and its variations in males and females. the usefulness of alginate impression and dental cast in obtaining the lingual impression was also evaluated.materials and methods : the study sample included twenty participants. the participants were subjected to visual examination following which digital photographs of the dorsal surface of the tongue were taken. alginate impressions of the tongue were made, and casts were prepared using dental stone. the photographs and the casts were analyzed by two observers separately for the surface morphology including shape, presence or absence of fissures and its pattern of distribution. three reference points were considered to determine the shape of the tongue.results:the most common morphological feature on the dorsum of the tongue was the presence of central fissures. multiple vertical fissures were observed in males whereas single vertical fissure was a common finding in females. the fissures were predominantly shallow in males and deep in females. the tongue was predominantly u shaped in males and females. v - shaped tongue was observed in 25% of females.conclusion:tongue prints are useful in biometric authentication. the methodology used in the study is simple, easy and can be adopted by dentists on a regular basis. however, large - scale studies are required to validate the results and also identify other features of the tongue that can be used in forensics and biometric authentication process. |
niniejszy retrospektywny przegld dotyczy chorych, ktrych poddano pneumonektomii w ramach leczenia niedrobnokomrkowego raka puca pomidzy styczniem 2005 a grudniem 2011 r. przeanalizowano istotne powikania oraz miertelno operacyjn, a nastpnie porwnano je pomidzy pacjentami leczonymi za pomoc chemioterapii neoadiuwantowej oraz pacjentami leczonymi wycznie chirurgicznie. w okresie objtym badaniem wykonano 177 pneumonektomii (77 prawych i 100 lewych), a 49 spord pacjentw (27,7%) zostao poddanych chemioterapii neoadiuwantowej. wskanik przetok oskrzelowo - opucnowych wynis 26,5% (13/49) w grupie poddanej terapii neoadiuwantowej w porwnaniu z 3,1% (4/128) w grupie leczonej wycznie chirurgicznie (p = 0,029). wskanik przetok oskrzelowo - opucnowych wynis 16,9% (13/77) w grupie poddanej prawej pneumonektomii w porwnaniu z 4% (4/100) w grupie poddanej lewej pneumonektomii (p = 0,004). miertelno w grupie neoadiuwantowej wyniosa 8,2% w porwnaniu z 4,7% w grupie leczonej wycznie chirurgicznie (p = 0,37). pneumonectomy is associated with increased morbidity and mortality rates when compared to other pulmonary resections. there are several controversial results in the literature about the effect of induction therapy on morbidity and mortality of pneumonectomy applied for the resection of non - small cell lung cancer, which questions the feasibility and benefit of performing such a high - risk procedure [210 ]. bronchopleural fistula (bpf) after pneumonectomy is a major problem in thoracic surgical practice. some risk factors are defined which might be the cause or a predictor of this complication [1115 ]. in this study, a retrospective review of patients who underwent pneumonectomy for non - small cell lung cancer from january 2005 to december 2011 was undertaken. the patients received neoadjuvant chemoradiation and patients having incomplete resection were excluded from the study. the data were obtained from the electronic and paper medical records, patients charts and follow - up records. informed consent was obtained from all the patients before the operation. computed tomography (ct) scan, positron emission tomography (pet) invasive mediastinal staging procedures such as endobronchial ultrasound (ebus) biopsy of mediastinal lymph nodes, mediastinoscopy, and video - assisted thoracoscopic surgery (vats) were used when there was a suspicion of a metastatic lymph node on pet scan. the physiologic evaluation of the patients considered for resection was performed according to the published guidelines. three cycles of platinum based chemotherapy were applied to patients having histologically proven ipsilateral mediastinal lymph node (n2) metastases or locally advanced tumor when there was a doubt of resectability. single - lung ventilation was obtained through a double - lumen endotracheal tube and epidural analgesia was performed routinely for pain management. a serratus anterior muscle sparing posterolateral thoracotomy was performed and mediastinal lymph node dissection was added to pneumonectomy in all cases. the bronchial stump was not reinforced routinely according to the surgeon 's preference, and mediastinal pleura was used most frequently when reinforcement was applied. the patients were taken to a postoperative intensive care unit after being extubated in the operating room if possible and then transferred to a thoracic ward whenever cardiopulmonary functions were stable. major cardiopulmonary complications such as arrhythmia requiring medical treatment, respiratory failure defined as reintubation, placement of tracheostomy and need for ventilation more than 48 hours after surgery and bronchopleural fistula, as well as operative mortality defined as death from any cause within 30 days of surgery were recorded and compared between the patients having neoadjuvant chemotherapy and patients having surgery only. statistical package for the social sciences (spss) version 19 was used for analyzing the data. the associations among the categorical variables and numerically coded qualitative variables between the two groups were examined by using fisher 's exact test. the descriptive statistics of the quantitative variables were reported as means, standard deviations, medians, minimums and maximums while the qualitative variables were summarized as counts (n) and percentages (%). the obtained statistics were reported with their 95% confidence intervals and the analysis results with p < 0.05 were considered as statistically significant. there were 168 (94%) male and 9 (6%) female patients in the study group with a median age of 60 years (range, 32 to 80). of the 177 pneumonectomies (77 right and 100 left) performed during the study period, 49 of these patients (27.7%) received neoadjuvant chemotherapy. neoadjuvant chemotherapy was applied to 12 patients for having histologically proven ipsilateral mediastinal lymph node (n2) metastases and to 37 patients for having a locally advanced tumor and there was a doubt of resectability. the characteristics of patients who received neoadjuvant chemotherapy are shown in table i. characteristics of the patients (n = 49) who received neoadjuvant chemotherapy the major cardiopulmonary complication rate was higher in the neoadjuvant group (44.8% vs. 4.6%) (p = 0.002) (table ii). the bronchopleural fistula rate was 26.5% (13/49) in the neoadjuvant group versus 3.1% (4/128) in the surgery alone group and the difference between the two groups was statistically significant (p = 0.029). additionally the bronchopleural fistula rate was 16.9% (13/77) in the right pneumonectomy group versus 4% (4/100) in the left pneumonectomy group (p = 0.004). comparison of major cardiopulmonary complications between the two groups overall 30-day operative mortality was 5.6% (10/177). mortality rates were higher in the neoadjuvant group (8.2% versus 4.7% in the surgery only group) but the results were not statistically significant (p = 0.37). the mortality rate was 6.5% (5/77) in the right pneumonectomy group versus 5% (5/100) in the left pneumonectomy group (p = 670). despite the recommendation by the american college of chest physicians to avoid pneumonectomy after induction therapy, there are several retrospective studies and clinical trials published investigating the morbidity and mortality after this treatment and most of them focused on the mortality rates [1820 ]. d'amato revealed a mortality rate of 21% and concluded that induction chemotherapy increases the risk of operative mortality after pneumonectomy. kim also reported a 20% 90-day mortality after right - sided pneumonectomy. on the other hand, there are also some recent studies published showing that this high - risk procedure can be performed with acceptable mortality rates from 3 to 10% [2, 3, 58, 10, 23 ]. more recently kim. reported a meta - analysis of perioperative mortality after neoadjuvant therapy and pneumonectomy. mortality rates were higher in the neoadjuvant group (8.2% versus 4.7% in the surgery only group), which was in accordance with the recently published data, but the difference was not statistically significant (p = 0.37). low preoperative serum albumin level, hyperglycemia, previous steroid therapy, poor predicted postpneumonectomy forced expiratory volume in 1 second, long bronchial stump, mechanical ventilation, right pneumonectomy, adjuvant radiotherapy and pneumonia after the operation are shown to be some of the risk factors [1115 ]. although some bronchopleural fistula rates have been revealed in several studies focusing on the mortality of pneumonectomy after induction therapy, the effect of neoadjuvant treatment on formation of postpneumonectomy bronchopleural fistula has not been evaluated thoroughly. refai reported that there was no increase in the postpneumonectomy bronchopleural fistula rate after induction treatment. d'amato reported a bronchopleural fistula rate of 8.8% in the induction therapy group versus 7.3% in the surgery alone group without any significant difference between the two groups. there are several studies in the literature that have shown an association between induction treatment and increased risk of bronchopleural fistula after pneumonectomy. ucvet and panagopoulos also found that neoadjuvant therapy is a significant risk factor for bronchopleural fistula. in our previous ex vivo study, we found that the tensile strength of pulmonary structures such as the pulmonary artery, pulmonary vein, and bronchus decreases and fibrosis grade increases after neoadjuvant chemotherapy, which may be a cause of the bronchopleural fistula. in our present study, | introductionperforming pneumonectomy after neoadjuvant chemotherapy is still controversial. bronchopleural fistula is a major complication after pneumonectomy. in this study the effect of neoadjuvant chemotherapy on postpneumonectomy bronchopleural fistula was investigated.material and methodsa retrospective review of patients who underwent pneumonectomy for non - small cell lung cancer from january 2005 to december 2011 was undertaken. the major complications and operative mortality were analyzed and compared between the patients having neoadjuvant chemotherapy and patients having surgery only.resultsone hundred and seventy - seven pneumonectomies (77 right and 100 left) were performed during the study period and 49 of these patients (27.7%) received neoadjuvant chemotherapy. median age was 60 years (range, 32 to 80). the bronchopleural fistula rate was 26.5% (13/49) in the neoadjuvant group versus 3.1% (4/128) in the surgery alone group (p = 0.029). the bronchopleural fistula rate was 16.9% (13/77) in the right pneumonectomy group vs. 4% (4/100) in the left pneumonectomy group (p = 0.004). overall operative mortality was 5.6%. mortality in the neoadjuvant group was 8.2% vs. 4.7% in the surgery only group (p = 0.37).conclusionsneoadjuvant chemotherapy and right pneumonectomy is a major risk factor for bronchopleural fistula. especially right pneumonectomy should be avoided after induction therapy. |
since various forms of fluorides have met wide acceptance for use in the prevention of dental caries, the metabolism of fluoride is of considerable interest.13 the human organism is exposed to fluoride in a number of ways. ingestion of fluoride is accomplished through various foods ; drinking water and fluoride containing products comprising dentifrices, mouth rinses, tablets, drops, etc.46 hard tissues are known to be the major sites of fluoride accumulation in the human body. approximately 99% of the total body burden of fluoride is retained in bones and teeth, with the remainder distributed in highly - vascularized soft tissues.4,5 kidneys are the primary route for the removal of fluoride from the body. other routes of fluoride excretion are sweat, feces, saliva and breast milk of lactating mothers.4,5,7 breast milk is the major dietary intake of infants in the early stage of life. the level of fluoride in breast milk plays an important role as a fluoride supplement to the infant.8 conversely, the concentration of fluoride could be deemed critical regarding the potential dental fluorosis that may result from high concentrations of dietary fluoride.9,10 plasma is the biological fluid into which fluoride must pass for its distribution elsewhere in the body as well as its elimination from the body. for these reasons, plasma is often referred to as the central compartment of the body.6 factors that include fluoride intake from various sources may affect plasma fluoride levels, and thus fluoride content of breast milk. the aim of this pilot study was to determine the fluoride levels of breast milk and plasma of lactating mothers and the correlation between breast milk and plasma fluoride levels in mothers who regularly consume drinking water with low levels of fluoride. one hundred twenty five mothers aged between 2030 years old with hospitalized newborns due to icterus neonatorum were included in the study. the human ethic committee of selcuk university experimental research center (sudam) approved this study (approval no:2004034). besides being otherwise healthy, the primary selection criteria stipulated the absence of fluoride supplement consumption one month before delivery. the participants regularly consumed drinking water from the same city supply which has been previously shown to contain low levels of fluoride (approx. milk and plasma samples were collected from lactating mothers within 5 to 7 days after delivery. for milk samples, the mother was instructed to press the breast gently to facilitate collection of 5 ml of milk into a polyethylene tube. at the same appointment, 5 ml of blood was obtained and transferred into a fluoride - free heparinized polyethylene tube. thereafter, the plasma was separated from the blood by centrifugation for 3 min at 3500 g. milk and plasma samples were further stored at 18c until analyses. before fluoride measurements, the samples were thawed at room temperature. to determine fluoride concentrations, equal volumes of tisab ii buffer (orion research, u.s.a.) an ion - selective electrode (model 9609, orion research, usa) was used in conjunction with a model ea 910 ion analyzer (orion research, usa) to measure the fluoride concentrations of the breast milk and plasma samples. paired t test was used to determine the differences between fluoride concentration of breast milk and plasma. pearson correlation analysis was used to assess any possible relationship between plasma and breast milk fluoride levels.12 the mean fluoride concentration of the plasma samples was 0.0170.011 ppm (range 0.0060.054 ppm). the mean fluoride concentration of the breast milk samples was 0.0060.02 ppm (range 0.0030.011 ppm). paired t test showed that the fluoride concentrations of plasma were significantly higher than those of the breast milk (p=.000). pearson analysis revealed a significant correlation between the fluoride concentrations of breast milk and of plasma (p=.000). when a mother s plasma fluoride concentration was above (or below) the mean plasma fluoride level of the entire study group, the breast - milk fluoride levels were affected accordingly. several methods are used to determine fluoride levels in biologic tissues that include spectrophotometry,13 gas chromatography,14 capillary electrophoresis,15 micro diffusion,16 and ion analysis in conjunction with ion - selective electrodes.17 as utilized in the present study, the most common procedure used to quantify free fluoride anion is the ion - selective electrode.18 the plasma fluoride concentration displays an increase along with fluoride intake. this increase is, however, attenuated due to distribution to the interstitial and intracellular fluid uptake by calcified tissues and renal excretion.5 the literature contains a wide range (0.0080.045 ppm) of reported normal plasma fluoride concentrations.6,18 the diversity of values may have been due to the inclusion of fasting individuals as subjects in contrast to other studies employing non - fasting participants.18 certainly, other factors that include methodological variations as well as the fluoride levels of drinking - water consumed by subjects should have a strong impact on the reported values.18 li reported a mean plasma fluoride concentration of 0.1060.076 ppm in 127 subjects. in their study, the subjects were selected from a region with the drinking water fluoride concentra tions of 5.03 ppm. in the present study the mean plasma fluoride concentration was 0.0170.011 ppm. our finding corroborates those of fejerskow and world health organization (who).18 breast milk possesses unique nutritional, biochemical, anti - infective and anti - allergic properties. as breast - fed infants obtain fluids almost exclusively from their mothers, breast milk represents an important way for delivering fluoride with certain levels to infants.20 the level of fluoride in human milk has been a topic of investigation for many years. according to the who,18 the breast milk fluoride levels range from < 0.002 to about 0.1 ppm, with most values being between 0.0050.010 ppm. the mean breast milk fluoride concentrations obtained here in (0.0060.002 ppm) are in line with the who.18 dabeka showed that the concentration of fluoride in breast milk was related to the content of the drinking - water consumed by the mothers. the mean concentration of fluoride in breast milk obtained from 32 women consuming drinking water that contained < 0.16 ppm was 0.004 ppm, whereas breast milk obtained from 112 women consuming drinking water containing 1 ppm reportedly was 0.009 ppm.8 similar levels of fluoride concentrations of breast milk and colostrum (0.008 ppm) have been reported by spak.1 however, spak found no significant difference in breast milk fluoride concentrations of mothers living in areas with low and high drinking - water fluoride concentrations. in the present study, the strict selection criteria which stipulated absence of recent use of fluoride supplements was a preventive measure to control variables that could interfere with the results. additional limitations included selection of patients from a region with low drinking water fluoride levels (< 0.3 ppm). in light of previous work,8 however, it is apparent that the fluoride concentration of breast milk in mothers regularly consuming higher concentrations of fluoridated water is still within normal limits. theoretically, a limited transfer of fluoride from plasma to breast milk should occur.21 the mechanism(s) responsible for the selective transfer of fluoride into breast tissue is obscure.21 it is thought that a physiological plasma - milk barrier functions against to fluoride.1,2,6 despite high doses of supplementary fluoride administered to the mother, the child receives a maximum dose of only 0.2% of the mother s fluoride intake.1,2 the results obtained in our study confirmed these conclusions. it should, however, be noted that the fluoride content transferred through breast milk is less than those present in cow s milk and in infant formulas, utilized as routine substitutes for breast milk. rahul found that fluoride concentrations of various commercially available infant milk formulations ranged from 1.95 ppm to 7.45 ppm and fluoride content of cow s milk samples was 0.12 ppm ; values exceeding those of breast milk. it has been verified by the positive correlation between plasma and breast - milk fluoride concentrations in subjects selected meticulously in terms of low drinking water fluoride levels. while the results of the present study confirm previous data regarding the difference between plasma and breast - milk fluoride concentrations, it is evident that more research, coupled with a wider study population is indicated to clarify the exact interactions between plasma fluoride levels and those of breast milk. | objectivesthe aim of this study was to determine the fluoride levels in breast milk and plasma of lactating mothers who regularly consumed drinking water with low levels of fluoride.methodsone hundred twenty five healthy mothers aged between 2030 years old who had given birth within 57 days were included in the study. besides being otherwise healthy, the primary selection criteria stipulated the absence of fluoride supplement consumption one month before delivery. approximately 5 ml breast milk and 5 ml blood samples were obtained from each participating mother at a hospital setting, where the mothers were scheduled for a regular hospital diet. the blood samples were centrifuged in fluoride - free heparinized polyethylene tubes and stored at 18c until measurements were made. breast milk samples were directly refrigerated as with blood samples until measurements. the fluoride concentrations of milk and blood samples were assessed using an ion - selective fluoride electrode combined with an ion analyzer.resultsthe fluoride levels of the plasma and breast milk samples were measured as 0.0170.011 ppm and 0.0060.002 ppm, respectively. the fluoride concentration of plasma was significantly higher than that of breast milk (p<.01). correlation analysis revealed a significant relation between the groups (p<.01).conclusiona limited level of fluoride transmission from plasma to breast milk was detected. nevertheless, a significant correlation between the fluoride concentrations of breast milk and plasma was evident. |
pregnancy anxiety is a powerful factor in prediction of negative outcomes in birth and infant. various studies have indicated that pregnant women are faced to new concerns, and numerous mental questions about developing fetus and future child stressed her. these issues have a devastating effect on the mental health of pregnant women ; studies have indicated neuro immunology models that stress during pregnancy affect maternal mental health and fetal growth and can lead to preterm birth. for many women, pregnancy represents a time of increased vulnerability to mental disorders. a significant number of women experience the very first anxiety disorders or mood changes during pregnancy or in the first few months after delivery ; and those, who have a history of psychiatric disorders, are at increased the risk of repetition or recurrence of symptoms. psychological distress during pregnancy has negative impact on women 's life quality and their personal well - being ; and if remain untreated or undiscovered, it may have a negative effect on the growth of fetuses and infants. despite it, anxiety during pregnancy and childbirth also plays a role in the baby suffering from schizophrenia and emotional disorders in the future, autism, hyperactivity, and shortness of breath during neonatal period. there is a direct relationship between anxiety of mother and abnormal fetal brain development, fetal distress, and a reduction in mental development at the age of 2. prenatal anxiety of mother affect on sleep disorders of children, and behavioral problems in early childhood ; and in the importance of mental health in pregnancy this issue is notable that the root of poor attachment of parent - child during postpartum depression lies in pregnancy period. many studies have shown the relationship between high levels of cortisol (which is influenced by the mother 's anxiety and stress) with the risks of low birth trauma and psychological disorders. the existence of anxiety in pregnancy and also in childbirth, have harmful effects and in the long - term anxiety, smooth muscles are constricted by stimulating the autonomic nervous system and in result, the uterus - placental blood flow and oxygenating to the uterus is decreased, and as a result, fetal heart rate pattern become abnormal and risk of preterm delivery will increase. hence, premature birth and low birth weight leads to negative consequences in physical and cognitive development of children and these babies are more likely to suffer from cerebral palsy, learning disabilities, and other disabilities. moreover, anxious and depressed mothers are not sensitive to the messages sent by their children. therefore, the prevalence of anxiety disorders may have reverse effects on results of midwifery, embryonic, and infancy. it is also noteworthy to mention that mental health problems during pregnancy has been studied in 90% of high - income countries ; whereas in this field, only 10% of low and middle income countries have available information. studies have indicated that pregnant women may have severe concerns about the failure of the embryonic period, concerns about pain and worry about changes in their personal lives as a result of pregnancy and childbirth. the shortcomings in measuring mental state of the mother during pregnancy can estimate their mental turmoil far less than the actual values. while, the early detection, prevention and the management of anxiety during pregnancy make women able to meet the challenges of pregnancy. therefore, testing the fears and concerns specific to pregnancy and reviewing changes of pregnancy related anxiety and clinical factors associated with it seems essential. the population included all pregnant women who visited medical health centers in kerman during the second half of 2014 for medical cares. from the above population, 170 women were selected and were evaluated with available sampling method over 1 month. for this reason, kerman city is divided into four regions of north west, northeast, southwest, and southeast and one health center randomly selected from each region. criteria for inclusion were being iranian, having enough knowledge to fill in questionnaires of the study, having general health as well as having a normal pregnancy and healthy fetus. determining the sample size in the exploratory analysis follows the general principle of sampling (i.e., the number of subjects should always be greater than the number of the questions of the questionnaire) ; a range of 520 participants for each question is considered. for this reason, based on stevens theory and according to the number of questions, 17 questions, the sample size was determined as 170 individuals (10 individuals per question) ; by taking a 10% probability of loss, the questionnaires were distributed among 187 pregnant women and finally 170 questionnaires were completed by the pregnant women. demographic information such as age, number of pregnancies, and education were collected using demographic questionnaire. this questionnaire was the main research instruments that evaluates fears and concerns about related to pregnancy and has been made in 1989 by vanden berg. exploratory factor analysis of the questionnaire revealed five - factors : fear of childbirth (three items), fear of giving birth to a child with physical or mental health issues (four items), fear of change in the marital relationship (four items), fear of changes in mood and its consequences on the child (three items), self - centered fear or fear of the changes in personal life of mother (three items). it is necessary to say that, first, the questionnaire was translated to persian by permission of its designer and in continue, it was returned to english by two english language experts. existence differences between english and persian versions were evaluated and these differences were decreased to minimum using from frequent review process. then, three experts of psychology field confirmed the content validity and cultural adaptation of pregnancy anxiety questionnaire. the questionnaire consists of 21 items, which covers the most common symptoms of anxiety. the phrase reflects one of the symptoms of anxiety that usually people who are clinically anxious or who are in a state of great anxiety experience it. the ratings are classified as not at all (0), mildly (1), moderately (2) and severely (3). if participant have a score of 07, no anxiety. if between 8 and 15 is mild anxiety, if between 16 and 25, the moderate anxiety and if between 26 and 63 indicate severe anxiety. its internal consistency coefficient is 0.92, test - retest reliability is within a week of 75/0, and the correlation of its items varies from 0.300.70. five types of content validity, concurrent, construct, diagnostic and a factor for this test measured which all of them indicative the effectiveness of this tool in measuring of anxiety in iranian population. this study was conducted regarding to permission from the health centers of kerman and principles of morality and ethics. all participants participated in the study consciously, and they were assured that the information gathered will be used only for the purpose of research. participants are also noted that are able to withdraw from the research process any time they want. to analyze the data, correlation matrix is used. to evaluate the reliability of questionnaires, cronbach 's alpha and split - half method and to evaluate validity and factor structure of questionnaires, confirmatory factor analysis and model parameters in the amos (analysis of moment structure) spss software version 20 (spss inc., demographic information such as age, number of pregnancies, and education were collected using demographic questionnaire. this questionnaire was the main research instruments that evaluates fears and concerns about related to pregnancy and has been made in 1989 by vanden berg. exploratory factor analysis of the questionnaire revealed five - factors : fear of childbirth (three items), fear of giving birth to a child with physical or mental health issues (four items), fear of change in the marital relationship (four items), fear of changes in mood and its consequences on the child (three items), self - centered fear or fear of the changes in personal life of mother (three items). it is necessary to say that, first, the questionnaire was translated to persian by permission of its designer and in continue, it was returned to english by two english language experts. existence differences between english and persian versions were evaluated and these differences were decreased to minimum using from frequent review process. then, three experts of psychology field confirmed the content validity and cultural adaptation of pregnancy anxiety questionnaire. the questionnaire consists of 21 items, which covers the most common symptoms of anxiety. the phrase reflects one of the symptoms of anxiety that usually people who are clinically anxious or who are in a state of great anxiety experience it. the ratings are classified as not at all (0), mildly (1), moderately (2) and severely (3). if participant have a score of 07, no anxiety. if between 8 and 15 is mild anxiety, if between 16 and 25, the moderate anxiety and if between 26 and 63 indicate severe anxiety. its internal consistency coefficient is 0.92, test - retest reliability is within a week of 75/0, and the correlation of its items varies from 0.300.70. five types of content validity, concurrent, construct, diagnostic and a factor for this test measured which all of them indicative the effectiveness of this tool in measuring of anxiety in iranian population. this study was conducted regarding to permission from the health centers of kerman and principles of morality and ethics. all participants participated in the study consciously, and they were assured that the information gathered will be used only for the purpose of research. participants are also noted that are able to withdraw from the research process any time they want. to analyze the data, correlation matrix is used. to evaluate the reliability of questionnaires, cronbach 's alpha and split - half method and to evaluate validity and factor structure of questionnaires, confirmatory factor analysis and model parameters in the amos (analysis of moment structure) spss software version 20 (spss inc., chicago, il, usa) are used. this questionnaire was the main research instruments that evaluates fears and concerns about related to pregnancy and has been made in 1989 by vanden berg. exploratory factor analysis of the questionnaire revealed five - factors : fear of childbirth (three items), fear of giving birth to a child with physical or mental health issues (four items), fear of change in the marital relationship (four items), fear of changes in mood and its consequences on the child (three items), self - centered fear or fear of the changes in personal life of mother (three items). it is necessary to say that, first, the questionnaire was translated to persian by permission of its designer and in continue, it was returned to english by two english language experts. existence differences between english and persian versions were evaluated and these differences were decreased to minimum using from frequent review process. then, three experts of psychology field confirmed the content validity and cultural adaptation of pregnancy anxiety questionnaire. the questionnaire consists of 21 items, which covers the most common symptoms of anxiety. the phrase reflects one of the symptoms of anxiety that usually people who are clinically anxious or who are in a state of great anxiety experience it. the ratings are classified as not at all (0), mildly (1), moderately (2) and severely (3). if participant have a score of 07, no anxiety. if between 8 and 15 is mild anxiety, if between 16 and 25, the moderate anxiety and if between 26 and 63 indicate severe anxiety. its internal consistency coefficient is 0.92, test - retest reliability is within a week of 75/0, and the correlation of its items varies from 0.300.70. five types of content validity, concurrent, construct, diagnostic and a factor for this test measured which all of them indicative the effectiveness of this tool in measuring of anxiety in iranian population. this study was conducted regarding to permission from the health centers of kerman and principles of morality and ethics. all participants participated in the study consciously, and they were assured that the information gathered will be used only for the purpose of research. participants are also noted that are able to withdraw from the research process any time they want. to analyze the data, correlation matrix is used. to evaluate the reliability of questionnaires, cronbach 's alpha and split - half method and to evaluate validity and factor structure of questionnaires, confirmatory factor analysis and model parameters in the amos (analysis of moment structure) spss software version 20 (spss inc., in this study, 187 pregnant women participated in the confirmatory factor analysis which 170 women filled out the praq questionnaire. table 1 shows participants details (with a mean age of 29.5 5.7 years). demographic characteristics (n=170) in this study, to evaluate the validity of praq factor analysis and concurrent validity are used. a confirmatory factor analysis was conducted in order to verify the assumed factor structure in the measurement of anxiety in pregnant women. our purpose in doing this was a comparison between the initial structural matrix and the new structural one through which the content of each factor as well as the initial structural matrix was reconfirmed. table 2 presents loading factor on each of the questions that shows the correlation of each question with the desired area. as mentioned in table and figure 1 show, the factor loadings on subjected factors are significant. in factor analysis, the minimum load factor was considered as 0.30. factor structure short pregnancy related anxiety questionnaire the output model for the pregnancy related anxiety questionnaire factors in amos table 3 results shows the results of indicators model for examining the factor structure of the questionnaire through confirmatory factor analysis. according to the values of indicators, confirmatory factor analysis fit indexes for the 5-factor model in amos beck anxiety inventory (bai) was used to evaluate concurrent validity that its correlation coefficient with the subjected tools and its related factors have been reported in table 3. as can be seen, based on the pearson correlation coefficients, there is a positive correlation (p < 0.01) between the total score of the praq questionnaire and its factors and bai. internal correlation of factors with each other and with the total score shows the reliability of factor structure. as much the correlation of factors with the total score is higher, and then the internal validity of the questionnaire is increased. in this study, according to rendal. for tucker lewis index (tli) significant, tli above 0.9 (tli 0.95) and comparative fit index near to 1 are supposed as significant value [table 3 and figure 2 ]. the confirmatory five - factor model of pregnancy related anxiety questionnaire based on the result of second - order factor analysis, correlation or loadings of each factor with total scale are : child with physical or mental issues factor (0.74), fear of childbirth (0.70), fear of changes factor (0.75), change in marital relationships (0.72), and self - centered fears (0.77) ; that all of the factors were statistically significant (p < 0.01) [table 4 and figure 2 ]. correlation coefficients and internal consistency between the scales and original questionnaire to estimate the stability coefficient of instruments using from test - retest method, sixty participants were selected from statistical sample. they filled out the pregnancy anxiety scale and they were examined again with the same scale 14 days later. cronbach 's alpha is used to calculate reliability, split - half coefficient (split method) and test - retest method is used by pearson correlation. reliability of pregnancy related anxiety questionnaire as the above table shows the results of cronbach 's alpha coefficient of praq has a high internal consistency (cronbach 's alpha = 0.78). cronbach 's alpha for the scales are (self - centered fear = 0.69), (fear of child with health issues = 0.74), (fear of childbirth = 0.76), (fear of change = 0.72) and (fear of change in marital relationships = 0.70). retest reliability within a month for the praq is 0.74 and for its dimensions are varied from 0.65 to 0.72. in this study, to evaluate the validity of praq factor analysis and concurrent validity are used. a confirmatory factor analysis was conducted in order to verify the assumed factor structure in the measurement of anxiety in pregnant women. our purpose in doing this was a comparison between the initial structural matrix and the new structural one through which the content of each factor as well as the initial structural matrix was reconfirmed. table 2 presents loading factor on each of the questions that shows the correlation of each question with the desired area. as mentioned in table and figure 1 show, the factor loadings on subjected factors are significant. in factor analysis, factor structure short pregnancy related anxiety questionnaire the output model for the pregnancy related anxiety questionnaire factors in amos table 3 results shows the results of indicators model for examining the factor structure of the questionnaire through confirmatory factor analysis. according to the values of indicators, confirmatory factor analysis fit indexes for the 5-factor model in amos beck anxiety inventory (bai) was used to evaluate concurrent validity that its correlation coefficient with the subjected tools and its related factors have been reported in table 3. as can be seen, based on the pearson correlation coefficients, there is a positive correlation (p < 0.01) between the total score of the praq questionnaire and its factors and bai. internal correlation of factors with each other and with the total score shows the reliability of factor structure. as much the correlation of factors with the total score is higher, and then the internal validity of the questionnaire is increased. in this study, according to rendal. for tucker lewis index (tli) significant, tli above 0.9 (tli 0.95) and comparative fit index near to 1 are supposed as significant value [table 3 and figure 2 ]. the confirmatory five - factor model of pregnancy related anxiety questionnaire based on the result of second - order factor analysis, correlation or loadings of each factor with total scale are : child with physical or mental issues factor (0.74), fear of childbirth (0.70), fear of changes factor (0.75), change in marital relationships (0.72), and self - centered fears (0.77) ; that all of the factors were statistically significant (p < 0.01) [table 4 and figure 2 ]. a confirmatory factor analysis was conducted in order to verify the assumed factor structure in the measurement of anxiety in pregnant women. our purpose in doing this was a comparison between the initial structural matrix and the new structural one through which the content of each factor as well as the initial structural matrix was reconfirmed. table 2 presents loading factor on each of the questions that shows the correlation of each question with the desired area. as mentioned in table and figure 1 show, the factor loadings on subjected factors are significant. in factor analysis, the minimum load factor was considered as 0.30. factor structure short pregnancy related anxiety questionnaire the output model for the pregnancy related anxiety questionnaire factors in amos table 3 results shows the results of indicators model for examining the factor structure of the questionnaire through confirmatory factor analysis. according to the values of indicators, beck anxiety inventory (bai) was used to evaluate concurrent validity that its correlation coefficient with the subjected tools and its related factors have been reported in table 3. as can be seen, based on the pearson correlation coefficients, there is a positive correlation (p < 0.01) between the total score of the praq questionnaire and its factors and bai. internal correlation of factors with each other and with the total score shows the reliability of factor structure. as much the correlation of factors with the total score is higher, and then the internal validity of the questionnaire is increased. in this study, according to rendal. for tucker lewis index (tli) significant, tli above 0.9 (tli 0.95) and comparative fit index near to 1 are supposed as significant value [table 3 and figure 2 ]. the confirmatory five - factor model of pregnancy related anxiety questionnaire based on the result of second - order factor analysis, correlation or loadings of each factor with total scale are : child with physical or mental issues factor (0.74), fear of childbirth (0.70), fear of changes factor (0.75), change in marital relationships (0.72), and self - centered fears (0.77) ; that all of the factors were statistically significant (p < 0.01) [table 4 and figure 2 ]. to estimate the stability coefficient of instruments using from test - retest method, sixty participants were selected from statistical sample. they filled out the pregnancy anxiety scale and they were examined again with the same scale 14 days later. cronbach 's alpha is used to calculate reliability, split - half coefficient (split method) and test - retest method is used by pearson correlation. reliability of pregnancy related anxiety questionnaire as the above table shows the results of cronbach 's alpha coefficient of praq has a high internal consistency (cronbach 's alpha = 0.78). cronbach 's alpha for the scales are (self - centered fear = 0.69), (fear of child with health issues = 0.74), (fear of childbirth = 0.76), (fear of change = 0.72) and (fear of change in marital relationships = 0.70). retest reliability within a month for the praq is 0.74 and for its dimensions are varied from 0.65 to 0.72.. early diagnosis of pregnant women stress and also providing the necessary guidelines, can cause pregnant women to have a pleasant experience. so, a tool with high reliability and validity seems necessary to achieve this goal. the results showed that the psychometric properties of the persian version of vanden berg pregnancy anxiety questionnaire for use in iranian population and using it in clinical practice and research is acceptable. the results of the factor analysis of questionnaire showed that the results in iran to support the five - factor structure. this finding in total was consistent with the results of the factor analysis of the original developers of questionnaire, it can indicate that despite cultural differences in psychological variables in different societies, concerns about pregnancy related issues is public and global. furthermore, the concurrent validity of the questionnaire showed that this tool has a high correlation with its parallel questionnaire (bai), and it seems that they measure similar structure. the analysis focused on reliability of questionnaire that examined two aspects of test - retest reliability and internal consistency of questionnaire had led to good results. in terms of reliability and stability of the test that examined through test - retest coefficient, the results showed that the questionnaire is valid and its result can be trusted in multiple implementations. also, high coefficient rates of internal consistency show that questionnaire has a consistent structure ; which is consistent with the results of huizink. and babanazari and kafi in this regard. from obtained correlations, relationship during pregnancy oneself and the partner concern for subscale which is equal to r = 0.77. this indicate important and vital role of marital relations and social support in experienced anxiety level among pregnant women. perhaps it can be said when women experience the pregnancy, they are exposed to excitement and worried about how to relate with others, particularly their wives. the lowest correlation was related to the subscale of fear for delivery that is equal to r = 0.70. so, it may be said that the spouse 's support, having enough information about the process of delivery and the positive acceptance of delivery have been effective in reducing the subscale of fear for delivery. generally speaking, due to the obtained correlations the questionnaire is of high factor validity. the high coefficients of internal similarity also indicated that the questionnaire has a coherent structure ; this shows that almost all dimensions of pregnancy anxiety have a near relationship with each other so that any change in one of them affects the other dimensions as well as the total amount of pregnancy anxiety. so, regarding to results of present study, it can be suggested that assessment of anxiety and response of pregnant women to the anxiety during pregnancy, along with monitoring and evaluation of health care is very beneficial. special educational and therapeutic approaches to identify stressors and training coping strategies to them can be useful in pregnant women. | background : pregnancy is an acute period in the lifetime of women, during which numerous excitatory physical and social changes occur. the purpose of this study is confirmatory factor analysis of pregnancy related anxiety questionnaire (praq) that is designed in iranian pregnant women population.methods:a total of 170 pregnant women in health centers of kerman city were chosen through random sampling method and completed praq questionnaire and beck anxiety inventory (bai). in this study, confirmatory factor analysis and concurrent validity are used to evaluate the validity of models ; and to test - retest and cronbach alpha were used for evaluating external and internal reliability in spss-19 and the amos software to evaluate reliability of models.results:confirmatory factor analysis gave an acceptable value for the latent praq in the question scale and 5 micro - scale level. furthermore, significant correlation between the components and the overall scale of the praq questionnaire with the bai confirmed concurrent validity of questionnaire. the reliability of questionnaire is confirmed based on cronbach 's alpha coefficient value of 0.78 that calculated 0.690.76 for the five - factors. a month later, reliability coefficient amplitude of test - retest on forty pregnant women was between 0.65 and 0.72 which shows the reliability of praq over time.conclusions:the short form of anxiety during pregnancy questionnaire has the essential psychometric properties. in this study, five - factors extracted in the praq were adapted with the factors extracted from the original version. this study introduces an instrument that can be benefit in measuring anxiety and concerns of women during pregnancy. |
we analyzed deidentified data obtained from two identical double - blind, placebo controlled, multicenter, 52-week clinical diabetic neuropathy trials of two alc doses (1.5 and 3.0 g / day), conducted and supported by sigma - tau research (13). eligibility criteria included a1c > 5.9%, age between 18 and 70 years, diabetes duration of > 1 year, and diabetic neuropathy as defined by the san antonio conference (13,14). because the data analyzed in this report were deidentified, the university of michigan institutional review board concluded that no human subjects were involved in this project. blood samples were collected at baseline, and a1c, triglycerides, cholesterol, albumin, and hematocrit were recorded. clinical symptoms, including pain, numbness, paresthesia, muscle weakness, postural dizziness, problems with sweating, gastrointestinal problems, and sexual dysfunction were recorded at baseline and at 52 weeks and scored on a scale of 0 (no symptoms) to 3 (incapacitating symptoms). in addition, the participants ' own assessment of their most troublesome symptom at baseline was recorded. vibration perception thresholds of the index finger and great toe were assessed bilaterally in triplicate (15) at baseline and at 52 weeks using a vibratron (physitemp instruments, clifton, nj) (16). these measures were completed during a 4-week run - in period prior to randomization (13). electrophysiological measurements included bilateral sural nerve amplitude and conduction velocity, peroneal amplitude and ncv on the dominant side, and median motor and sensory amplitudes and ncvs on the nondominant side. a biopsy was taken from one ankle at the beginning of the study, and a second biopsy was taken from the opposite ankle after 52 weeks. morphometric parameters measured included total myelinated fiber number, fascicular area, mean fiber size, mfd, fiber occupancy, and axon - to - myelin ratio (17). mfd (fibers / mm) in the largest fascicle was determined in semi - thin, paraphenylene diamine the primary outcome measure of the present study was the difference between the initial and 52-week sural nerve mfd. participants without both a primary and secondary sural nerve biopsy or blood chemistry data were excluded from our current data analyses. a total of 427 participants were included in the primary data analysis of the present study (fig. this flow diagram represents the decision process for including or excluding participants at each stage of the analysis. the analysis of these data were divided into two stages. in the primary data analysis however, a simple correlation assumes a consistent, linear progression of diabetic neuropathy, which may not be the case. in the secondary analysis, we balanced groups based on initial diabetic neuropathy status and tested the significant variables correlated with divergent outcomes. in the cohort of 427 participants, 99.5% of clinical symptoms, vibration perception, and electrophysiological measures were available. the small number of missing values were imputed by the k - nearest - neighbor technique (18). the o'brien rank - sum (19) of each patient was calculated at baseline using the values for ncv, amplitude, vibration perception, and the clinical symptom score. continuous variables (e.g., a1c) from the initial time point were correlated with change in sural nerve mfd using the spearman nonparametric method, and a significance value of the correlation was calculated. categorical variables (treatment, sex, diabetes type, most bothersome symptom at baseline, and insulin treatment) were tested for significant differences in sural nerve mfd by a mann - whitney test (20) (two categories) or kruskal - wallis (21) (more than two categories). to identify factors driving diabetic neuropathy progression, two groups of participants with a similar sural nerve mfd and diabetic neuropathy at baseline, but differing degrees of sural nerve mfd at 52 weeks, were defined. a perl program evaluated the change in sural nerve mfd and identified participants with an absolute loss of 500 fibers / mm over 52 weeks as having rapidly progressing diabetic neuropathy. participants with a loss of 100 fibers / mm or less over 52 weeks were identified as having nonprogressing diabetic neuropathy. participants with a 52-week sural nerve mfd > 1,000/mm greater than baseline were excluded. diabetic neuropathy was also estimated in the participants using the o'brien rank - sum score, a nonparametric combination of neuropathy measures. the o'brien rank - sum is composed of a linear combination of the variables listed in table 1, excluding the most bothersome symptom at baseline, demographic data and drug treatment information. these 16 variables describe the ncv in three nerves, along with the corresponding nerve amplitudes, vibration perception thresholds in the fingers and toes, and the total clinical symptom score. the program matched each participant with rapidly progressing diabetic neuropathy with a nonprogressing diabetic neuropathy participant with similar sural nerve mfd and o'brien score at baseline. the maximum difference in mfd between matching participants was set as 1,000 fibers / mm, and the maximum difference in the o'brien was set as 1,000. this o'brien threshold required that at least 3 of 16 measures of neuropathy differ by a large degree between the participants at baseline. when multiple participants were under the similarity thresholds defined, the samples with the most similar initial fiber densities were matched. significance of correlation or association between possible risk factors and mfd loss based on these unbiased criteria, type 1 diabetic and insulin - treated participants were overrepresented in the nonprogressing group. this enrichment was not statistically significant (diabetes type p = 0.19, insulin treatment p = 0.11). however, because insulin is known to be neuroprotective (22,23), the two groups were then explicitly balanced for diabetes type and insulin treatment to prevent a potential confounding effect. after balancing, 104 rapidly progressing and 104 nonprogressing participants were identified for further analysis, for a total n = 208 (fig. variables that were significantly correlated or associated with decreased sural nerve mfd in the primary analysis were advanced to the secondary analysis. the variables were tested for significant differences between the rapidly progressing and nonprogressing groups using the mann - whitney nonparametric test (20). machine - learning analysis was performed according to the american diabetes association consensus statement on computer modeling of diabetes (24). 1) were used as a training set for seven machine - learning techniques (nave bayes, k - nearest - neighbor, support vector machine, linear regression, random forest, classification tree and cn2 rule based) (25). the accuracy, sensitivity, and specificity of each model was estimated using leave - one - out cross validation (26). to ensure that overfitting did not take place, the highest - performing and most sensitive models were then tested on an independent dataset from the same population. the dataset was taken from 56 participants who fit the criteria of rapidly progressing (28 participants) or nonprogressing (28 participants) but who were not included in the secondary analysis cohort. a classification confidence threshold was chosen using this independent set, creating a third category of unclassified participants that the model lacked confidence to classify. all analyses were performed by one investigator (t.d.w.) using graphpad prism 5.01 for windows (san diego, ca) and orange (ljubljana, slovenia) (25). blood samples were collected at baseline, and a1c, triglycerides, cholesterol, albumin, and hematocrit were recorded. clinical symptoms, including pain, numbness, paresthesia, muscle weakness, postural dizziness, problems with sweating, gastrointestinal problems, and sexual dysfunction were recorded at baseline and at 52 weeks and scored on a scale of 0 (no symptoms) to 3 (incapacitating symptoms). in addition, the participants ' own assessment of their most troublesome symptom at baseline was recorded. vibration perception thresholds of the index finger and great toe were assessed bilaterally in triplicate (15) at baseline and at 52 weeks using a vibratron (physitemp instruments, clifton, nj) (16). these measures were completed during a 4-week run - in period prior to randomization (13). electrophysiological measurements included bilateral sural nerve amplitude and conduction velocity, peroneal amplitude and ncv on the dominant side, and median motor and sensory amplitudes and ncvs on the nondominant side. a biopsy was taken from one ankle at the beginning of the study, and a second biopsy was taken from the opposite ankle after 52 weeks. morphometric parameters measured included total myelinated fiber number, fascicular area, mean fiber size, mfd, fiber occupancy, and axon - to - myelin ratio (17). mfd (fibers / mm) in the largest fascicle was determined in semi - thin, paraphenylene diamine stained sections. the primary outcome measure of the present study was the difference between the initial and 52-week sural nerve mfd. participants without both a primary and secondary sural nerve biopsy or blood chemistry data were excluded from our current data analyses. a total of 427 participants were included in the primary data analysis of the present study (fig. this flow diagram represents the decision process for including or excluding participants at each stage of the analysis. the analysis of these data were divided into two stages. in the primary data analysis, variables were tested for a simple correlation with the rate of mfd loss. however, a simple correlation assumes a consistent, linear progression of diabetic neuropathy, which may not be the case. in the secondary analysis, we balanced groups based on initial diabetic neuropathy status and tested the significant variables correlated with divergent outcomes. in the cohort of 427 participants, 99.5% of clinical symptoms, vibration perception, and electrophysiological measures were available. the small number of missing values were imputed by the k - nearest - neighbor technique (18). the o'brien rank - sum (19) of each patient was calculated at baseline using the values for ncv, amplitude, vibration perception, and the clinical symptom score. continuous variables (e.g., a1c) from the initial time point were correlated with change in sural nerve mfd using the spearman nonparametric method, and a significance value of the correlation was calculated. categorical variables (treatment, sex, diabetes type, most bothersome symptom at baseline, and insulin treatment) were tested for significant differences in sural nerve mfd by a mann - whitney test (20) (two categories) or kruskal - wallis (21) (more than two categories). to identify factors driving diabetic neuropathy progression, two groups of participants with a similar sural nerve mfd and diabetic neuropathy at baseline, but differing degrees of sural nerve mfd at 52 weeks, were defined. a perl program evaluated the change in sural nerve mfd and identified participants with an absolute loss of 500 fibers / mm over 52 weeks as having rapidly progressing diabetic neuropathy. participants with a loss of 100 fibers / mm or less over 52 weeks were identified as having nonprogressing diabetic neuropathy. participants with a 52-week sural nerve mfd > 1,000/mm greater than baseline were excluded. diabetic neuropathy was also estimated in the participants using the o'brien rank - sum score, a nonparametric combination of neuropathy measures. the o'brien rank - sum is composed of a linear combination of the variables listed in table 1, excluding the most bothersome symptom at baseline, demographic data and drug treatment information. these 16 variables describe the ncv in three nerves, along with the corresponding nerve amplitudes, vibration perception thresholds in the fingers and toes, and the total clinical symptom score. the program matched each participant with rapidly progressing diabetic neuropathy with a nonprogressing diabetic neuropathy participant with similar sural nerve mfd and o'brien score at baseline. the maximum difference in mfd between matching participants was set as 1,000 fibers / mm, and the maximum difference in the o'brien was set as 1,000. this o'brien threshold required that at least 3 of 16 measures of neuropathy differ by a large degree between the participants at baseline. when multiple participants were under the similarity thresholds defined, the samples with the most similar initial fiber densities were matched. significance of correlation or association between possible risk factors and mfd loss based on these unbiased criteria, type 1 diabetic and insulin - treated participants were overrepresented in the nonprogressing group. this enrichment was not statistically significant (diabetes type p = 0.19, insulin treatment p = 0.11). however, because insulin is known to be neuroprotective (22,23), the two groups were then explicitly balanced for diabetes type and insulin treatment to prevent a potential confounding effect. after balancing, 104 rapidly progressing and 104 nonprogressing participants were identified for further analysis, for a total n = 208 (fig. variables that were significantly correlated or associated with decreased sural nerve mfd in the primary analysis were advanced to the secondary analysis. the variables were tested for significant differences between the rapidly progressing and nonprogressing groups using the mann - whitney nonparametric test (20). machine - learning analysis was performed according to the american diabetes association consensus statement on computer modeling of diabetes (24). 1) were used as a training set for seven machine - learning techniques (nave bayes, k - nearest - neighbor, support vector machine, linear regression, random forest, classification tree and cn2 rule based) (25). the accuracy, sensitivity, and specificity of each model was estimated using leave - one - out cross validation (26). to ensure that overfitting did not take place, the highest - performing and most sensitive models were then tested on an independent dataset from the same population. the dataset was taken from 56 participants who fit the criteria of rapidly progressing (28 participants) or nonprogressing (28 participants) but who were not included in the secondary analysis cohort. a classification confidence threshold was chosen using this independent set, creating a third category of unclassified participants that the model lacked confidence to classify. all analyses were performed by one investigator (t.d.w.) using graphpad prism 5.01 for windows (san diego, ca) and orange (ljubljana, slovenia) (25). the dataset included 748 participants in the alc clinical trials, but blood chemistries, initial sural nerve mfd, and 52-week sural nerve mfd were only available from 427 participants (fig. 1). there were no significant demographic, treatment, or metabolic differences between the excluded cohort (321 participants) and those with the necessary data for the primary analysis (427 participants). alc treatment did not affect sural nerve mfd loss (p = 0.87) ; therefore, data from alc - treated participants were pooled with placebo - treated participants in tests related to outcome. the participants included in the primary analysis were primarily male (67%), the majority had type 2 diabetes (78%), and the majority (59%) were treated with insulin (table 2). patient characteristics at baseline data are means sd for continuous variables, unless otherwise indicated. in the primary analysis, the baseline values of the patient symptoms, functional neurological exams, blood chemistries, and demographics were tested for association with change of sural nerve mfd between the initial and 52-week sural nerve biopsies (table 1). five baseline variables were significantly correlated with a loss of sural nerve mfd over the 52 weeks of the study. they were dominant peroneal motor ncv (r = 0.13, p = 0.005), nondominant median motor ncv (r = 0.11, p = 0.02), sural sensory ncv (r = 0.10, p = 0.05), a1c (r = 0.12, p = 0.02), and triglyceride level (r = 0.11, p = 0.02). the primary analysis was potentially confounded by the effect of initial sural nerve mfd on mfd change. there was a positive correlation between initial sural nerve mfd and the size of the decrease of sural nerve mfd over 52 weeks (r = 0.14). to account for this confounding factor, the variables with a nominal p value 25% over the course of the study) (p 56%) were assigned a prediction, overall accuracy increased to 63% (fig. a : important variables in the model are triglycerides, cholesterol, and clinical symptom score. b : the model assigns a probability of progressing to each participant. when the probability is > 56% or 500/mm) exhibited elevated triglycerides and greater deficits in peroneal ncv at baseline than the nonprogressing participants. the diabetes control and complications trial and its continuation, the epidemiology of diabetes and its complications (7,8), established hyperglycemia as the primary cause of diabetes complications. consistent with these studies, we initially found that elevated a1c correlated with loss of sural nerve mfd. however, when directly comparing participants with a similar degree of baseline diabetic neuropathy (i.e., similar sural nerve mfd), a1c did not differ between rapidly progressing and nonprogressing participants ; it was not a specific marker for diabetic neuropathy progression in this study. this suggests that other factors may underlie variation in the progression of diabetic neuropathy. in the last decade, abnormalities in insulin signaling, caused by insulin deficiency, as in type 1 diabetes, or insulin resistance, as in type 2 diabetes, have been invoked as additional pathogenetic components in diabetic neuropathy. this is underscored by the data from the longitudinal rochester study, in which type 1 diabetes was found to be a major risk factor for severity of diabetic neuropathy (33). experimental studies also suggest that insulin deficiency is a major contributor to diabetic neuropathy, because of the prominent neurotrophic effects of insulin (22,23). for this reason, the number of participants with type 1 diabetes and those treated with insulin was balanced when defining the progressing and nonprogressing groups. in contrast to a1c, baseline serum triglycerides were significantly elevated in the rapidly progressing compared with the nonprogressing groups. when measured in serum, free triglycerides are a surrogate marker of endogenous lipid transport pathway activity. free triglycerides are released from vldl, leading to their conversion to ldl (34). our findings support the emerging idea that dyslipidemia contributes to the development of diabetic neuropathy. this hypothesis may explain the earlier incidence of diabetic neuropathy in individuals with type 2 diabetes compared with type 1 diabetes. dyslipidemia develops later in the course of type 1 diabetes, and the delayed development of an abnormal lipid profile coincides with the delayed onset and progression of diabetic neuropathy (35,36). in this study, triglycerides were significantly elevated in those participants exhibiting diabetic neuropathy progression independent of diabetes type or insulin treatment. these data confirm reports from several large - scale trials of participants with type 2 diabetes that also point to early dyslipidemia as a major independent risk factor for the progression of diabetic neuropathy (37,38). correction of dyslipidemia with statins has an ameliorative affect on the development and progression of diabetic neuropathy (39,40). this finding is more indicative of concordant damage to peroneal and sural nerve function than of a specific mechanism for that damage. multiple studies (13,2729) agree with our findings and report a correlation between mfd and ncv. however, in the current study, decreased peroneal motor ncv was detectable prior to the loss of a significant amount of sural nerve sensory fibers, as assessed by sural nerve mfd. this most likely reflects metabolic nerve dysfunction in the peroneal nerve rather than earlier nerve fiber loss and is consistent with experimental models of diabetic neuropathy (31). while ncv and fiber density are closely related, factors other than fiber density, such as acute metabolic disruption (41,42), affect ncv without resulting in nerve fiber loss. modeling done on this dataset was motivated by the desire to identify noninvasive predictors of the loss of mfd. the american diabetes association has issued guidelines (24) for the use of modeling and machine learning that specify that validation of a model should be done in three ways : the model should first be validated on the initial dataset, then the data should be validated on an independent set from the same experiment, and finally an independent set from a different experiment from which the same parameters were collected. in this study, only the first two parts of the recommended validation could be completed due to the lack of additional published datasets with serial sural nerve biopsies. we found that a model for predicting the progression of diabetic neuropathy using the american diabetes association guidelines for modeling and machine learning performed with 63% overall classification accuracy. the three most influential measures in this model for predicting patient outcome were triglycerides, cholesterol, and the clinical symptom score. interestingly, despite being significantly different between progressing and nonprogressing patients, ncv was not a major contributor to this predictive model. this may be because the difference between the two groups, while significant, was 5%. the baysean model used may not be sensitive enough to include this subtle change. a specialized learning algorithm or a measure with greater dynamic range may allow us to include this important predictor in future modeling. future informatics studies on diabetic neuropathy hold promise and are being proposed on the diabetes control and complications trial / epidemiology of diabetes and its complications cohort. in summary, both elevated triglycerides and reduced peroneal motor ncv are predictive of a dramatic decrease in sural nerve mfd over a 1-year period. the correlation between triglycerides and diabetic neuropathy progression suggests that hyperglycemia and aberrant glucose metabolism are not the only factors contributing to nerve damage. the exact mechanism underlying triglyceride mediated injury has yet to be elucidated but may dysregulated lipid metabolism within motor and/or sensory neurons. these same factors, along with acute metabolic flux, may explain the correlation between reduced peroneal motor ncv and rapidly progressing diabetic neuropathy. we have also demonstrated that given an adequate dataset, predictive models of diabetic neuropathy progression may be trained using standard machine learning techniques. | objectiveto evaluate mechanisms underlying diabetic neuropathy progression using indexes of sural nerve morphometry obtained from two identical randomized, placebo - controlled clinical trials.research design and methodssural nerve myelinated fiber density (mfd), nerve conduction velocities (ncvs), vibration perception thresholds, clinical symptom scores, and a visual analog scale for pain were analyzed in participants with diabetic neuropathy. a loss of 500 fibers / mm2 in sural nerve mfd over 52 weeks was defined as progressing diabetic neuropathy, and a mfd loss of 100 fibers / mm2 during the same time interval as nonprogressing diabetic neuropathy. the progressing and nonprogressing cohorts were matched for baseline characteristics using an o'brien rank - sum and baseline mfd.resultsat 52 weeks, the progressing cohort demonstrated a 25% decrease (p < 0.0001) from baseline in mfd, while the nonprogressing cohort remained unchanged. mfd was not affected by active drug treatment (p = 0.87), diabetes duration (p = 0.48), age (p = 0.11), or bmi (p = 0.30). among all variables tested, elevated triglycerides and decreased peroneal motor ncv at baseline significantly correlated with loss of mfd at 52 weeks (p = 0.04).conclusionsin this cohort of participants with mild to moderate diabetic neuropathy, elevated triglycerides correlated with mfd loss independent of disease duration, age, diabetes control, or other variables. these data support the evolving concept that hyperlipidemia is instrumental in the progression of diabetic neuropathy. |
odontogenic tumor has been a topic of considerable interest to oral pathologists who have studied and catalogued them for decades. this constitutes a group of heterogeneous lesions that range from hamartomatous or non - neoplastic tissue proliferation to malignant neoplasm with metastatic capacity. a marked geographic variation is apparent in the relative incidences of various odontogenic tumors, particularly ameloblastoma. ameloblastoma was the most common tumor in studies done on chinese, japanese, and african populations, while in american and canadian populations, the most frequent tumor was odontome. on the basis of world health organization (who) classification, a retrospective study was undertaken of ameloblastoma in the central region of india, nagpur city (vidharbha region). this would enhance the understanding of prevalence and occurrence of this unique tumor limited to the odontogenic apparatus, thereby enabling us to treat them effectively. one hundred ninety - nine cases of odontogenic tumors were retrieved from files of department of oral pathology and microbiology, government dental college and hospitals, nagpur, from 1977 to 2003. a retrospective study of 91 cases of ameloblastoma was done considering parameters such as age, sex, location, duration, radiographic findings, and histopathological appearances and these were compared with other reported studies. the data were analyzed with unpaired t - test, chi - square goodness - of - fit test, analysis of variance (anova) tests for statistical significance. out of 7,700 surgical specimens received in the department, 199 were diagnosed as odontogenic tumors., 91 cases were diagnosed as ameloblastoma, thus accounting for 45.7% of odontogenic tumors and 1.18% of surgical specimens [figure 1 ]. percentage of occurrence of various odontogenic tumors the age at the time of presentation was in the range of 10 - 60 years, with a median at 30 years. out of the 91 patients, 49 (53.8%) were male and 42 (46.2%) were female. the male : female ratio was 1.2:1 [figure 3 ]. when the age was analyzed separately for male and female patients, the mean age of males and females was 31 and 34.2 years, respectively [figure 4 ]. two - tailed, unpaired student t - test was applied and a value of p > 0.2543 was found. hence, the difference in the average age of males and females was non - significant. age distribution of ameloblastoma sex distribution of ameloblastoma age distribution of ameloblastoma in male and female patients out of 91 patients, 55 (60.4%) reported with asymptomatic hard swelling. pain followed by swelling (n = 32, 35.2%), ulceration (n = 9, 9.9%), mobility of teeth (n = 10, 11%), displaced teeth (n = 4, 4.4%), and paresthesia (n = 5, 5.5%). the mean, median, and mode duration of symptoms are 16.4, 6, and 12 months, respectively [table 1 ]. duration of symptoms amoug ameloblastoma the clinical history was non - contributory in seven cases. about 51.6% of patients presented to the hospital within 11 months of symptoms and 22% reported within 2 years [figure 5 ]. duration of symptoms before clinical presentation of ameloblastoma the site distribution of various ameloblastomas among males and females is listed in table 2. the ratio of ameloblastoma occurring on the right side as compared to the left was 0.83:1. the highest incidence of ameloblastoma (46%) was seen in the posterior segment and vertical ramus of the mandible [figure 6 ]. site distribution of ameloblastoma in males and females site distribution of ameloblastoma in male and female patients the various histological subtypes of ameloblastoma are listed in table 3 and figure 7. unicystic ameloblastoma was the most common type with an incidence of 34.1% (n = 31), followed by plexiform ameloblastoma (22%, n = 20) and follicular ameloblastoma (19.8%, n = 18). the sex distribution [table 4 ], age distribution [table 5 and figure 8 ], and site distrib ution [table 6 ] were also assessed. histological variants of ameloblastoma distribution of various histological subtypes of ameloblastoma sex distribution of various ameloblastoma age distribution of various ameloblastoma age distribution of different variants of ameloblastoma site distribution of various of ameloblastoma radiographs of 85 cases were evaluated [table 7 ]. unilocular appearance was observed in 29 (34.1%) cases, while multilocular appearances were observed in 56 (65.9%) cases. other radiographic findings included embedded tooth (n = 7), root resorption (n = 12), missing tooth (n = 5), and egg shell crackling (n = 3). the most common embedded tooth was the third molar. the average age of 25.3 years was seen in the unilocular variety as compared to multilocular appearances at an average age of 34.8 years. radiographical assessment of ameloblastoma the management of ameloblastoma could be traced for 71 cases and is summarized in table 8, as 20 patients were not willing for treatment. curettage and enucleation was performed in 21 cases, marginal resection in 7 cases, while segmental resection was done in 43 cases. out of 43 cases, 23 management of ameloblastoma follow - up was done in 46 cases and recurrence was noted in 9 cases, accounting for 14.1% cases [figure 9 ]. recurrence developed at average time interval of 7.4 years, median was 7 years, and the maximum was 15 years. on recurrence, follicular variant was noted in four cases and unicystic ameloblastoma was found in five cases. out of these, two were plexiform unicystic type, one of follicular unicystic type, and one of unicystic ameloblastoma with granular cell metaplasia. the most frequent tumor in this review was ameloblastoma, with an incidence of 45.7% comparable to that found by lu. this finding contrasts with rates in series involving american and canadian population in whom ameloblastoma accounted for 12.2% and 14.8%, respectively. the average age of the patient at the time of initial diagnosis was 32.5 years, which is similar to that in the chinese population in whom tumors were presented at the mean age of 32.4 years. reichart and philipsen in their biological profile of 3,677 cases found the average age of 35.9 years at the time of initial diagnosis. female patients reporting with tumor had a mean age of 34.2 years, which was higher than that of male patients with a mean age of 31 years. in our series, 53.8% of the patients were was men and 46.2% were women comparable to reichart and philipsen study in which 53% were male and 47% were female. there is striking predilection for the mandible though maxillary lesion varies considerably among the reports. in the present series, a single case of tumor occurred in the maxillary region, a figure not comparable to corresponding data of asian / african countries (2 - 8%) and american series (16 - 22%). the predilection of ameloblastoma for the posterior segment is 25.3% and posterior segment and vertical ramus is 40.6%, which is consistent with reports elsewhere. our review revealed a multilocular appearance (60.4%) and unilocular appearance (31.9%), which was higher than that of reichart and philipsen 's study, in which multilocular appearance was noted in 49% and unilocular in 51% cases. statistically significant results were obtained when average age of unilocular and multilocular appearances was analyzed, indicating that unilocular occurred in younger age group as compared to multilocular ameloblastoma. in our review, unicystic ameloblastoma was the most frequently encountered histological subtype (34%) followed by follicular (19.8%), plexiform (22%), granular cell ameloblastoma (9.9%), and acanthomatous type (6.6%). our results are different from reichart and philipsen 's study (1995), which showed follicular (33.9%) plexiform (30.2%) acanthomatous (11.3%) and unicystic (6%) types. when different variants of ameloblastoma were analyzed separately for males and females, the results were non - significant in all variants of ameloblastoma. hence, no particular sex distribution was seen in different variants of ameloblastoma. when average ages of different variants of ameloblastoma were analyzed statistically using the anova test, it was found that plexiform ameloblastoma occurred in younger age group as compared to follicular, acanthomatous, and granular cell ameloblastoma. it is evident from our review that unicystic and plexiform variants occurred at a younger age and more frequently involved the body and ramus area of the mandible. in contrast, the acanthomatous type occurred in older patients and involved the anterior segment of jaws. granular cell type and desmoplastic type occurred in older patients and were seen involving both anterior and posterior segments of mandible. among the therapy modalities, follow - up was done in 46 patients, with a recurrence of 14.1%, which is less than the reichart and philipsen 's study (22.6%). on recurrence, the follicular variant (four cases), unicystic type (five cases), and granular type (one case) are not consistent with reichart and philipsen 's study, which reported follicular (29.5%), plexiform (16.7%), and unicystic types (13.7%). the decrease in recurrence rate in the last few decades could be attributed to early diagnosis and improved therapeutic approach. there is only one excellent review of 73 cases of ameloblastoma by krishnapillai r and angadi pv. our review adds valuable information on the incidence of ameloblastoma in indian population to the existing limited literature. our study was performed over a period of 26 years (1977 - 2003). out of 7,700 cases received in the department, odontogenic tumors comprise 2.5%. this is lower than the reported incidence of 2.97% by osterne. it must be stressed that our knowledge of biological behavior of ameloblastoma is still insufficient for drawing a definite conclusion. many more detailed reports including long - term follow - ups are needed for proper assessment of treatment modalities. | aim : to assess the cases of ameloblastoma retrospectively for various parameters.materials and methods : ninety - one previously reported cases of ameloblastoma from government dental college and hospitals, nagpur, were included in this study. data were collected considering parameters such as age, sex, symptoms, radiographic, histopathology, treatment modalities, and recurrence. our findings were also compared with world literature.results:the average age was 32.5 years. mostly men complained of an asymptomatic swelling and duration of less than one year. posterior segment and vertical ramus of mandible was the frequently involved site. radiographically, multilocular appearance was noted more than 60% of the cases. plexiform and unicystic ameloblastoma occurred frequently. surgery was the treatment of choice in this review. recurrence was noted in more than 10% of the cases.conclusion:there are variations in our review in comparison to reported literature. |
during winter 2014 and into the early spring 2015, we became aware of an unusual number of reported vibrio infections in northern europe. colleagues at the european centre for disease control relayed the initial information to the centre for environment, fisheries and aquaculture science (weymouth, uk) and the university of bath (bath, uk). the information suggested that an unprecedented number of vibrio infections had been observed in sweden and finland during summer 2014 and that many cases were reported in high - latitude coastal counties. to scrutinize cases of infection we initially contacted the public health agency of sweden (stockholm, sweden) and the national institute for health and welfare (helsinki, finland), as well as other northern europe reference laboratories, in december 2014. although vibriosis is not regionally notifiable in europe, finland and sweden maintain national databases of vibrio infections. in finland, v. cholerae is a notifiable infection, and isolates from persons with suspected infections are submitted to the reference laboratory for confirmation, serotyping, and pcr testing for the cholera toxin gene (ctx). also, other vibrio species (e.g., v. vulnificus, v. parahaemolyticus) may be sent to the reference laboratory for subsequent species - level confirmation. in sweden, diarrhea with ctx - producing v. cholera o1 or o139 is a notifiable disease, as is infection with other vibrio species, including v. cholerae not producing ctx that causes wound infections, septicemia, enteritis, and otitis. isolates of v. cholerae are sent to the public health agency of sweden for serotyping and confirmation of virulence factors, such as ctx, using appropriate molecular methods, such as pcr. for cases identified in 2014, the geographic location of each reported infection was established (e.g., town or city where the patient was treated). where possible, information relevant to disease transmission, such as possible water - associated activities, also was gathered ; however, for many cases, this information was not available. basic epidemiologic data on each case, including patient sex and age, was subsequently collated, as was the site of bacterial isolation (e.g., wound, ear, blood). the date the case was reported to regional authorities was determined, and for a subset of cases, data on the onset of reported symptoms also were established. to assess recent trends regarding infections, we collated vibrio cases identified in finland and sweden from 2005 onward and omitted from analysis cases we suspected of being foreign - acquired. to assess the possible role of extreme weather events on the emergence and dynamics of vibrio disease in finland and sweden, we analyzed the epidemiologic data alongside long - term sea surface temperature (sst) records (hadisst [hadley centre sea ice and sea surface temperature dataset ] and ersst [extended reconstructed sea surface temperature dataset, v3b from the us national oceanic and atmospheric administration (noaa) ]). we used satellite - derived data to scrutinize temperature conditions and changes in the baltic sea area using noaa s optimum interpolation v2 daily sst analysis dataset that integrates satellite sst data retrievals. noaa data (baseline period of 30 years [19712000 ]) was used to determine anomalies from this dataset. we also scrutinized daily sst and sst anomaly retrieval data from 6 fixed positions in the baltic sea area, which included the transitional waters between southern sweden and denmark, the southeastern and mideastern baltic coasts of sweden, and bay of bothnia (northern baltic) and southern coast of finland. to assess the significance of climatologic data from summer 2014, we also used long - term oceanographic datasets to analyze sst. in situ sst was provided by the finnish meteorological institute and was downloaded on november 14, 2014. we also used instrumental measurements of sst in coastal areas in the baltic sea area. we removed short - term fluctuations from the buoy data by applying a 1-hour wide median filter to the original dataset. statistical tests used to infer the relationship between maximum sst and annual vibrio case occurrence were investigated by using a generalized linear model that assumed a quasi - poisson error distribution (log link function) in r version 3.1.3 (http://www.r-project.org). we analyzed daily long - term sst and anomaly data (19812015) using a welch t test (which enables analysis of the unbalanced size of the 2 datasets). a total of 89 vibrio infections were reported in sweden and finland during the summer and autumn 2014, the largest yearly total number of cases, to our knowledge, identified in these countries. numerous cases were reported at extreme subarctic regions, and as far north as > 65n, 18c for several weeks beginning in mid - july and ceasing in mid - august (figure 3). sst s reported in the gulf of bothnia at the end of july were the most extreme reported during 19812016, exceeding 21.7c on july 29, 2014, and with several days of temperatures > 20c. the observed sst anomaly during this period was also the largest ever seen in this dataset, encompassing almost 13,000 data points, with an anomaly of 9.79c on july 29, 2014. the number of infections coupled with the extreme sst anomaly, particularly in northern latitude areas, is particularly noteworthy. sst anomaly data for coastal areas of sweden and finland. a) maximum sst anomalies during july and august 2014. the anomalies were substantially high throughout the region but especially in the northern baltic sea area. a statistical analysis between maximum sst and annual vibrio cases using a generalized linear model showed that maximum sst explained a significant amount of the variability in cases (as determined by a significant reduction in the residual deviance from 120.55 to 42.16). the model predicted that, as the maximum sst increases, the number of annual number of cases also will increase significantly (= 0.33002, se = 0.08045, t = 4.102, p = 0.00343). domestically acquired vibrio infections are rare in northern europe, and the spike in recorded cases of vibriosis reported in this region is particularly noteworthy. the cases in 2014 are the largest yearly total of reported vibrio infections in sweden and finland, more than double the number of reported cases than in other recent years (figure 4). in sweden, 2014 was the warmest year on record since recordkeeping began in 1860 ; in finland, 2014 was the second - warmest year on record (10,11). across finland, 50 days of hot summer weather (temperatures > 25c) were recorded during may august, which is 14 days more than the long - term average (10). the large number of reported infections corresponded closely with an intense and northerly sst anomaly, suggesting that these unusual oceanographic and climatic conditions drove this episode of waterborne disease. a subsequent quantitative and statistical analysis of sst data from this region revealed 3 further observations : 1) the peak ssts in late july 2014 were the most intense observed in the bay of bothnia ; 2) the anomaly is the most intense in almost 35 years of climate data (19812015) ; and 3) the likelihood of such an event occurring based on recent climate data (19812015) is highly unlikely the 2014 maximum observed temperature was significantly higher than the maximum expected based on the data for other years, and based on the distribution of maximum temperatures observed, a temperature this much higher than the mean would be expected only in 0.78% of years (once every 128 years). epidemiologic data were gathered from public health agencies in sweden and finland (see materials and methods). vibrio species such as v. cholerae grow preferentially in low - salinity warm water, and recreational exposure to water, which appears to have been responsible for a sizeable proportion of these reported infections, also increases substantially during heat waves. that 2014 followed several other recent heat wave years (e.g., 1994, 1997, 2003, 2006, and 2010), during which recorded domestically acquired vibrio cases spiked in northern europe (4,5), is particularly noteworthy. previous epidemiologic analysis regarding the emergence of vibrio infections in the region (5) indicated that sustained ssts > 18c were a notable risk factor, significantly increasing reported cases. the relation between maximum sst and annual vibrio case occurrence analyzed by using generalized linear model based methods demonstrated similarly to previous studies in the region (4) that maximum temperature correlates highly with risk, and cooler years (e.g., 2005, 2007, and 2012) indicate lower levels of reported infections than heat wave years (e.g., 2006, 2010, and 2014). in our study, the observation that a sizeable proportion of described cases were reported in subarctic latitudes (> 65n) and within 100 miles (160 km) of the arctic circle is striking. ten v. cholerae infections were reported above 63n, of which 6 cases were identified in the oulo area (65n). the cases recorded here are, to our knowledge, the most northerly reported instances of vibriosis documented, exceeding previous studies where cases have been reported at high latitudes, such as alaska (9) and previously in northern europe (5). disease data, such as those reported here, often are sporadic and usually grossly underreported. likewise, a major limitation of our investigation was the lack of detailed trace - back epidemiologic data, which limits the assessment of exposure and subsequent risk. for many reported cases, data about prior exposure (e.g., specific information about the timing and location of recreational exposure to water) and subsequent routes of transmission were absent. however, almost without exception, cases from finland and sweden were reported in coastal rather than inland medical centers. second, when prior transmission information was available from confirmed cases, most patients reported exposure to seawater in the days before symptom onset. these 2 factors, coupled with the striking climatic and oceanographic conditions during summer 2014, suggest that exposure to seawater was largely responsible for these episodes of disease emergence. the limitations underscore the need for a centralized system of surveillance and reporting. in the united states, the centers for disease control and prevention s covis (cholera and other vibrio illness surveillance) maintains a national database of vibriosis that contained detailed epidemiologic and transmission route information (12). a similar centralized reporting, monitoring, and surveillance system would greatly enhance risk assessment and risk management of vibriosis in europe. across the region, and with the exception of toxigenic v. cholerae infection, vibriosis is not a notifiable disease (5). given that these rare waterborne infections appear to have emerged and increased in northern europe recently (13) (e.g., 1994, 2006, 2014), this event underlies the need for clinicians to identify possible exposure to seawater. this event is particularly relevant for patients who have a history of conditions where progression of vibriosis to systemic infection is more likely, including diabetes, immune disorders, and liver dysfunction. climatic anomalies, such as the heat wave conditions during summer 2014 in northern europe, appear to be responsible for restructuring the geographic distribution of waterborne infectious diseases and resulted in major and far reaching consequences for the identification, treatment, and management of these pathogens. the greater number and intensity of large heat wave events in northern europe during the past 20 years or so (1994, 1997, 2003, 2006, 2010, 2014) further highlights the need for improved epidemiology and reporting, coupled with enhanced diagnostic capability in clinical settings to manage and ameliorate risk. | during summer 2014, a total of 89 vibrio infections were reported in sweden and finland, substantially more yearly infections than previously have been reported in northern europe. infections were spread across most coastal counties of sweden and finland, but unusually, numerous infections were reported in subarctic regions ; cases were reported as far north as 65n, 100 miles (160 km) from the arctic circle. most infections were caused by non - o1/o139 v. cholerae (70 cases, corresponding to 77% of the total, all strains were negative for the cholera toxin gene). an extreme heat wave in northern scandinavia during summer 2014 led to unprecedented high sea surface temperatures, which appear to have been responsible for the emergence of vibrio bacteria at these latitudes. the emergence of vibriosis in high - latitude regions requires improved diagnostic detection and clinical awareness of these emerging pathogens. |
in most outpatients with suspected community acquired pneumonia microbial diagnosis is not mandatory since empiric antibiotic treatment is usually succesful. immunosuppressed patients deserve special attention and invasive procedures are compulsory in order to obtain sputum to make specific diagnosis. empiric antimicrobial coverage should be tailored to treat the most likely pathogen in accordance to individual patient characteristics. a 79-year - old man presented to the emergency department of the regional hospital of bellinzona, switzerland with a 2-day history of fever and unproductive cough as well as dyspnea on exertion. previous history was remarkable for chronic corticosteroid use (10 mg daily of prednisone for eight months to treat polymyalgia rheumatica without giant cell arteritis). he was a non - smoker and did not have pre - existing pulmonary diseases. he had not travelled abroad recently, but was gardening during the last week before hospital admission. on initial presentation, his temperature was 38.5 c, heart rate was 95 beats / min, blood pressure was 128/64 mmhg and his respiratory rate was 18 breaths / min. physical examination revealed fine crackles of the left lower lobe whereas other findings were normal. laboratory values were remarkable for anemia (9.1 g / dl, normal range 14 - 18 g / dl) elevated c - reactive protein (crp 373 mg / l, normal 92%. the hemodynamic was supported with norepinephrine (doses between 10 - 15 mcg / min). a chest computer tomography revealed an airspace consolidation of the left lower lobe and bilateral infiltrates (figure 1). computed tomography of the chest one day after admission to the intensive care unit showing bilateral infiltrates and airspace consolidation of the left lower lobe. a transthoracic echocardiography excluded a left ventricular dysfunction, and mitral and aortic valves appeared normal. a bronchoalveolar lavage sample showed a neutrophil predominance (> 50%). despite regression of the inflammatory parameters (crp from 350 to 129 mg / l and procalcitonin from 6.3 to 2.2 g / l in three days) high fever persisted with abundant tracheal secretion requiring frequent aspirations and therefore we replaced clarithromycin with intravenous levofloxacin (500 mg/12 h). the bronchial sample obtained on the day of intubation was cultured on buffered charcoal - yeast extract agar medium on which legionella longbeachae surprisingly grew.. these results allowed us to make a definitive diagnosis of l. longbeachae community - acquired pneumonia (cap) complicated by acute respiratory distress syndrome with septic shock. the clinical course was characterized by a progressive improvement until the sixth day after admission to the icu. thereafter, we observed a new worsening of the respiratory parameters without an apparent cause requiring higher fio2 and peep levels. in the absence of clinical signs of ventilator associated pneumonia (vap) further bronchial samples were not obtained and antibiotic coverage remained unchanged. despite higher fio2 and peep levels the patient remained hypoxemic and, to protect the right ventricle from high intrathoracic pressure and to maintain an open - lung ventilation protective strategy (tidal volume of 6 ml per kg ideal body weight and peak airway pressure 2o), with a transient moderate permissive hypercapnia we decided to prone the patient with a consistent improvement allowing us to progressively reduce the respiratory support. on day thirteen after admission to the icu the patient developed again high fever with worsening respiratory parameters and hemodynamic instability consistent with a suspected impending vap. after discussion with the family members and in accordance with the patient s wishes, we decided not to perform the diagnostic evaluation of a suspected vap and to withhold further support and the patient died shortly after. the patient described herein with polymyalgia rheumatica represents a sporadic cap case with ards and septic shock due to l. longbeachae infection, diagnosed by culture from bronchial secretions. in accordance with the bal cultures, and sample with a neutrophil predominance excluding an acute eosinophilic pneumonia and a cryptogenic organizing pneumonia the delay between the onset of the symptoms and appropriate antibiotic coverage is probably responsible for the development of the ards diagnosed in accordance with the berlin definition. by admission to our icu supine low tidal volume ventilation the patient progressively improved rendering prone ventilation initially no more indicated. the only risk factor for legionella pneumonia that we could identify was the long - term systemic steroids treatment (10 mg daily of prednisone) associated with depression of - cell mediated immunity. the four days transient increase of prednisone dosage in order to prevent adrenal insufficiency seems unlikely to be responsible for the poor patient s outcome. to the best of our knowledge this case represents the first report of a l. longbeachae cap complicated by ards with septic shock in a patient with corticosteroid - dependent polymyalgia rheumatica. this infection is very uncommon in the united states and europe but it accounts for approximately 50% of all cases of legionnaires disease in australia and new zealand, and it is far more prevalent than l. pneumophila in southeast asia. l. longbeachae is one of the soil - dwelling pathogenetic legionella species and its transmission has been linked to gardening and exposure to potting soil. the present case was notified to the regional public health authorities, but because it was a sporadic case of non - pneumophila legionella infection, no additional epidemiological investigations were undertaken. l. longbeachae is not only less prevalent than l. pneumophila but also probably less virulent. pneumonia cases due to l. longbeachae reported from outside australia occurred in immunocompromized patients : those undergone splenectomy, under immunosuppressive drugs for systemic lupus erythematosus (sle) or in transplant recipients. garcia and coll. reported a fatal case of community - acquired pneumonia due to l. longbeachae in spain. their young patient with sle was also under corticosteroid treatment, but in contrast to our patient she had one important additional risk factor for legionella pneumonia : cigarettes smoking. moreover despite severe hypoxemia this patient did not fulfill all the criteria for ards diagnosis because hydrostatic pulmonary edema was formally not excluded (eg echocardiography). the widespread introduction of legionella urinary antigen testing into many hospital laboratories has resulted in a decreasing use of cultures and serology studies. although urinary antigen test remains positive even during administration of empiric anti - legionella antibiotics, it is helpful in patients with unproductive cough and the test - result is usually available within hours permitting an early diagnosis, it is only specific for l. pneumophila serogroup 1 [14, 15 ]. therefore it is likely that many infections caused by non - pneumophila species remain undiagnosed, as in our patient at hospital admission. does not grow on blood agar media, and are usually not detected by sputum gram stain or blood culture. the standard media for legionella isolation is buffered charcoal - yeast extract agar (bcye) eventually supplemented with anisomycin, dyes, polymycin and vancomycin to reduce sample contamination. our findings support the importance of examining sputum for legionella spp. by cultural methods when pneumonia is suspected, especially in the immunocompromized host, where early recognition of the responsible pathogen and appropriate antibiotic therapy are crucial for the patient s survival. furthermore, the sensitivity of legionella urinary antigen test depends on clinical severity of pneumonia, so that a mild pneumonia or an early - onset pneumonia may go undiagnosed if the test is used alone [14, 15 ]. in summary, this culture - proven case of l. longbeachae infection demonstrates that non - pneumophila legionella species must be considered as casual agents of pneumonia even in mild immunosuppressed patients. it should be noted that non - pneumophila legionella infections are probably underreported because urinary antigen testing and serology fail to detect these pathogens and because sputum culture on appropriate media is not always standard practice in the cap diagnosis. a delay in diagnosis and in appropriate antibiotic treatment could significantly affect patients outcome, especially in the immunocompromized hosts. since sputum cultures take several days, empiric anti - legionella antibiotic coverage and real - time pcr testing should be considered in high - risk patients. finally, we suggest to administer anti - legionella antibiotics in severe caps if the urinary antigen test is negative, unless another pathogen has been detected as the causal agent and the involvement of legionella has, definitively, been ruled out. | legionella longbeachae is a very uncommon cause of community acquired pneumonia in western countries. l. longbeachae does not grow on blood agar media and is usually not detected by sputum gram stain or blood culture. furthermore legionella urinary antigen testing fails to detect it. in this report we described a 79-year - old man with polymyalgia rheumatica under systemic corticosteroid treatment without other additional risk factors who developed a cultured - proven l. longbeachae community - acquired pneumonia complicated by an acute respiratory distress syndrome with septic shock. this case report demonstrates that non - pneumophila legionella species must be taken into account as casual agents of community acquired pneumonia even in mild immunosuppressed patients, and empiric anti - legionella antimicrobial coverage might be indicated until legionella has definitively been rule out by adequate testing. |
about 75,000 hip fractures occur annually in the united kingdom often as the result of trivial injury. the incidence of these fragility fractures is expected to increase to 91,500 by 2015 and 101,000 in 2020 with people > 85 years which are 1015 times more likely to sustain hip fractures than people aged 60 to 65 years. the seriousness of hip fractures is reflected by the 23-day postoperative hospital stay and 10% 30-day mortality, associated with an annual cost of medical and social care amounting to nearly 2 billion. these figures are multifactorial and are in part due to the complications that occur after a hip fracture in a population group with significant comorbidities (median asa grade 3). hip fractures are painful and inadequately controlled pain can have significant physiological and psychological effects such as an acute confusional state (delirium) seen in 1016% of hip fracture patients presenting to emergency department (ed). these factors make further pain assessment difficult and have the potential to delay surgical intervention, compound complications, and ultimately prolong hospital stay and increase the risk of nursing home placement. the delivery of effective pain relief for hip fracture patients at the first point of contact in the emergency department (ed) is therefore crucial. beside this, many studies suggest that pain management for limb fractures in the elderly is poor with as little as 2% of patients receiving adequate analgesia [810 ]. systemic analgesia including both opioids and nonsteroidal analgesia can have significant adverse effects especially in the elderly population due to age - related changes in pharmacokinetics and pharmacodynamics. furthermore, the long list of medications that accompany most patients also increases the risk of drug interactions. a lack of in depth knowledge about such issues and how to manage them by ed doctors may further hinder effective analgesia prescribing. fascia iliaca blocks (fibs) provide regional pain relief and can address many of the issues surrounding traditional forms of analgesia. they have traditionally been a remit of anaesthetic professionals perioperatively targeting the lateral cutaneous, femoral, and obturator nerves found beneath the fascia iliac. while doctors in other specialities, such as the ed, have carried out the blocks, routine administration in clinical practice has been staggered with only 19% of patients receiving a peripheral nerve blockade for hip fracture. the reason for this is not known but could be due to concerns over adequate training and the misconception that administration takes longer than systemic analgesia. this study describes our experience of fib applied in the emergency setting and compares outcomes to traditional systemic analgesia. in addition, we demonstrate the feasibility of junior orthopaedic and emergency doctors administering this form of pain relief at the point of need. a prospective case - control study was carried out over a six - month period in a large uk district general hospital involving the orthopaedic and emergency departments. all patients with hip fractures presenting to the ed from january to march 2012 received traditional forms of systemic analgesia according to the who pain ladder (controls). all patients with hip fractures presenting to the ed from april to june 2012 received fib (cases). the absolute contraindications to fib included patient refusal, bleeding diatheses, femoral grafts in the affected limb, and inflammation over the injection site and allergy to local anaesthetics. all junior doctors (foundation year 2core training year 2) in the trauma and orthopaedics department were trained on administering fib through a teaching presentation from an anaesthetic trainee, followed by supervised training in theatre by a senior anaesthetist. once deemed competent by the consultant anaesthetist, juniors were permitted to administer a single fib per patient during the day and night on - call period. once a robust orthopaedic service delivery was in place, ed junior doctors were also trained and administered blocks allowing for an earlier use of the block. after receiving the block, codeine and paracetamol were prescribed on the as - required section of the drug chart. regular systemic analgesia including paracetamol and opioids was commenced if operative intervention was delayed by more than 24 hours, if required. an 18 g tuohy needle was used to administer a weight - dependant volume of 0.25% chirocaine (levobupivicaine) as per local anaesthetic protocol (table 1) under aseptic technique. the injection site was located along the lateral one - third of a line joining the anterior superior iliac spine (asis) and pubic tubercle (pt) targeting the compartment between fascia iliaca and fascia lata (figure 1). demographic data (age, sex), abbreviated mental test score (amts) and fracture classification (intracapsular / extracapsular) was recorded (table 2). pain scores were measured on gentle pin rolling of the affected leg in both groups and assessed through a visual analogue scale of 010 preanalgesia / block and then at 15 minutes, 2, 8, 16, and 24 hours after analgesia / block. at t = 15 mins, monitoring was carried out by the clinician who performed the block and also included monitoring of blood pressure, heart rate, and oxygen saturation. thereafter, monitoring was carried out in orthopaedic wards by healthcare professionals who received appropriate education and training about the project and how to elicit pain scores and their documentation. block success was deemed as pain score < 50% to the preblock score at 2 hours after block administration as per the standard of care agreed by the national collaborative that a pain score should never exceed 50 per cent [13, 14 ]. the time of initial analgesia, total preoperative dose of analgesia required, and any complications were also recorded. analyses were carried out using the statistical package spss for windows (v.18.0, chicago, il, usa). for continuous data, student 's t - tests were used for two groups of variables and one - way analyses of variance (anova) for more than two groups, followed by post - hoc tukey analysis. for categorical data, pearson 's chi - square test was used with statistical significance reached by a p value of < 0.05. from january 2012 to june 2012, a total of 104 patients were included in the study. 52 received systemic analgesia as per the traditional methods (controls) and 52 patients received a fib as soon as possible after radiological diagnosis (cases). four patients were excluded from receiving a fib, 3 due to a femoral graft in the affected limb and one due to severe aggressive dementia preventing safe administration. the demographics of the two groups and their preoperative variables are shown in table 2. hip fractures are painful and pain left untreated can result in a host of complications that may delay operative intervention and complicate hospital stay. pain management in many hospitals in the uk is based upon the use of systemic analgesia according to local hip fracture protocols. with more than 313000 patients waiting more than 4 hours in the ed before being seen the busy nature of the ed and increased patient - to - staff ratio may delay pain assessment and treatment, with one study reporting mean time to pain assessment of 40 minutes and mean delay to treatment of 122 minutes. the national institute of clinical excellence guidelines suggest considering the use of neural blockade by trained personnel to limit opioid dosage. this study demonstrates the beneficial effect of fib, performed by junior doctors in ed, in management of pain in hip fracture patients. there was a significant reduction in pain scores at 2 hours (p = 0.03) following blockade which continued for up to 8 hours (p = 0.01). while this study did not find any significant difference at 15 min, two previous studies have reported reduced pain scores at 15 min after block [11, 18 ]. the pain scores were also reduced to half at 16 and 24 hours after block but they were not statistically significant (table 3). the time to initial analgesia was also reduced (25 mins versus 40 mins, p = 0.04) (table 3) with most patients and relatives verbally reporting satisfaction with the prompt pain service delivery by the admitting clinician (no quantifiable data available). systemic analgesic requirements were also significantly reduced within 24 hours of admission, a similar finding by monzon.. this rapid and long lasting effect of a single fib makes this form of analgesia attractive in the busy surroundings of the emergency department and acute orthopaedic wards. the beneficial effect of fibs in patients with radiologically confirmed hip fractures is well known with several studies reporting a good outcome [11, 2023 ], when compared to nsaids [11, 20 ], alfentanil, and placebo [22, 23 ]. its success is attributable to blocking pain sensation in the femoral nerve, lateral cutaneous nerve of the thigh, and obturator nerve. pain relief has been found to be both at rest and upon movement [18, 23 ]. this is beneficial in allowing patients to sit up more comfortably while they await surgery and can facilitate spinal anaesthesia. mouzopoulos. also found a reduction in the occurrence of delirium in hip fracture patients who have had a block, most likely as a result of the opioid sparing effect in this particularly vulnerable population group. our block success was 65%, which is on the lower site of the reported rates of 67%96% [12, 17, 23, 25 ]. this could be due to the fact that different authors have used different definitions for the block success, for instance, pain score reduction of <3, sensory loss over the thigh, and an increase in flexion [18, 24 ]. we defined our block success as a 50% reduction in pain score at t = 2 hr from preblock pain score based on the suggestion by counsell and the nhs fractured neck of femur collaborative (nhs modernisation agency, 2001). there are a number of ways to administer the fib such as the loss of resistance (2-pop techqnique) that we used, uss guided blocks, or the nerve stimulators to locate the femoral nerve. uss guided blocks are not practical for all eds due to training, timing, and resource limitations. the use of nerve stimulators has been shown to be no better than the loss of resistance technique. in fact the time for peripheral nerve stimulator block was significantly longer. while there are publications detailing the use of intradermal needles for the block, we used the blunt tuohy needles which provide a further margin of safety and give a better pop sensation as the needles traverse the fascia, thereby reassuring the junior doctor that they are in the right compartment. as there are no reported studies comparing this technique, it would seem reasonable that this form of analgesia may be administered in the ed according to local resource and expertise available. the main limitations of this study are that the patients were not randomised, although the two groups were comparable (table 2). furthermore, some patients were given analgesia by the ambulance staff prior to presentation in the ed ; however, this information was not always available. as a result this study might have underestimated the additional doses of analgesia required in some patients. since the guidelines for analgesia prescription in suspected hip fracture patients have not changed for the ambulance staff, it is quite possible that the two groups were also comparable for this variable. the implementation of european working time directive (ewtd), hospital at night, shift system has increased the burden on ed. as junior doctors are increasingly at the forefront of service delivery, administering fibs is an essential skill to possess that can extend beyond anaesthetic training and the theatre environment. this study demonstrates that the junior doctors without anaesthetic backgrounds can provide a rapid and effective pain service, supporting change to current clinical practice. with only 71.4% of hip fracture patients receiving their operation within the recommended 36 hours and the majority of delays occurring due to medical causes including complications of pain management, such a service has far - reaching effects and can also contribute to the provision of a streamlined high - quality perioperative hip fracture service that is necessary in the current climate of shift systems imposed by the ewtd. the use of continuous fib using indwelling catheters, as piloted by dulaney - cripe., may be of further benefit and is currently under ongoing investigation. in addition, utilising the expertise of an existing anaesthetic department could facilitate adequate training. not only will this be time and cost effective but | hip fractures are common and the incidence is expected to increase. systemic analgesics, often prescribed to relieve pain after hip fractures, have huge side effects and can delay surgery. we analyse the role and efficacy of alternative forms of analgesia like fascia - iliac blocks (fib) and assess the feasibility of a service delivered by junior doctors. 104 consecutive hip fracture patients were prospectively recruited and equally divided into cases (patients receiving fib) and controls (patients receiving systemic analgesia). outcome measures included time of initial analgesia, total preoperative dose of analgesia, pain scores from admission to 24 hours preoperatively, and complications. the pain scores were significantly lower (p 0.05) in patients receiving fib at 2 and 8 hours preoperatively. the timing of initial analgesia was also quicker in patients with fib (25 compared to 40 minutes). fib patients required fewer doses of systemic analgesia. the block was successful in 67% of patients. there were no complications. the implementation of ewtd, han, and shift - system and the reduction in the number of medical staff have increased the burden on emergency departments. this study demonstrates that fib performed by junior doctors are not only safe and effective analgesia but also provide an opportunity for junior doctors to improve current clinical practice. |
culex pipiens complex species have been known as important vectors of medical and veterinary arthropod - borne diseases (kasai. some vector borne diseases such as filariasis, west nile fever, western and eastern equine encephalitis, japanese encephalitis and st louis encephalitis are transmitted by these species complex (smith 1973, vinogradova 2000, kasai. pipiens transmits west nile virus among wild birds and plays an important role in enzootic cycles (hayes. some pathogens transmitted by culicine mosquitoes such as west nile and sindbis viruses, dirofilaria immitis (dog heartworm) and d. repens (dirofilariasis), and have been reported in iran (naficy and saidi 1970, saidi. 1976, azari - hamidian. 2007). moreover, potential outbreaks of some mosquito - borne arboviral diseases such as japanese encephalitis (je) and rift valley fever reported in the eastern mediterranean region (who 2004). the morphological and ecophysiological variations of cx. pipiens complex have been an important topic in extensive researches (harbach 1985, 1988, vinogradova 2000, 2003, azari - hamidian and harbach 2009, dehghan. because of the complicities, more than 75 synonyms have been proposed for this complex species (knight and stone 1977). barr (1982) reported a wide geographical distribution, and morphological variations among the cx. there are variations among the diagnostic characters of these species complex that attributed to some degree of expression such as species, subspecies, variety or forms (ishii 1991). culex quinquifasciatus is distributed in the tropical areas with various hosts, whereas, cx. pipiens found in the moderate areas with host preferences of nest maker birds (vinogradova 2000). shahgudian (1960) and lotfi (1976) provided identification keys for iranian anopheles and culex species, respectively. pipiens, the nominotypical and molestus form, was described in iran (lotfi 1970, 1973, 1976, amirkhanian 1974, zaim and cranston 1986). some years later a checklist and systematic key was provided for iranian culicinae by zaim and cranston (1986). although, in the previous study had not been mentioned about diagnostic characters of cx. quinquefasciatus in the systematic keys (zaim and cranston 1986), but azari - hamidian and harbach (2009) addressed these characters. pipiens complex species, collected in some parts of iran to facilitate conducting comprehensive research about systematics, ecology, medical and veterinary importance of the complex. until now, seven genera, 64 species, and three subspecies of iranian mosquitoes was reported (azari - hamidian 2007a). culex pipiens complex belongs to the pipiens group, and was divided to several subgroups and subtypes (harbach 2011, 2013). quinquefasciatus have been reported in iran (azari - hamidian 2007a, azari - hamidian and harbach 2009). the polygene chromosomal pattern of the autogenous tehran strain of culex pipiens molestus was described by amirkhanian in 1974. pipiens have been reported in iran (zaim 1987, azari - hamidian. more studies reported distribution of this species in southern parts of iran (zaim 1987, mousa - kazemi 2000, azari - hamidian. 2005, moosa - kazemi. 2010, moosa - kazemi. 2010, khoobdel. 2012). the distribution of this species has been reported in tropical areas in south of iran based on molecular identification (azari - hamidian. pipiens complex, revealed that, male genitalia and dv / d ratio are the main morphological characters for distinguishing adults of cx. the main reliable character for identification in larval stage has been known as the number of branches of seta 1 on the abdominal iii iv segments (harbach 1988, dehghan. 2013). since there are scatter studies about the morphological variations and distribution of cx. pipiens complex in iran, (zaim 1987, azari - hamidian and harbach 2009, azari - hamidian. 2010) it is necessary to obtain more accurate data on the variability of the species. on the other hand, the final decision about taxonomic status needs more complete data that will be obtained from further studies in different geographical areas. pipiens complex species, as well as designing plans for vector control programs in the future. chabahar (2517n, 6037e) and nikshahr (2604n, 6037e) counties were selected from sistan and baluchistan province with tropical warm and humid climate. in southern iran, jiroft (28.5n, 57.8e) from kerman, borazjan (2915n, 5112e) from bushehr, ahvaz (3119n 4841e) and bostan (3127n 4804e) counties from khuzistan province with subtropical warm and humid climate were selected. yazd, zarch (5404n 3159e) from yazd and kerman (3017n 5704e) from kerman province with hot and dry desert climate were selected. for cool and moist mediterranean climate neka (3642n 5333e) county from mazandaran and mashhad (3618n 5936e) from khorasan - e - razavi province were chosen. hamedan (3448n 4831e) from hamedan, and tehran (3545n 5135e) from tehran province were selected from cold and dry climate (fig. the study areas and distribution of culex pipiens and culex quinquefasciatus in different stratum of iran, 2010 this cross sectional study was conducted in 13 randomly selected areas in iran. larval stages of the mosquitoes were collected, using standard dipping technique from april 2009 to october 2010. the larvae were collected from different regions of the country using who standard dipping method (who 1992). the samples were transferred to the entomology laboratory, department of entomology and parasitology, school of medical sciences, tarbiat modares university. the mosquitoes larvae maintained in specific cage for rearing in insectarium condition (2225 c, 7075% rh). microscopic slides were used to mounting of some parts of the adult body such as wings and maxillary palps using canada balsam diluted with xylen. the caudal abdominal segment of males were removed, and placed in koh 10% for 20 to 30 minutes, then washed with distilled water and placed in ethanol 96% for dehydration (barr 1957, jakob. the taxonomic figures were drawn using light zeiss microscope with a nikon drawing tube accessory long arm (9.1 inches) (22.5 cm). 3) (harbach 1988, azari - hamidian and harbach 2009). identification of culex pipiens and culex quinquefasciatus larvae based on abdominal segments of iii iv identification culex pipiens and culex quinquefasciatus larvae based on siphon characters dv / d : the ratio used for adult identification, also used as a confirmation for molecular studies. dv was described as the distance between two tips of dorsal and ventral arms and d was defined the distance between two tips of dorsal arms (fig. 4) (mattingly. 1951, barr 1957, kamura 1959, vinogradova 2003, smith and fonseca 2004). identification of adult male in culex pipiens and culex quinquefasciatus by male genitalia and different of dorsal and ventral arms costa and subcosta intersection with r2 + 3 furcation (fig. identification of adult female in culex pipiens and culex quinquefasciatus based on vegetation of wings data were analyzed using spss ver. chabahar (2517n, 6037e) and nikshahr (2604n, 6037e) counties were selected from sistan and baluchistan province with tropical warm and humid climate. in southern iran, jiroft (28.5n, 57.8e) from kerman, borazjan (2915n, 5112e) from bushehr, ahvaz (3119n 4841e) and bostan (3127n 4804e) counties from khuzistan province with subtropical warm and humid climate were selected. yazd, zarch (5404n 3159e) from yazd and kerman (3017n 5704e) from kerman province with hot and dry desert climate were selected. for cool and moist mediterranean climate neka (3642n 5333e) county from mazandaran and mashhad (3618n 5936e) from khorasan - e - razavi province were chosen. hamedan (3448n 4831e) from hamedan, and tehran (3545n 5135e) from tehran province were selected from cold and dry climate (fig. the study areas and distribution of culex pipiens and culex quinquefasciatus in different stratum of iran, 2010 larval stages of the mosquitoes were collected, using standard dipping technique from april 2009 to october 2010. the larvae were collected from different regions of the country using who standard dipping method (who 1992). the samples were transferred to the entomology laboratory, department of entomology and parasitology, school of medical sciences, tarbiat modares university. the mosquitoes larvae maintained in specific cage for rearing in insectarium condition (2225 c, 7075% rh). microscopic slides were used to mounting of some parts of the adult body such as wings and maxillary palps using canada balsam diluted with xylen. the caudal abdominal segment of males were removed, and placed in koh 10% for 20 to 30 minutes, then washed with distilled water and placed in ethanol 96% for dehydration (barr 1957, jakob. the taxonomic figures were drawn using light zeiss microscope with a nikon drawing tube accessory long arm (9.1 inches) (22.5 cm). 3) (harbach 1988, azari - hamidian and harbach 2009). identification of culex pipiens and culex quinquefasciatus larvae based on abdominal segments of iii iv identification culex pipiens and culex quinquefasciatus larvae based on siphon characters dv / d : the ratio used for adult identification, also used as a confirmation for molecular studies. dv was described as the distance between two tips of dorsal and ventral arms and d was defined the distance between two tips of dorsal arms (fig. 4) (mattingly. 1951, barr 1957, kamura 1959, vinogradova 2003, smith and fonseca 2004). identification of adult male in culex pipiens and culex quinquefasciatus by male genitalia and different of dorsal and ventral arms 5) (harbach 1985, azari - hamidian and harbach 2009). identification of adult female in culex pipiens and culex quinquefasciatus based on vegetation of wings data were analyzed using spss ver. overall, 304 larvae and 419 adults (177 males and 242 females) were randomly selected. the branch number of seta 1 on abdominal segments iii iv, 1a s tuft, 1b s tuft and siphon shape are showed in table 1. culex pipiens larvae were identified in the samples of mashhad, tehran, neka, yazd and zarch1 areas whereas, cx. quinquefasciatus was found in zarch 2 and kerman based on the seta 1 abdominal segments iii iv. the findings showed variations at the mentioned character among the samples collected from borazjan, chabahar, jiroft, and nikshahr. quinquefasiatus larvae, islamic republic of iran, 20092010 all of the species identified as culex pipiens all of the species identified as culex quinquefasciatus the mean average of 1a s tuft branches were calculated 2.7 to 4.7 and range of 2 to7 and 6.2 to 7.9, range 2 to 10 for cx. pipiens and cx. the mean average of 1b s tuft branches were counted 2.8 to 4.4, rage 2 to7 and 6 to 7.8, range 3 to 13 for cx. pipiens larvae were found gradually narrowing toward the end of siphon in all samples that were collected from mashhad, tehran, neka, and zarch1 whereas, this character included 84.2 % of cx. quinquifasciatus larvae in borazjan, chabahar, jiroft, kerman, and nikshahr samples (table 1). pipiens were identified based on the morphological characters of female wings vegetation in hamedan, yazd and bostan, while it was found varied among the samples collected from neka, zarch, ahvaz, chabahar, nikshahr and jiroft. culex quinquefasciatus was identified in chabahar (97%), ahvaz (88.9%), and nikshahr (73.7 %) (table 2). the variations of some morphological characters of wings in the females of culex pipiens and cx. quinquefasciatus, islamic republic of iran, 20092010 all of the species identified as culex pipiens all of the species identified as culex quinquefasciatus table 3 shows the dv / d and d / v ratios. the dv / d ratio calculated 1.230.12 in mean average and range 0.210.2 in cx. pipiens. in addition, mean average of this ratio was calculated 0.350.64 and the range was 0.170.78 in cx. quinquefasciatus, islamic republic of iran, 20092010 all of the species identified as culex pipiens all of the species identified as culex quinquefasciatus harbach (1988) and azari - hamidian and harbach (2009) described this character in cx. our finding showed, siphonal seta 1a - s and 1b - s of cx. these results are supported by previous study, knight and malek (1951) reported an average 4 and a range 29 branches for cx. pipiens. results of this study showed the branch number of seta 1a - s and 1b - s had a mean average of 67.9 and range of 213 for cx. shape of the siphon was studied among 165 larvae samples and confirmed vast most of the cx. as mentioned above, there are character variations among the samples which were collected from south of iran. generally, the length of siphon in cx. according to our observations, the seta 1 branches of abdominal segments iii iv and shape of the siphon in larval samples were found as valuable characters, which can easily used to distinguish of cx. quinquefasciatus species, therefore, it is recommended the whole characters should be evaluated to accurate identification. culex pipiens complex is considered cosmopolitan species. although, the distribution patterns of the complex species have been reported in iran (zaim 1987, harbach 1988, azari - hamidian. pipiens in bostan, yazd, neka, mashhad, hamedan and tehran. nevertheless, the distribution of cx. quinquefasciatus was limited in chabahar, nikshahr, jiroft, kerman, borazjan, ahvaz and zarch. quinquefasciatus in the iranian persian gulf islands had been reported previously (azari - hamidian. in addition this species was reported in kermanshah, western iran (ghaffary 1954), and in bandar - e anzali northern iran (harbach 1988). as mentioned, the male genitalia reported as the main morphological characters to identification of the cx. quinquefasciatus was more than 0.4 (0.561.89, mean 1.03), but in cx. pipiens, calculated at mean average of 1.230.12 and range of 0.210.2 while, calculated at mean average of 0.51.09 and ranges 0.332.37 for cx. the d / v ratio was calculated at mean average of 0.951.35, and range of 0.71.75 for cx. pipiens samples collected in south of the country, whereas it was calculated at mean average of 0.350.64 and range of 0.170.78 for cx. further supports for these results also came from some previous studies, sasa (1967) express the d / v ratio in range of 0.40.9 in cx. moreover, choochote (1987) reported the d / v ratio at average of 0.35 for cx. pipiens form molestus and 0.337 for cx. quinquefasciatus. in recent research, dorsal arms of male genitalia of cx. quinquefasciatus described as narrow, sharp apex and parallel as the base toward the end. whereas, in cx. pipiens dorsal arms were described quite broad, truncate at the apex, and diverges in the base toward the end of arms, which, indicated the occurrence of cx. further support to these results also came from a previous study, harbach (1988) described the dorsal arms of phalosoma as divergent, broad and nearly truncate at the apex for cx. pipiens complex species using morphological key have some difficulties because of occurring variations among closely related complex species. in present pictorial key, the colors of the siphon and male s genitalia are not referring to the color of original samples. however, the described taxonomical characters in this article should be included in other characters, which were reported in the previous literatures. pipiens complex should be identified by morphological characters in the first step, although some researchers prefer molecular studies such as ace. 2 gene, microsatellite loci and coi gene for solving the morphological taxonomic problems. comprehensive studies such as phylogenic and molecular are necessary to obtain new information for identification of cx. | background : the aim of this study was to design pictorial key and taxonomic literature of culex pipiens complex in iran.methods:larvae were collected using standard dipping methods in 13 randomly selected areas of bushehr, hamedan, kerman, khorasan - e - razavi, khuzistan, mazandaran, tehran, sistan and baluchistan and yazd provinces from april 2009 to october 2010. the data were analyzed using spss ver. 11.5.results:culex pipiens larvae were identified based on the seta 1 of the abdominal segments iii iv in north and central parts of iran. this diagnostic character had some variation among the cx. quinquefasciatus collected from south of the country. the identification value of intersection of costa, subcosta and bifurcation of r2 + 3 of female veins, was calculated as 90100 % for cx. pipiens. this diagnostic character was varied among the cx. quinquefasciatus specimens. the male genitalia found as the main characters to distinguish of cx. quinquefasciatus from cx. pipiens.conclusion:it is necessary more studies on the behavior and genetic variations of cx. pipiens complex in iran. |
transformation of normal oral mucosa (nom) to scc represents a complicated process involving numerous etiologic factors. during progression to invasive carcinoma, neoplastic cells activate the underlying connective tissue and generate a phenotypically altered and specific tumor stroma, which may influence the cancer cells. tumor stroma comprises of immunocompetent and inflammatory cells, endothelial cells, fibroblasts and a subtype specific of fibroblasts called myofibroblasts. myofibroblasts are derived mainly from fibroblasts and also from smooth - muscle cells, pericytes, macrophages, hepatic stellate cells, epithelium and bone marrow. the tumor - promoting effect of myofibroblast is based on the direct cytokine stimulation of cancer cells, maintenance of vascularity and on their capacity to produce enzymes which degrade molecules like lysyl oxidase which enhance the structural integrity of matrix. myofibroblasts create a physical barrier between carcinoma cells and immune system of the : body against cancer. an increase in myofibroblasts may be useful to predict the prognosis of oscc patients, since cervical node metastasis is one of the major prognostic factors in patients with oscc. earlier studies using alpha - smooth muscle actin (-sma) have been done to compare the distribution of myofibroblasts in nom and histological grades of oscc. the present study uses h1 calponin to compare the distribution of myofibroblasts in nom, early invasive carcinoma and histological grades of oscc. calponins are components of the smooth muscle thin filament that are suggested to regulate interactions between actin and myosin ii. the study included the archival tissues of 18 oscc cases of well, moderate and poorly differentiated grades, three early invasive carcinomas and five normal mucosa selected from the department of oral and maxillofacial pathology, rajah muthiah dental college and hospital, annamalai university. two sections of 35 m thickness were sliced from each tissue block for routine hematoxylin and eosin staining and immunohistochemical staining. the hematoxylin and eosin stained sections of oscc were examined to confirm the diagnosis of oscc histopathologically. the other sections were mounted on glass slides coated by aminopropyl triethoxy silane (apes ; sigma chemical co., usa) and processed for subsequent immunohistochemical study. the sections were deparaffinized at 60c for 1 h. the sections were dewaxed in xylene and rehydrated in descending grades of alcohol. the sections were covered with 3% hydrogen peroxide for 10 min followed by treatment with protein block for 10 min. the sections were covered completely with optimally diluted mouse monoclonal primary antibody to h1 calponin (biogenix) for 30 min. then the slides were kept in phosphate - buffered saline (pbs) buffer bath for 5 min. then the slides were treated with poly horseradish peroxidase (hrp) enzyme (dako real envision, denmark) for 30 min. the slides were then washed with pbs and immunostaining was carried out by staining with dab (3,3-diaminobenzidene tetrahydrochloride) for 5 min. the slides were immersed in mayer 's hematoxylin for 7 min and blueing was done for 10 min. the sections were dehydrated in ascending grades of alcohol and air dried thoroughly and mounted using dpx. presence of brown - colored end product at the site of target antigen was considered as positive immunoreactivity. cytoplasmic staining of stromal spindle cells observed in the experimental slides of well, moderate and poorly differentiated scc was considered as positive immunoreactivity. immunostaining was assessed by the evaluation of the staining intensity and percentage of positive - staining stromal cells, according to the method proposed by tuxhorn. the percentage of immunopositive cells in the non - inflammatory and non - endothelial stromal cells immediately adjacent to the carcinomatous islands and normal was recorded as : 0% = no positive cells, 1 = 133% positive cells, 2 = 3466% positive cells and 3 = 67100% positive cells. staining intensity was considered 0 when there was no staining ; 1, in parts where positivity was observed only at a magnification of 40 ; 2, in cases where staining was obvious at 10, but not 4 ; and 3, in fields where immunopositive cells were seen even at 4. multiplication of the percentage and intensity scores comprised the staining index of each specimen. this index was classified as zero (0), low (12), moderate (34) and high (69). dark brown stained stromal spindle cells of each representative area were selected and cell counting was performed using the cell counter in image j software. differences in the presence of myofibroblasts between groups were analyzed using kruskal wallis and mann whitney tests. histopathological observations of the study samples were done and grading was confirmed. according to the invasive tumor front grading system by bryne., (1969), the study samples were grouped into three grades. the sample comprised of 10 cases of oscc of grade i, seven cases of oscc of grade ii and four cases of oscc of grade iii. the study samples were evaluated immunohistochemically for myofibroblast expression by h1 calponin. the staining index in histological grades of oscc and invasive patterns of oscc are calculated. expression of calponin in myofibroblast was compared in 21 oscc and five nom using kruskal statistical analysis showed a significant difference in the expression of calponin between normal and oscc (p < 0.001) [table 1 ]. expression of myofibroblast in normal and oscc there was no statistically significant difference in the expression of calponin among well, moderate and poorly differentiated and early invasive oscc (p < 0.812). statistical analysis showed a nearing significance (p < 0.070) [table 2 ] in the expression of calponin among the invasive patterns of oscc. between grades i and iii, there was significant difference (p < 0.02) [table 3 ] in the expression of calponin. expression of myofibroblast in invasive patterns of oscc mann - whitney test for comparison between grade i and grade iii with approximately 500,000 new cases annually, scc of the head and neck represents one of the sixth most common cancers in the world. according to the tissue organization field theory, cells are normally in a proliferative state and do not tend to be quiescent. thus, mutated epithelial or stromal cells and disturbed stromal epithelial interactions may be equally responsible for the induction of carcinogenesis. during progression to invasive carcinoma, neoplastic cells activate the underlying connective tissue and generate a phenotypically altered and specific tumor stroma, which may influence the cancer cells. carcinomas induce a modified stroma through expression of growth factors that promotes angiogenesis, altered ecm expression, accelerated fibroblast proliferation and increased inflammatory cell recruitment. some stromal events such as fibroblast 's activities, myofibroblast 's differentiation and presence of some specific stromal proteins like proteolytic enzymes, fibronectins and laminin-5 have been reported as the main features of stromal tumor. fibroblasts play a major role in regulating and maintaining extracellular homeostasis and when activated after tissue injury, are responsible for wound contraction, fibrosis, scaring and regulation of inflammatory reactions. in addition they differentiate into contractile and secretory fibroblasts with abundant endoplasmic reticulum, pronounced golgi apparatus and -sma fibers. these -sma - positive fibroblasts, termed myofibroblasts, synthesize extracellular matrix components and several proteinases, growth factors and cytokines. derek., 2007 suggested that exposure of epithelial cells to matrix metalloproteinases (mmps) can lead to increased levels of cellular reactive oxygen species (ros) that stimulate transdifferentiation to myofibroblast - like cells. myofibroblasts were originally identified in granulation tissue as modified fibroblasts with prominent rough endoplasmatic reticulum. later, myofibroblast were most often defined as fibroblasts positive for -sma and containing actin microfilaments and vimentin as identified by immunohistochemistry. tumor growth factor (tgf)- induces expression of -sma and is considered the major growth factor promoting myofibroblast development. squamous carcinoma cells may directly induce a myofibroblast phenotype in primary fibroblasts through the secretion of tgf-1. oral fibroblasts undergo tgf--induced myofibroblast differentiation and this effect is enhanced by il-1. myofibroblasts are present in the stroma of most human oscc and the two dominant patterns, spindle and network, have been described by several authors (vered., 2009 and kellermann., 2007) myofibroblast appears to be a key player in the carcinogenesis and progression of osccs. in the present study, the expression of myofibroblast between five normal mucosal specimen [figure 1 ] and 21 oscc specimens [figures 2 to 4 ] were analyzed using calponin. the study showed a consistently increased calponin expression levels (p < 0.001) in almost all oscc tissues compared to normal mucosa which was devoid of myofibroblasts [figures 3 and 5 ]. the finding suggests that the increase in myofibroblasts in the stroma of osccs may be an important event in the invasion of epithelial cells. this finding is in agreement with those reported by etemad., in 2009, eliene., in 2011 who found that the presence of myofibroblasts was significantly higher in osccs compared to normal mucosa and dysplastic epithelium which were devoid of it. barth., in 2004 investigated tumor - free mucosa and sccs of the oral cavity, the pharynx and larynx and showed the presence of -sma positive myofibroblasts in sccs and their absence in tumor - free mucosa. 2007 in a correspondence article reported myofibroblasts at the invasive front of the osccs and no myofibroblast were found in the stroma of normal mucosa and epithelial dysplasia. zidar., in 2002 found increased presence of myofibroblast in squamous carcinoma of larynx and lack of myofibroblast in the stroma of normal laryngeal mucosa and laryngeal epithelial hyperplastic lesions. chaudhary., in 2012 compared the presence of myofibroblasts in oscc, verrucous carcinoma (vc), high - risk epithelial dysplasia (hred), low - risk epithelial dysplasia (lred) and nom. the -sma positive myofibroblast was expressed in 97.29% of oscc, 86.66% of vc and 46.66% of hred but not in nom and lred. in the present study, the association between expression of calponin in myofibroblast and the parameters such as tumor grade and invasive patterns of oscc were also evaluated. the expression of calponin among early invasive scc and well, moderate and poorly differentiated osccs was compared. all the grades showed significant expression of calponin compared to nom but there was no significant difference in calponin expression among the grades of oscc. this is in accordance with the results obtained by etemad., in 2009 who were unable to find significant difference between the three histologic grades of oscc. these findings may suggest that the transdifferentiation of myofibroblasts is induced somewhere in the invasive stage of oscc irrespective of the tumor cell differentiation. the histological features of oscc may differ widely from area to area within the same tumor and it is believed that the most useful prognostic information can be deduced from the invasive front of the tumors, where the deepest and presumably most aggressive cells reside. the invasive front, defined as the band of tissue between the tumor front and adjacent normal tissue, may better reflect tumor prognosis than other parts of the tumor. according to bryne 's invasive tumor front grading system, a remarkable difference was observed between the grades of invasion when the oscc samples were classified by their invasive patterns, (p < 0.070) in the presence of stromal myofibroblasts. the calponin expression was significantly higher in grade iii invasive pattern when compared to grade i. in a similar study by eliene., in 2011, presence of myofibroblast was higher in tumors with a more diffuse histological pattern of invasion. these findings suggest that myofibroblasts are associated with the creation of a permissive environment for tumor invasion in oscc and play an active role in oscc invasion and metastasis., 2007 demonstrated that an abundance of myofibroblasts leads to more aggressive behavior of the sccs, including an elevated proliferative potential. the abundant presence of myofibroblasts, particularly at the invasive tumor front, was significantly associated with shorter overall survival. in 2010 concluded that the expression of myofibroblasts was an index of invasive behavior of odontogenic lesions and they suggested that target therapy can be beneficial as an auxiliary method for treatment of more invasive lesions. low power magnification showing normal mucosa with negative immunoexpression of calponin (ihc stain, x100) low power magnification showing poorly differentiated squamous cell carcinoma (h&e stain, x40). h&e = hematoxylin and eosin low power magnification showing poorly differentiated squamous cell carcinoma with calponin expression in stromal spindle cells (ihc stain, x40) high power magnification showing moderately differentiated squamous cell carcinoma (h&e stain, x400) high power magnification showing moderately differentiated squamous cell carcinoma expressing calponin (ihc stain, x400) in view of the observance of the presence of myofibroblasts in scc and their absence in normal, it seems that the genetically altered epithelium (carcinomatous epithelium) may have an inductive effect on the adjacent stroma to produce myofibroblasts. also lack of myofibroblasts in normal epithelium and their appearance in early invasive carcinoma indicates that transdifferentiation of myofibroblasts is induced somewhere in the invasive stage of scc irrespective of the epithelial cell differentiation. | background : oral squamous cell carcinoma (oscc) is the most common malignancy of the oral mucosa. stromal myofibroblasts play an important role in tumor invasion and metastasis, due to its ability to modify the extracellular matrix. the purpose of this study was to evaluate and compare the presence of myofibroblasts in normal mucosa, early invasive carcinoma and different grades of oscc.materials and methods : the study included the archival tissues of 18 oscc of well, moderate and poorly differentiated grades, three early invasive carcinomas and five normal mucosa. myofibroblasts were identified by immunohistochemical detection of h1 calponin.results:the percentage and intensity of h1 calponin were examined and positive immunostaining was observed in the myofibroblasts of all sccs and early invasive carcinomas ; however, these cells did not stain in the normal epithelium specimens. the presence of myofibroblasts was significantly higher in invasive pattern of osccs compared to normal mucosa cases (p < 0.070). a significant difference was not observed between the different grades of oscc (p 0.812).conclusion : these findings show the presence of myofibroblasts in oscc but not in normal mucosa, suggesting that the genetically altered epithelium (carcinomatous epithelium) may have an inductive effect on the adjacent stroma to produce myofibroblasts. also transdifferentiation of myofibroblasts is induced somewhere in the invasive stage of scc irrespective of the epithelial cell differentiation. |
iridoschisis is an uncommon condition characterized by separation of the anterior iris stroma from the posterior stroma.1 iridoschisis is a rare disease, of which a only few cases have been reported.2 most patients are aged over 65 years and the condition is usually bilateral,2,3 associated with angle - closure glaucoma,35 trauma,4 and syphilitic interstitial keratitis.6 iridoschisis presents with two or more layers of iris tissue that usually is characterized by iris fibers flowing in the anterior chamber.7 in 2001, agrawal reported the rare association of unilateral iridoschisis and ipsilateral lens subluxation.5 in 2004, adler reported unilateral iridoschisis and bilateral lens subluxation.8 to the best of our knowledge, the current report is the first about unilateral iridoschisis and ipsilateral lens displacement into the vitreous cavity. a 67-year - old woman presented with visual loss in the right eye of two months duration. her spherical equivalent refractive error was + 11.75 diopters (d) in the right eye and 0.75 d in the left eye. the intraocular pressure was 15 mmhg in the right eye and 19 mmhg in the left eye. slit - lamp examination showed sectoral schisis of the right iris stroma inferonasally from the 4 to 6 oclock position (figure 1). a normal peripheral anterior chamber was seen in the right eye, but no lens was present (figure 1). postmydriatic examination showed lens displacement into the vitreous cavity in the right eye (figure 2), and no abnormalities in the left eye. the endothelial cell densities in the center were 1,263 cells / mm in the right eye and 2,849 cells / mm in the left eye (figure 3a, b). a 25-gauge pars plana vitrectomy and lens removal were performed immediately in the right eye. iris tissue flowing into the anterior chamber was cut with a vitreous cutter during surgery. one month postoperatively, the va was 1.0, and the spherical equivalent refractive error was + 11.25 d. the endothelial cell density inferotemporally was 763 cells / mm, 2,525 cells / mm superotemporally, and 1,623 cells / mm in the center (figure 4a c). the postoperative progress has been satisfactory and we are planning scleral fixation of an intraocular lens. we considered that lens subluxation had worsened to lens displacement into the vitreous cavity in this patient. based on previous reports, it is plausible that iridoschisis is related to lens subluxation or lens displacement into the vitreous cavity.5,8 however, adler reported unilateral iridoschisis in the right eye and bilateral lens subluxation;8 that the patient presented with lens subluxation without iridoschisis in the left eye and had a longstanding history of periocular eczema.8 our current patient had no such general complications. future studies will clarify the connection between iridoschisis and lens subluxation or lens displacement into the vitreous cavity. the corneal endothelial cell density decreased to 1,263 cells / mm preoperatively in our patient. it is interesting to confirm the presence of iridocorneal contact overlying the area of iridoschisis using ultrasound biomicroscopy.1 although there was no clinical evidence of iridocorneal contact in the current case, we speculated that intermittent iridocorneal touch might have a normal distribution in the left eye. have caused this change, as srinivasan reported,1 as the free end of the iris fibers sometimes seemed to be in contact with the corneal endothelium before surgery. in addition, there was a report that the iris fibers touched the corneal endothelium, and caused decreasing corneal endothelial cell density.9 finally, the inferonasal endothelial cell density, which corresponds to iridoschisis, decreased extremely after surgery. we cut iris tissue flowing into the anterior chamber during surgery, with care not to injure the corneal endothelial cells. patients with iridoschisis should undergo specular microscopy to determine the status of the corneal endothelial cells. we report a case of iridoschisis with lens displacement into the vitreous cavity of the right eye. the postoperative progress has been good but the corneal endothelial cell density decreased from preoperatively | we report the case of a 67-year - old woman with a lens that was displaced into the vitreous cavity in one eye and ipsilateral iridoschisis. she was free from a history of ocular trauma or of heritable ocular disease. her best - corrected visual acuity was 1.2 bilaterally and right eye showed signs of iridoschisis. the corneal endothelial cell density decreased to 1,263 cells / mm2 in the right eye preoperatively. we speculated that iris tissue flowing in the anterior chamber might have intermittently touched the corneal endothelium. 25-gauge pars plana vitrectomy and lens removal were performed immediately. free - floating iris tissue was cut during surgery with care not to injure the corneal endothelial cells. the postoperative progress was satisfactory and scleral fixation of an intraocular lens is planned. iridoschisis is an uncommon cause of lens displacement into the vitreous cavity. |
surgical treatment is the best option for the treatment of many pulmonary diseases but many potentially curable diseases, first of all bronchopulmonary carcinoma, occurs in patients with impaired pulmonary function due to the associated chronic obstructive pulmonary disease (copd), which is caused by common etiologic factors primarily smoking (1, 2). patients with airway obstruction are limited in their daily lives ; additional loss of lung tissue contributes to their disability. loss of lung tissue in them can severely deteriorate the function of ventilating the lungs which may lead to respiratory failure, various cardiopulmonary complications including death. these patients are at increased risk of perioperative immediate and postoperative complications such as acute respiratory failure, need for mechanical ventilation for more than 48 hours, or the need to re - intubation, atelectasis of lung tissue of different volumes, pneumonia, pulmonary embolism, and cardiac complications as acute myocardial infarct, heart rhythm disorders, heart failure, pulmonary edema, and also chronic pulmonary insufficiency because of the removal of functional lung tissue (1 - 9). the aim of the surgeon in performing resection surgery is curative resection with preservation of the maximum amount of functional lung tissue. functional loss that results due to pulmonary resection depends on the extent of resection, the relative functional status of removed in relation to the remaining tissue and on the degree of damage of lung function preoperatively (1 - 3, 8 - 11). the assessment of possibility of the planned operation is possible after a detailed analysis of the clinical, laboratory and radiological findings, and pulmonary function tests (1 - 24). high risk can be justified if patients are suffering from cancer and there may be placed a question -what is the risk of postoperative complications in relation to the disease that is certainly fatal if not operated. the possible extent of resection is carefully planned preoperatively, depending on the patient s cardiovascular and pulmonary functional status and there is a need for a close coordination of pulmonologists and surgeons (1 - 24). several authors dealt with predicting lung function after the planned surgical resection of lung parenchyma and monitored the accuracy of the forecasts according to the methods of forecasting, and less of them compared the lung function before and after resection and determined the functional loss. to determine how big the reduction of lung function is after surgical resection of lung parenchyma of different volumes. the study was done on 58 patients operated at the clinic of thoracic surgery of kcu sarajevo, who had previously treated at the clinic of pulmonary diseases podhrastovi in a period from 01.06.2012. to 01.06.2014. patients were undergone to following resection surgery : pulmmectomy (left, right), lobectomy (left and right : upper, lower). pulmonary function tests were done when the patient was clinically stable, and after taking a complete bronchodilator therapy if it was needed. all patients did a complete spiropletizmographic processing as follows : fvc (forced vital capacity), fevl (forced expiratory volume in one second), and the flow - volume curve, total pulmonary resistance (r tot) including bronchodilator test, rv (residual lung volume), tlc (total lung capacity). the patients were divided into 12 groups of which can be seen in table 1. type of surgical resection, the number of cases for each operation, sex and average age of patients table1. the study included 45 males average age of 60.49 and 13 females average age of 55.65 years. the degree of lung function impairment is preferably determined based on fev1 as this is the most objective parameter of lung function, and indicates the obstructive and restrictive disorders ie. indicates the type and degree of impairment of ventilatory lung function. for normal spirometric parameters standards established under the auspices of european coal and steel community, which was the forerunner of today s european union, taking into account height, weight, age and sex of the patient and which are entered into the computer of each spiroplethyzmographic apparatus. complete spiroplethyzmographic processing was done on the device master lab jaeger, a determination of blood gases on the device radiometer abl 505 in the laboratory of clinical physiology of breathing of clinic for pulmonary diseases and tb podhrastovi. pulmonary functional tests were done 7 and 10 days before surgery, and after the patient has taken a bronchodilator (if it was required), and 2 months after the operation, when it is considered that there is the stabilization of the resulting functional lung status. the results are shown in the tables and figure, where it can be seen how fev1 decreased after the surgical lung resection of different volumes. it is showed how much the average values of fev1 were in each group of patients, that is : what is the normal value in liters for that group, how much they have achieved before surgery in liters and as a percentage of their normal value, what are the actual postoperative fev 1 values in liters and as percentage of the normal values, and how much the postoperative decrease in fev1 in liters, and as percentage of actual preoperative values and as percentages of the average norm for the group is. fev1 decreased as compared to preoperative values for 0.98l or 36.70% in men, and 0.90l or 44.12% in women. reduction in pulmonary function after left pulmectomy legend : fev1 = forced expiratory volume in one second. normal = norm. -average normal value of fev1 (according to gender, age, height, weight) for this group of patients in liters (l). % of normal - achieved (realized) average value of fev1 in per cents of normal values for this group of patients loss of function reduction of fev1 compared to preoperative values expressed in liters (l), in per cents of preoperative values, and in per cents of normal values m - males f - females table 3. fev1 decreased as compared to preoperative values for 1.38 l or 43.81% in men, and 0.54 l or 34.84% in women. fev1 decreased as compared to preoperative values for 0.51l or 17.65% in men, and 0.51l or 22.08% in women. fev1 decreased as compared to preoperative values for 0.55l or18.46% in men, and 0.38l or 17.02% in women. fev1 decreased as compared to preoperative values for 0.41 l or 15.07% in men, and 0.41l or 20.27% in women readuction in pulmonary function after right upper lobectomy table 7. fev1 decreased as compared to preoperative values for 0.60 l or 22.14% in men, and 0.22 l 13.10% in women reduction in pulmonary function after right lower lobectomy figure 1. fev1 decreased as compared to preoperative values for each type of lung resection reduction in pulmonary function after different volumes of lung resection compared to preoperative values expressed as per cents although surgical treatment is the best option for treating many lung diseases including bronchopulmonal cancer, many lung diseases, tumors in the first place, occur in patients with impaired pulmonary function due to the associated copd, what is caused by the common etiological factors, especially cigarette smoking (1, 2). the decision about surgery in these patients is difficult because of the lack of respiratory reserve causing postoperative morbidity and mortality (3 - 5). leading cause of postoperative morbidity and mortality are pulmonary complications (5, 6, 8, 9). indicating of thoracic surgical operation depends on the type, localization and extent of the lung process (3, 4, 6 - 9, 12 - 16). in the assessing the possibility of performing each thoracic surgical operation there is necessary close cooperation between pulmonologists and thoracic surgeons (6 - 9). the possible extent of lung resection is planned preoperatively depending on the cardiovascular and pulmonary functional status (1 - 24). for each patient preoperative staging is essential that, if carefully designed, may be correct in 70 - 80% of cases (17). (18) in a series of 54 patients examined functional loss 3 months after surgery. according to them pulmectomy reduces lung volume by 30 to 40%, and less resection to 15%. in our study, the functional loss was, at left pulmectomy for men for fev1 0.98 l or 36.7% compared to preoperative values, and in women, 0.9 l, or 44.12%. with the right pulmectomy functional loss in men is for fev1 1.38 l, or 43.81%, while for women it is 0.54 l or 34.84%. in the left upper lobectomy in men fev1 is reduced to 0.51 l, or 17.65%, in women it is also 0.51 l, but 22.08%. in the left lower lobectomy in men fev1 decline of 0.55 l, or 18.46%, and 0.38 l in women or 17.92%. in the right upper lobectomy men and women lost in fev1 0.41 l but compared to preoperative values, the decrease was 15.97% for men and for women 20.30%. in the right lower lobectomy in men fev1 decreased by 0.60 l, or 22.14%, while for women it is 0.22 l, or 13.10%. bolliger ct and colleagues (19) have been doing tests of lung function in 68 patients before and 3 and 6 months after lung resection. in 50 patients lobectomy and in 18 pulmectomy was done three months after lobectomy fvc, fev1, tlc, dlco, and maximum oxygen consumption were significantly lower than the preoperative values, grew between 3 and 6 months, and 6 months later still showed significantly less values than preoperative ones. in pulmectomy six months after resection a functional deficit has persisted in comparison with preoperative values for fev1 and so 9% for lobectomy and 34% for pulmectomy. the results were identical regardless of whether the preoperative pulmonary function was normal or damaged. 20) did testing on 20 patients with lobectomy whose average preoperative fev1 was 1.73 l. preoperative tests were performed within a month before the surgery and post - operative after 1 week to 3 years (average 1.5 years). the average loss of fev1 in lobectomy was 150 ml and the biggest fall was 870 ml. these authors suggest that up to 70% of lung function may be present in diseased parts of the lung and therefore they require careful preoperative tests. in our study the biggest drop occurred after lobectomy in a patient 69 years old who had right lower lobectomy and in whom the fev1 decreased by 0.93 l, but as compared to preoperative values expressed as% of normal for the patient it made a reduction of 25.2%. the largest decline after pulmectomy occurred in a patient who had right pulmectomy where fev1 decreased by 1.66 l but as compared to preoperative values expressed as% of normal for the patient it was 52% (it was the patient with advanced copd who did not take the recommended bronchodilator therapy) and patient with left pulmectomy where fev1 decreased by 1.71 l but as compared to preoperative values expressed as% of normal for the patient it was 49%. zether bh. have determined the effects of pulmonary resection on lung function in 62 patients. of the patients who had lobectomy eight had irradiation treatment after surgery. they lost an average of 5.47% fev1 per resected segment, although it is not clear whether this is a transient phenomenon due to regional postirradiative pneumonitis or permanent loss, and patients who had not irradiation have lost on average 2.84% of fev1 by resected segment. these authors worked postoperative pulmonary function tests, on average, 8.5 months after lobectomy and 7.2 months after pulmectomy (from 24 days to 5 years). in 49 patients who had lobotomy mean preoperative value for fev1 were 2.49 0.13 l, mean post - operative value of fev1 was 2.12 0.1. fev1 is reduced for 13.3 2.2% and 3.27 0.55% for the resected segment. middle fev1 of 13 patients with pulmectomy before surgery was 2.24 0.52 l. middle postoperative fev 1 was 1.56 0.37 l, it was 29.0 3.94% decrease in fev1 or 3.07 0.42% for the resected segment. the remaining lung tissue, after lung resection, adapts to the loss of part of the area for gas exchange by creating a new functional area of ventilation and perfusion with an increase of relationship this adaptation occurs in the first few months after surgery, and for 6 - 12 months remaining lung tissue receives its definitive form. however, in patients with copd distension is not likely to happen because the lung before surgery was maximally stretched and can not be further stretched. therefore the creation of new functional area is limited (24). in pulmectomy unilateral obstruction due to tumor or partially destroyed parenchyma can reduce postoperative forced expiratory flow rate less than expected. alli mk (22) showed that lung function was relatively stable after pulmectomy but in lobectomy it is reported to exist disproportionate early loss, and afterward it came to significant functional improvement as found in the study bolliger j. (19). pulmonary function was significantly increased during the long follow - up (more than 3 months). clinicians should be aware of this phenomenon, especially in patients with marginal lung function and be ready to intervene to prevent the development of acute respiratory failure shortly after operation. alli mk (22) found that, in the early postoperative period, a decline in fev1 of 30 % for lobectomy existed with subsequent recovery. in a study of marcos j (15) it is not confirmed, he found the mean decrease for 3 months for fev1 of 17% compared to preoperative values. today there is an increasing need for resective thoracosurgical operations, primarily due to an increase in the number of patients with lung cancer, which often develops in patients with already impaired pulmonary function due to associated chronic obstructive pulmonary disease (copd), that is caused by the common etiological factors primarily cigarette smoking. patients with airway obstruction are limited in their daily lives before surgery, and an additional loss of lung tissue after resection contributes to their inability. each resection of lung tissue leads to a decrease in lung function and according to our study in pulmectomy up to 44%, and lobectomy and up to 22% compared to preoperative values which should be taken into account in preoperative assessment. during the preoperative functional assessment two questions should be asked : whether cardiopulmonary reserve is sufficient to make the patient survive the operation and whether the patient will be chronically unable of daily life due to insufficient lung function. potential benefit from pulmonary resection should be carefully balanced in relation to postoperative morbidity and mortality. | introduction : in recent years an increasing number of lung resections are being done because of the rising prevalence of lung cancer that occurs mainly in patients with limited lung function, what is caused with common etiologic factor - smoking cigarettes.objective:to determine how big the loss of lung function is after surgical resection of lung of different range.methods:the study was done on 58 patients operated at the clinic for thoracic surgery kcu sarajevo, previously treated at the clinic for pulmonary diseases podhrastovi in the period from 01.06.2012. to 01.06.2014. the following resections were done : pulmectomy (left, right), lobectomy (upper, lower : left and right). the values of postoperative pulmonary function were compared with preoperative ones. as a parameter of lung function we used fev1 (forced expiratory volume in one second), and changes in fev1 are expressed in liters and in percentage of the recorded preoperative and normal values of fev1. measurements of lung function were performed seven days before and 2 months after surgery.results:postoperative fev1 was decreased compared to preoperative values. after pulmectomy the maximum reduction of fev1 was 44%, and after lobectomy it was 22% of the preoperative values.conclusion:patients with airway obstruction are limited in their daily life before the surgery, and an additional loss of lung tissue after resection contributes to their inability. potential benefits of lung resection surgery should be balanced in relation to postoperative morbidity and mortality. |
despite multi - modality therapy, cancer of the head and neck region carries recurrence rates as high as 50%, depending on the initial stage [1, 2 ]. available treatment modalities include, either alone or in combination, chemotherapy, surgical resection, and, less commonly, radiation. as many patients have already been treated with a full radiation dose, treatment with external beam radiation is either contraindicated, or at least requires special techniques. although localized delivery of high - dose radiation remains a viable option in appropriately selected patients, the attendant soft tissue deficits in the area of previous resection, combined with the poor wound healing inherent to previously radiated tissues make the likelihood of wound healing complications significantly greater (satisfactory perioperative outcomes are especially important in the neck, where poor wound healing can lead to exposure of vital structures, fistula, carotid blowout, and also the risk of death). brachytherapy is delivered either via removable catheters secured to the area of surgical resection through which radioisotopes are inserted (often referred to as high - dose radiation, or hdr), or with permanent radioisotope - containing seeds placed into the wound bed at the time of surgical resection (often referred to as low - dose radiation, or ldr). as external beam radiation (ebr) alone or in combination with surgery and chemotherapy, is a first line treatment for nearly all head and neck cancer, most patients with recurrence are not candidates to receive further ebr. brachytherapy can mitigate more global toxicity associated with ebr by allowing precise targeting of the radiation to a limited area (often less than 1 cm in radius), making many patients with local recurrence candidates for surgical resection. because re - resection usually involves previously radiated tissues, the likelihood of wound healing complications becomes significant unless vascularized flaps are utilized in the reconstruction. historically, there has been some hesitation to perform concurrent flap coverage and brachytherapy because of concern regarding the toxic effects of the directly adjacent radiation to the newly manipulated tissue [4, 5 ]. the safety and potential for morbidity of flap reconstruction and brachytherapy has been studied in the past, but the cohort of particularly high risk patients with recurrent head and neck cancer have not yet been specifically evaluated, nor has the relative safety of hdr versus ldr been compared. this leaves an important gap in our understanding of how best to treat this difficult clinical scenario. therefore, the purpose of this study was to compare the safety and efficacy of flap coverage of surgical wounds in patients with rh&nc who concurrently undergo either hdr or ldr. a retrospective chart review was performed of patients between 2007 and 2016 with recurrent head and neck cancer with metastases in the cervical lymph node field who underwent surgical treatment and concurrent brachytherapy. the head and neck surgery team performed the surgical ablation, which involved resection of the recurrent mass, as well as in almost every case some form of cervical lymphadenectomy (either selective neck dissection, modified radial neck dissection, or radical neck dissection). in order to minimize exposure of the surgical team to the brachytherapy radiation, the plastic surgery team would then elevate their chosen flap prior to assisting the radiation oncologist in precise placement of the hdr catheters or ldr mesh / beads (figure 1). high - dose radiation was delivered via temporary implantation of catheters that delivered ir for 4 - 6 days and then removed at the bedside, and ldr was delivered via permanent cs radioactive seeds (loaded into polyglactin suture or mesh). intraoperative photographs of a) brachytherapy catheters (hdr) in place, b) catheters covered by pedicled pectoralis major muscle flap (note catheters emerging from skin), and c) overlying split thickness skin graft the ldr dose was 80 gy at 0.5 cm from the implant. implantation was done with seeds placed 1 cm apart to achieve the prescribed dose. the hdr dose was 20 gy in 2 gy per fraction, delivered twice daily. high - dose radiation catheters were also placed on the tumor bed 1 cm apart and planning was done using brachyvision software (varian ; palo alto, ca, usa), and the treatment started within a week of implantation. after securing the brachytherapy catheters / seeds, all pedicled flaps in this series were the pectoralis major muscle (with or without overlying skin), and the free flaps were either rectus abdominus muscle, or anterolateral thigh fascia and overlying skin. a split - thickness skin graft (10 - 12/1000 of an inch) was placed on top of the flap if needed in order to achieve complete wound closure at the completion of the operation. the primary endpoint of this study was flap viability, defined as survival of the flap sufficient to maintain wound coverage and obviate the need for future procedures in order to obtain would coverage. secondary endpoints included wound healing complications (classified as minor, which resolved with wound care, and major, which required return to the operating room), seroma, bleeding complications, and surgical site infections. all values are presented as (mean) (standard error of the mean). statistical analysis was performed using fisher 's exact test (graphpad software, inc ; la jolla, california), and statistical significance was set at p 0.05). patient demographics and operative procedures scc squamous cell carcinoma, hdr high - dose - rate, ldr low - dose - rate hdr high - dose - rate, ldr low - dose - rate hdr the concept of local delivery of radiation as a treatment modality nearly immediately followed the discovery of radioactivity by henri becquerel in 1896. while the initial iterations of brachytherapy were permanently implanted (and therefore ldr), the development of radiation afterloaders led to the advent of removable catheters (enabling short courses of hdr). this and other innovations in the second half of the twentieth century led to improved patient and healthcare provider safety and efficacy, and therefore a resurgence in interest in brachytherapy. high - dose and low - dose radiation are important elements of the multi - disciplinary and multi - modality treatment of a number of forms of cancer, including breast, prostate, endometrial, and cervical cancer, in addition to head and neck cancers. unlike ebr, brachytherapy is able to deliver a high - localized dose with relative sparing of critical normal tissues due to rapid tissue falloff, thereby leading to a high rate of local tumor control with limited long - term morbidity [7, 8, 9 ]. in particular, pham. recently showed that cs ldr treatment in rh&nc led to survival rates comparable to that of ebr, with a reduced rate of radiation - induced toxicity. similarly, kishan. showed good local recurrence rates and low long - term morbidity with hdr. almost all patients with rh&nc who are deemed repeat surgical candidates have been previously treated with ebr. these locally recurrent tumors are usually difficult to resect cleanly, given the extensive tissue changes such as fibrosis, edema, and loss of tissue planes from prior ebr. attaining wide clean margins is difficult in such situations, and this poses a significant risk of local recurrence of tumor despite a complete surgical resection. it is for this reason that brachytherapy is used at our institution in patients with suspicious, close, or positive margins based on frozen section pathological analysis. the quality of the skin and soft tissue within and adjacent to the site of resection and neck dissection site is usually extremely poor. therefore, in these cases it is nearly impossible to achieve reliable coverage of the resulting wound with primary closure alone. the pectoralis major muscle flap, first described by ariyan in 1979, remains the most reliable means of providing well - vascularized tissue for coverage of exposed vital structures in the head and neck region. the pectoralis major muscle flap is usually sufficient to provide coverage, but in circumstances where the pectoralis muscle alone is insufficient or unavailable, free tissue transfer is mandated. while coverage using well - vascularized muscle of this kind is thought to be robust, reliable, and resistant to most local insults, there are rare circumstances when flaps are exposed to conditions as noxious as targeted local radiation. few reports that exist in the literature that assessed the safety of brachytherapy in combination with flaps suggest that the two modalities can be combined. ross. reviewed their series of patients with head and neck cancer (both primary and recurrent) who were reconstructed using microvascular free tissue transfer, and compared those who had received ldr and those who had not. their study found that the overall complication rate was higher in the ldr group (38.3% vs. 15.9%). despite the higher complication rate, they concluded that the survival benefits conferred by ldr outweighed the manageable increase in complications, and therefore advocated its use. retrospectively studied the use of ir hdr catheters in 18 patients with either primary or recurrent squamous cell carcinoma, and determined that local hdr did not increase flap morbidity.. looked at ir hdr catheters in 13 subjects with scc, and panchal. looked at ir hdr catheters in 10 subjects with scc, parotid tumors, or sarcoma. in addition to the heterogeneous histology, the study populations combined subjects with primary and recurrent cancer, and had a mixed population of patients who had undergone prior radiation and those who had not. these 4 studies make important contributions to the literature, but they leave important questions unanswered including the safety of brachytherapy in the patients with rh&nc (which is the group at highest risk for complications), and whether there is a difference in healing when patients are treated with hdr versus ldr. the data presented herein shows 100% flap viability regardless of the modality of brachytherapy used. additionally, in this high risk cohort of 23 patients, the overall complication rate was low (only 3 occurrences of minor wound breakdown in the hdr group, and 2 hematomas, 1 superficial surgical site infection, and 2 occurrences of minor wound breakdown in the ldr group). the only complication that required a return to the operating room was the hematoma, which was drained. taken together, the results of our study indicate that either hdr or ldr can be safely delivered with concomitant flap reconstruction. the data does not indicate that either form of brachytherapy is more likely to cause flap morbidity (although this finding may be limited by the relatively small number of patients included in the study), and therefore one form of brachytherapy can not be recommended over the other. in conclusion, in patients who have flap reconstruction and immediate postoperative radiotherapy following salvage procedures for recurrent head and neck cancer, neither hdr nor ldr impacted long term flap viability or skin graft survival. flap coverage of defects (with or without skin grafting) in combination with brachytherapy is a safe and effective means of providing soft tissue coverage in these challenging patients. | purposewhile brachytherapy is often used concurrently with flap reconstruction following surgical ablation for head and neck cancer, it remains unclear whether it increases morbidity in the particularly high risk subset of patients undergoing salvage treatment for recurrent head and neck cancer (rh&nc).material and methodsa retrospective chart review was undertaken that evaluated patients with rh&nc who underwent flap coverage after surgical re - resection and concomitant brachytherapy. the primary endpoint was flap viability, and the secondary endpoints were flap and recipient site complications.resultsin the 23 subjects included in series, flap viability and skin graft take was 100%. overall recipient site complication rate was 34.8%, high - dose radiation (hdr) group 50%, and low - dose radiation (ldr) group 29.4%. there was no statistically significant difference between these groups.conclusionsin patients who undergo flap reconstruction and immediate postoperative radiotherapy following salvage procedures for rh&nc, flap coverage of defects in combination with brachytherapy remains a safe and effective means of providing stable soft tissue coverage. |
epithelial cells have been recognized as playing an important role in mucosal immunity through the expression of proinflammatory cytokines in response to microbial injury. among cytokines produced in the intestinal mucosa during sepsis and endotoxemia, interleukin (il)-6 is particularly important because of its multiple significant biological effects. although commonly considered a proinflammatory cytokine, there is also evidence that il-6 has important antiinflammatory properties and may exert protective effects in various tissues [46 ]. il-6 plays an essential role in the intestinal barrier in l. monocytogenes - infected human intestinal epithelial cells [7, 8 ]. because of the multiple biological effects of il-6, a better understanding of the molecular regulation of enterocyte il-6 production and methods to modulate il-6 production in intestinal mucosa in infected enterocytes may have important clinical implications. over the last decade, a multitude of studies have verified the role of parp-1 activation in a wide range of pathophysiologic conditions, such as arthritis, asthma, inflammatory bowel disease, lung inflammation, multiple organ failure, and septic shock. the marked beneficial effect of parp inhibitors in these animal models of various diseases also suggests that parp inhibitors can be exploited to treat human inflammatory diseases. the recent studies in a variety of rodent models of experimental colitis support the role of parp-1 activation in the pathogenesis of the disease [1014 ]. pj-34, a novel and highly potent (the in vitro ic50 is 10000 times lower than that of the prototypical compound 3-aminobenzamide) parp-1 inhibitor, is suitable for mechanistic investigations into the regulatory roles of parp. furthermore, pj-34 treatment improved survival in septic shock induced by bacterial peritonitis in pigs. however, the role of parp in the pathogenesis of salmonella enteritis and the effect of the parp-1 inhibitor pj-34 and genetic knock down of parp-1 sirna on the inflammatory response of enterocytes to salmonella infection are not known, prompting us to investigate the role of pj-34 in salmonella - induced intestinal inflammation and its mechanisms. in this study, we aimed to examine the effect of pj-34 on salmonella - induced il-6 production in caco-2 cells in vitro and the intracellular signaling pathways regulating the effect. pj-34 was purchased from inotek corporation (beverly, ma) and stock solutions made in dimethylsulfoxide (dmso). the inhibitor was added to cells at the specified concentrations about 3060 minutes before infection. the wild - type s. typhimurium strain sl1344 has been described previously [17, 18 ]. bacteria were grown overnight in static cultures with minimal aeration in luria - bertani (lb) medium. the bacteria were collected by centrifugation at 14000 g for 5 minutes, washed with sterile phosphate - buffered saline (pbs), and resuspended in tissue culture medium without antibiotics at a density of 4 10/ml. twenty - five l aliquots of this suspension (10 bacteria) were used to infect the cells. caco-2 cells (atcc, rockville, md), a transformed human colonic epithelial cell line, were grown in dulbecco modified eagle medium (dmem) supplemented with 10% heat - inactivated fetal calf serum, 100 units / ml penicillin, 100 g / ml streptomycin sulfate, and 20 mm hepes (sigma) in a 5% co2 atmosphere at 37c. cells were seeded in 12-well tissue culture plates (4 cm / well ; bd biosciences) and used at 60%80% confluence. cytosolic, nuclear, and membranous extracts from uninfected, infected, or pj-34-treated caco-2 cells were prepared by the method of wang. with slight modifications. cells were washed twice with ice - cold phosphate - buffered saline, lysed in buffer a (10 mm hepes - koh, ph 7.8, 10 mm kcl, 2 mm mgcl2, 0.1 mm edta, 0.1 mm egta, 0.7% nonidet p-40) with protease and phosphatase inhibitors for 30 minutes on ice, vortexed vigorously for 15 s, and centrifuged at 3000 g at 4c for 10 minutes (the supernatants are the cytosolic fractions). the pelleted nuclei and membrane were resuspended in buffer b (40 mm hepes - koh, ph 7.8, 350 mm nacl, 2 mm mgcl2, 1 mm edta, 0.2 mm egta, 20% glycerol, 1% nonidet p-40) with protease and phosphatase inhibitors for 60 minutes on ice, mixed vigorously for 10 s at 15, 30 and 45 minutes, and centrifuged at 15,000 g at 4c for 30 minutes. caco-2 monolayers were infected (apically, in the case of the polarized monolayers) in triplicate for 1 hour at 37c. the medium was aspirated at the end of the infection period, the cells were washed twice with sterile pbs, and medium containing gentamicin at 100 g / ml was added. after incubating for 5 hours at 37c, the supernatant medium was collected and il-6 concentrations were determined by enzyme - linked immunosorbent assay (elisa) as described below. the cells were washed with pbs and lysed with 0.2 ml of 1% triton x-100. an aliquot of the lysate was used to determine protein concentration by the dc protein assay (bio - rad, hercules, ca) following instructions provided by the manufacturer. the il-6 concentration was assessed using an opteia human il-6 enzyme - linked immunosorbent assay (elisa) set (bd biosciences) as described by the manufacturer. to allow comparison between multiple experiments, the amount of il-6 produced was normalized to the protein content of the cell monolayer. because of variations in baseline il-6 production, the results were expressed as fold increase, representing the normalized il-6 produced by infected monolayers divided by the normalized il-6 produced by control, uninfected monolayers. total rna was prepared from control or infected cells with the trizol reagent (invitrogen corporation, carlsbad, ca), following the manufacturer 's directions. the rna was reversetranscribed with random hexamers using the geneamp kit (roche, nutley, nj) as described in detail earlier [17, 18 ]. real - time reverse - transcription pcr analyses were performed in a fluorescence temperature cycler (lightcycler ; roche diagnostics) as described previously. this technique continuously monitors the cycle - by - cycle accumulation of fluorescently - labeled pcr product. briefly, cdna corresponding to 10 ng of rna served as a template in a 10 l reaction containing 4 mm mgcl2, 0.5 m of each primer, and 1 lightcycler - faststart dna master sybr green i mix (roche diagnostics). samples were loaded into capillary tubes and incubated in the fluorescence thermocycler (light - cycler) for an initial denaturation at 95c for 10 minutes followed by 45 cycles, each cycle consisting of 95c for 10 s, 58c for 5 s, and 72c for 20 s. at the end of each run, melting curve profiles were produced by cooling the sample to 65c for 15 s and then heating slowly at 0.20c / s up to 95c with continuous measurement of fluorescence to confirm amplification of specific transcripts. cycle - to - cycle fluorescence emission readings were monitored and analyzed using lightcycler software (roche diagnostics). the specificity of the amplification products was further verified by subjecting the amplification products to electrophoresis on a 2% agarose gel. the fragments were visualized by ethidium bromide staining, and the specificity of pcr products was verified by sequencing of representative samples. the following primers were used : il-6, 5- atg aac tcc ttc tcc aca agc gc-3 (forward primer) and 5-g aag agc cct cag gct ggactg-3 (reverse primer, 628bp) ; and glyceraldehyde-3-phosphate dehydrogenase, 5-ccagccgagccacatcgctc-3 (forward primer) and 5-atgagccccagccttctccat-3. standard curves were obtained for each primer set with serial dilutions of cdna. relative expression was given as a ratio between target gene expression and glyceraldehyde-3-phosphate dehydrogenase expression. equal amounts of total protein were separated by sds - page and then transferred to nitrocellulose membranes by semidry blotting as previously described [17, 18 ]. after blocking the membranes with 5% nonfat dry milk, they were probed with antibodies to phosphorylated akt (cell signaling, beverly, ma), phosphorylated ib (new england biolabs, beverly, ma), p65 nf-b or phosphorylated erk (santa cruz biotechnology, santa cruz, ca), and phosphorylated jnk or p38 (new england biolabs, beverly, ma). the membrane was continuously incubated with appropriate secondary antibodies coupled to horseradish peroxidase and developed in the ecl western detection reagents (amersham pharmacia biotech, piscataway, nj, usa). appropriate exposures to x - ray film were made, and the filters then stripped and reprobed with antibodies to total akt (cell signaling, beverly, ma), total erk, p38, ib, -actin, lamina / c, or e - cadherin (santa cruz biotechnology, santa cruz, ca) as appropriate. in figure 1, we found that enhancement of the il-6, either in protein secretion or mrna expression, was increased as higher concentration of pj-34 was applied (statistically significant after 20 m pj-34). then, we proceeded to study the mechanisms of the effect of pj-34 on il-6. to further elucidate the mechanism by which pj-34, parp-1 inhibitor, increased salmonella - induced il-6 production in intestinal epithelial cells, we examined the intracellular signaling pathways that have been implicated in expression of the cytokine. transcription factor nf-b is a key transcription factor in the regulation of cytokines and chemokines, including il-6 gene expression. activation of the nf-b pathway, as well as the mitogen - activated protein kinases (mapk) ; extracellular growth factor - regulated kinase (erk) and p38, has all been shown to be involved in salmonella - induced cytokines and chemokines production [17, 23, 24 ]. to determine the involvement of these signals in the effect of pj-34 on salmonella - infected intestinal epithelial cells, caco-2 cells were left untreated, or treated with 40 m pj-34, and then infected with the wild - type salmonella strain sl1344. activation of the nf-b pathway was assessed by examining nuclear translocation of nf-b and degradation of the inhibitor protein ib-. as shown in figure 2(a), salmonella infection resulted in degradation of ib- and nuclear translocation of nf-b. pj-34 enhances and prolongs the activation of nf-b by increasing ib- degradation and nuclear translocation of nf-b (figure 2(b)), resulting in subsequent upregulation of il-6 gene transcription. we also examined activation of mapks (erk, jnk, and p38 kinases) (figure 3), using antibodies specific to either the phosphorylated (activated) or total forms of these proteins. while inhibition of parp with pj-34 had no effect on salmonella - dependent phosphorylation of the p38 kinase and jnk, it had a clear and reproducible enhancing effect on activation of the erk kinase. these findings suggest that mapk pathways are involved in the regulatory effect of pj-34 on salmonella - induced il-6 production in intestinal epithelial cells. nf-b is a key transcription factor in the regulation of proinflammatory cytokines and chemokines. besides, an intimate relationship between that nf-b and parp-1 is demonstrated by the multiple lines of evidence showing that the synthesis of poly(adp - ribose) promotes nf-b transactivation and inhibition of parp-1 can attenuate this activation and subsequent cytokine expression. some reports have also demonstrated that parp inhibitors induced the phosphorylation and activation of akt in lipopolysaccharide - treated mice or cultured cells during oxidative stress, raising the protective effect of parp inhibition mediated through the pi3k - kinase / akt pathway. in our previous studies, we demonstrated nf-b and pi3k / akt pathways play important roles in the pathogenesis of salmonella enteritis [17, 18 ]. however, we found inhibition of parp-1 with pj-34 had no effect on salmonella - induced phosphorylation of akt (figure 4). our study gave first insight into the regulatory effect on the inflammatory responses by a novel parp-1 inhibitor pj-34 in salmonella - infected intestinal epithelial caco-2 cells. in this study, we found that wild - type s. typhimurium induced il-6 production in caco-2 cells, whereas pj-34 enhanced il-6 expression, either secreted il-6 or il-6 mrna. although commonly considered a proinflammatory cytokine, there is also evidence that il-6 has important antiinflammatory properties and may exert protective effects in various tissues [46 ]. the studies from hasselgren. suggest that il-6 produced by enterocytes may have antiinflammatory and cell - protective effects and that increased il-6 levels in gut mucosa may counteract some of the injurious effects of sepsis and endotoxemia [25, 26 ]. intense inflammatory response induced by salmonella infection results in destruction of the epithelial layer of the intestinal mucosa that may lead to translocation of bacteria and absorption of endotoxins into the circulation [27, 28 ]. consequently, translocation of bacteria and absorption of endotoxins may have profound systemic effects and may result in bacteremia as well as endotoxemia. however, il-6 has been described to be a mediator of epithelial barrier protection and endogenous il-6 plays an essential, nonredundant role in limiting intestinal injury and cell death. probiotic bacterium l. paracasei may exert some of their beneficial effects by enhancing il-6 production in enterocytes subjected to an inflammatory stimulus. in our study that might explain the marked beneficial effect of pj-34 in various models of local inflammation in rodents, in which the levels of il-6 were not measured. pj-34 may provide the antiinflammatory and protective effects on intestinal epithelial cells to counteract the invasion and injurious effects of salmonella endotoxemia through the upregulation of enterocyte il-6 production. to the best of our knowledge, up to now, no report has demonstrated that pj-34 upregulated salmonella - induced il-6 expression in intestinal epithelial cells. it has been previously reported that genetic deficiency or pharmacological inhibition of parp-1 confers beneficial effects in experimental models of colitis [1014, 32, 33 ]. blockade of parp inhibits intercellular adhesion molecule 1 (icam-1) or cyclooxygenase-2 expression [13, 33 ], neutrophil recruitment [13, 14, 33 ], oxidant generation, and mucosal injury in murine colitis. however, one (rare) report has demonstrated the effect of pj-34 on il-6 production. analysis of local expression of the il-6 in skeletal muscle after ischemia and 48 h of reperfusion showed significantly higher levels in the pj-34 treated group when compared with saline. activation of the nf-b pathway, as well as the mitogen - activated protein kinases (mapks) : extracellular growth factor - regulated kinase (erk) and p38, has been shown to be involved in salmonella - induced cytokines and chemokines production [17, 22, 23 ]. besides, the diversity of signal pathways involved in the protective effect of parp inhibitors depends on the experiment models and inhibitors used. most studies [1014 ] have shown reduced activation of transcription factors nf-b and ap-1, while very few studies have shown increased phosphorylation of mapk and pi3k / akt pathways except jnk. l-2286, a novel parp inhibitor, facilitated the ischemia - reperfusion - induced activation of akt, erk, and p38-mapk in both isolated hearts and in vivo cardiac injury. to further elucidate the mechanism by which pj-34 upregulated il-6 production, we examined various signaling pathways that have been implicated in expression of the cytokine. while inhibition of parp-1 with pj-34 had no effect on salmonella - induced phosphorylation of the p38 kinase, jnk, or akt, it had a clear and reproducible enhancing effect on activation of the erk kinase and nuclear translocation of nf-b. these findings are in contrast with previous reports in a549 lung epithelial cells or wrl-68 human liver cells. they found that pj-34 suppressed nf-b activation but not ap-1 in cytokine - stimulated a549 cells and had no effect on the expression of most chemokines. they also showed that pj-34 had no effect on phosphorylation of all mapks. in wrl-68 cells transfected with parp sirna or pharmacologically inhibited by pj-34, the phosphorylation of akt (ser) increased during oxidative stress compared with wild type. nevertheless, in accordance with our results, kameoka. demonstrated an inverse correlation between parp and nf-b activities. they showed that cl-3527 cells with the lowest parp content expressed 35-fold greater activity of nf-b than wild - type l1210 cells. however, a discrepancy seems to exist with cl-3527 and the parp-1-gene disrupted cells [39, 40 ], which showed markedly suppressed nf-b - dependent signaling. these authors explained that the discrepancy may be due to the difference in the residual poly(adp - ribosylating) activity in these mutants. it suggested that transcription factors and signal pathways varied between different cell types and may also vary within the same cell depending on stimulus. our results suggest that might also modulate a diverse array of signaling cascades beside gene expression. to our knowledge, this is the first in vitro report, which attributes a critical role to nf-b and erk in the salmonella - induced upregulation of il-6 in caco-2 cells, conferred by parp-1 inhibitor pj-34. in conclusion, the present study provides the first evidence that pj-34 may enhance il-6 production in enterocytes subjected to salmonella infection and that the effect of pj-34 is, at least in part, through nf-b and erk signal pathways. because other studies have shown that il-6 has antiinflammatory and protective effects in the intestinal mucosa, the present results offer a novel mechanism by which pj-34 may exert some of its beneficial effects, although additional in vivo experiments will be needed to define the role of il-6 in cell - protective effects provided by pj-34 treatment. | following salmonella invasion, intestinal epithelial cells release a distinct array of proinflammatory cytokines. interleukin (il)-6 produced by enterocytes may have anti - inflammatory and cell - protective effects, and may counteract some of the injurious effects of sepsis and endotoxemia. recent studies in a variety of rodent models of experimental colitis by using pj-34, a potent poly (adp - ribose) polymerase-1 (parp-1) inhibitor, support the concept that the marked beneficial effect of pj-34 can be exploited to treat human inflammatory diseases. the present study was to investigate the effect of pj-34 on salmonella - induced enterocyte il-6 production and its mechanisms. we found that pj-34 enhanced salmonella - induced il-6 production in caco-2 cells, either secreted protein or mrna expression. pj-34 treatment enhanced the activity of nf-b in salmonella - infected caco-2 cells. besides, the involvement of pj-34 in up - regulating il-6 production in s. typhimurium - infected caco-2 cells might be also through the erk but not p38 mapk, jnk or pi3k / akt pathways, as demonstrated by western blot of phosphorylated erk, p38, jnk and akt proteins. it suggests that pj-34 may exert its protective effect on intestinal epithelial cells against invasive salmonella infection by up - regulating il-6 production through erk and nf-b but not p38 mapk, jnk or pi3k / akt signal pathways. |
iron deficiency (i d) affects an estimated two billion people worldwide and is one of the most common nutrient deficiencies in all regions, including europe [2, 3 ]. if no corrective action is taken, i d can manifest as iron deficiency anemia (ida), which has been linked to fatigue, weakened immunity, poor work performance, and a decreased quality of life. moreover, infants with ida have been shown to achieve lower scores on mental and motor development tests than infants with normal iron status. inadequate intestinal absorption of nutritional iron to meet physiological requirements may occur due to inadequate iron intake, increased iron requirement (e.g., during periods of rapid growth), or chronic blood loss. infants, preschool children, and adolescents are among the groups most susceptible to development of ida. providing adequate iron supplementation, ideally before the development of anemia, can prevent the systemic neurological and developmental disorders that result from ida in infancy and childhood. long - term oral iron is frequently used as a first - line therapy, but iron salts such as ferrous sulfate are associated with a high incidence of gastrointestinal side effects such as nausea, vomiting, constipation, and diarrhea. polynuclear preparations based on the ferric form of iron, such as iron hydroxide polymaltose complex (ipc), have been developed to improve tolerability. ipc provides similar iron bioavailability to ferrous sulfate but has a stable structure that confers more controlled absorption of iron. a recent meta - analysis has confirmed that ipc and ferrous sulfate provide similar improvements in hemoglobin (hb) levels in adult patients with iron deficiency anemia, but with superior tolerability. the available data comparing ipc versus ferrous sulfate in children suggest that efficacy is similar with the two preparations, but randomized trials are more rare than in adults and long - term data are lacking. two randomized studies, one in 30 iron deficient children with or without anemia aged 2481 months and the other in 49 children with ida aged 640 months, reported no difference between ipc and ferrous sulfate for the improvement in hb or other efficacy markers over a two - month period, although one trial observed a more rapid improvement in hb with ferrous sulfate. tolerability was superior with ipc in both studies [11, 12 ]. the current study evaluated the efficacy, tolerability, and acceptability of ipc and ferrous sulfate in a cohort of 103 pediatric patients with ida during a four - month treatment period. this was a prospective, randomized, open - label, four - month study undertaken in children with ida at the department of pediatric health and diseases outpatient clinics of the university of istanbul during 2009. patients were eligible for enrollment if they were older than six months of age and presented with at least one of the symptoms of fatigue, faintness, or getting tired quickly, without known underlying chronic disease. diagnosis of ida was based on age - dependent lower limits of normal for hb and iron status parameters (table 1) [13, 14 ]. patients with hb values below normal were tested for transferrin saturation (tsat), serum iron, and serum ferritin levels. if any of these iron parameters were below normal, the patient was included in the trial and randomized to iron treatment with ferrous sulfate (twice daily ; ferro sanol syrup, adeka, turkey) or ipc (once daily ; ferrum hausmann syrup, abdi ibrahim, turkey) at a total dose of 5 mg iron / kg / day. randomization was performed by alternating treatment allocation of newly recruited patients on a weekly basis, that is, patients who were recruited during one week were allocated to one treatment group and those recruited during the following week to the other treatment group. the study was conducted in accordance with the declaration of helsinki and good clinical practice guidelines. the study protocol was approved by the local ethics committee (registration number 2009/1897) and legal representatives of the children provided informed consent before enrollment in the study. baseline measurements comprised the erythrocyte - related hematologic markers hb, hematocrit (hct), mean corpuscular volume (mcv), mean corpuscular hemoglobin (mch), mean corpuscular hemoglobin concentration (mchc), and red blood cell (rbc) count as well as the iron status markers serum iron, serum iron binding capacity (sibc), tsat, and serum ferritin. outcome assessments comprised the percentage of reticulocytes at day 7, erythrocyte markers at months 1 and 4, and iron status markers at month 4. iron parameters were assessed with standard laboratory methods using cobas integra 800 and cobas e autoanalyzers. gastrointestinal adverse events with a possible relation to study medication (e.g., nausea, abdominal pain, diarrhea, and constipation) that developed during the course of treatment and were reported at any study visit. treatment acceptability was assessed using the wong - baker scale, which scores facial expressions on a scale of 05 points that reflect difficulties during administration of iron treatment. happy face (0) stands for no difficulties, while a sad face (5) indicates that the child refused or was forced to take the medicine. statistical analyses were performed with the ncsd (number cruncher statistical system) 2007 and pasd 2008 statistical software (utah, usa). student 's t - test was used for group comparison of parameters with normal distribution. mann - whitney u test was used for group comparison of parameters that did not show normal distribution. variance analysis was used for the detection of difference between repeated measurements of parameters showing normal distribution. wilcoxon test was used for repeated measure analysis of parameters that did not show normal distribution. one hundred and three children were screened for eligibility, all of whom met the criteria for inclusion and were recruited to the study (42 girls, 61 boys ; mean age 6.4 5.1 years, range 7 months to 17 years). the patients were evenly distributed between the two treatment groups (ipc, n = 52, 49.5% ; ferrous sulfate, n = 51, 50.5%). baseline characteristics were comparable between both groups except for serum ferritin levels, which were significantly higher in patients randomized to ipc compared to ferrous sulfate (table 2). however, baseline ferritin levels were below the age - dependent lower limit of normal in both groups. the percentage of reticulocytes at day 7 was similar with ipc (1.41 1.31%) and ferrous sulfate (1.57 1.29% ; p = 0.905). all erythrocyte - related hematologic parameters at months 1 and 4 and all iron parameters at month 4 showed a significant improvement from baseline with both treatments (table 2). a significant improvement in hb was observed by month 1 in the ipc group (9.5 1.1 g / dl to 10.6 1.0 g / dl, p = 0.001) and the ferrous sulfate group (9.4 1.6 g / dl to 11.2 0.9 g / dl, p = 0.001), with an increase of more than 2 g / dl in both treatment arms by month 4 (ipc 11.7 0.8 g / dl, ferrous sulfate 12.4 1.0 g / dl ; both p = 0.001 versus baseline). the changes in hb and hct levels from baseline to months 1 and 4 were not significantly different between treatment groups, although at month 1 there was a nonsignificant trend to a greater increase in hb in the ferrous sulfate group (ipc 1.2 0.9 g / dl versus 1.8 1.7 g / dl, p = 0.060). in terms of iron status parameters, tsat improved from approximately 5% in each group at baseline to > 20% at month 4 (ipc 5.1 3.3% to 20.2 15.5%, p = 0.001 ; ferrous sulfate 5.4 3.5% to 22.4 13.2%, p = 0.001) with no significant difference between the groups. the increase in serum ferritin level from baseline to month 4 was almost twofold lower in the ipc group versus ferrous sulfate (22.7 26.1 g / ml versus 42.5 62.0 g / ml, p = 0.001). overall, 38.8% patients (40/103) reported one or more gastrointestinal adverse event typical for oral iron supplementation, with a significantly lower frequency of events in the ipc group (26.9% [14/52 ]) compared to the ferrous sulfate group (50.9% [26/51 ], p = 0.012) (table 3). the frequencies of nausea / abdominal pain and of constipation were comparable between groups, but 25.4% of patients receiving ferrous sulfate experienced both types of adverse events compared to 1.9% of ipc - treated patients. treatment acceptability at day 7 was comparable for both groups, but at months 1 and 4, the children found it significantly easier to accept ipc administration than ferrous sulfate (figure 1). at the end of the four - month study period, the mean facial expression score on the five - point wong - baker scale was 0.51 points lower in the ipc group compared to the ferrous sulfate group (1.63 0.56 versus 2.14 0.75, p = 0.001). results from this large, randomized study show that improvements in hematologic parameters and the availability of iron for erythropoiesis are comparable with ipc and ferrous sulfate over a four - month period in children with ida but are achieved with fewer adverse events and improved acceptability using ipc. a number of studies have previously demonstrated that ipc achieves a significant increase in hb levels in children with ida [11, 12, 1618 ]. the rate of the erythropoietic response to ipc appears to be dose dependent. in a study of 63 adults with ida, the mean time to achieve target hb level was 6.6, 8.3, and 11.3 weeks, respectively, for patients receiving 200, 400, or 600 mg iron. at a dose of 200 mg iron / day, langstaff. observed hb increases to be higher with ferrous sulfate than ipc at weeks 3 and 6, but not at week 9. similarly, murahovschi. found in a randomized trial of 49 ida infants that patients treated with ferrous sulfate showed a faster increase in hb during the first month of treatment compared to those given ipc at a dose of 4 mg / kg / day, but that the increase was then slower with ferrous sulfate. this may explain the lack of response to ipc 100300 mg / day after one month described in a small study of 16 iron - depleted adults. it has been suggested that the bioavailability of iron may be lower to ipc than iron salts [21, 22 ], but evaluation of iron bioavailability from orally administered compounds is complex, and conventional pharmacokinetic measurements of serum iron concentration are largely irrelevant in this setting [23, 24 ]. the true measurement of iron bioavailability is uptake of iron into the erythrocytes, which peaks at 2 - 3 weeks after the start of oral iron administration, and which is similar with ipc and ferrous salts including ferrous sulfate [9, 25 ]. in our population, the equivalent reticulocyte response at day 7 suggests that ipc rapidly provides adequate iron bioavailability for effective erythropoiesis. by month 1, there was a significant increase in hb in the ipc cohort compared to baseline and no significant difference was seen between the ipc and ferrous sulfate arm. of the other five hematologic parameters that were measured, only mcv showed a significantly greater improvement in the ferrous sulfate arm at month 1. at month 4, although absolute hb was lower in the ipc arm, the change in hb from baseline was similar between treatment groups. while levels of the storage iron ferritin were higher with ferrous sulfate, tsat exceeded 20% in both groups, indicating that adequate iron was available for erythropoiesis. a drawback of oral iron supplementation, particularly ferrous sulfate, is the high incidence of gastrointestinal adverse events such as nausea, vomiting, abdominal cramps, constipation and diarrhea, and tooth staining [7, 26 ]. randomized studies in adults have confirmed a lower rate of gastrointestinal symptoms with ipc versus ferrous sulfate [8, 20, 27, 28 ]. in children, comparative data are more sparse, but there are reports of fewer gastrointestinal adverse events and less frequent tooth staining in ipc - treated children compared to those given ferrous sulfate. the differences in safety profiles between the two preparations are attributed to a slower release of iron from the stable ipc complex. rapid iron release from ferrous sulfate within the gastric lumen can overload the active, control uptake mechanism in the enterocytes, leading to local gut reactions and symptoms such as vomiting and dyspepsia. overload of the active uptake mechanism also leads to passive absorption via the intercellular route and absorption of iron from the gut directly into the bloodstream, with a consequent increase in nontransferrin bound iron (ntbi). ntbi iron is known to induce oxidative stress that can cause systemic adverse events including nausea. the rise in ntbi thus is negligible after ipc dosing since the size of the hydroxide complex means that there is almost no passive diffusion and the slow release of iron avoids overload of the active transport mechanism, but when iron is given in the form of ferrous salts, rapid release of iron means that there is a dose - dependent passive absorption of iron. as a consequence, ferrous sulfate is associated with increased levels of ntbi and increased oxidative stress [2830 ], whereas ipc administration is not [28, 29 ]. the significantly lower rate of gastrointestinal adverse events seen with ipc compared to ferrous sulfate in the current study is in line with earlier clinical experience in children and with the difference in iron absorption patterns. taking ferrous salts at mealtimes improves gastrointestinal tolerance, but markedly reduces iron bioavailability such that it is recommended to take ferrous sulfate between meals. ipc, in contrast, can be taken at meal times without compromising bioavailability or effectiveness. the good tolerability of ipc was confirmed in a randomized trial of ipc versus ferrous gluconate in a series of 105 healthy infants to assess their efficacy in the prevention of anemia. adverse effects such as vomiting, diarrhea, constipation, and discolored teeth were significantly less frequent in the ipc treatment group, although mean hb levels were higher in the ferrous gluconate arm. the progressive increase in erythrocyte - related hematologic parameters between months 1 and 4 confirms the benefit of a long - term treatment schedule in patients given oral iron supplementation. compliance is inevitably an issue for any long - term treatment regimen, but the high rate of gastrointestinal adverse events in infants and children given ferrous sulfate [12, 33 ] is likely to be an additional barrier. limited data from studies in children and infants have suggested that compliance and adherence with a ferrous sulfate regimen may be as low as 3040% over a one - week period. two randomized studies in pregnant women have shown significantly higher compliance with ipc than ferrous sulfate [36, 37 ], but comparative data are not available in children. our evaluation of the acceptability of ipc versus ferrous sulfate showed a progressive increase in the unfavorable attitude of the infants and children to ferrous sulfate compared to ipc over the four - month study period, which would tend to discourage compliance. in conclusion, the results of this study show that ipc is as effective as ferrous sulfate when used as an oral iron replacement therapy in pediatric patients with iron deficiency anemia. the superior tolerability of ipc compared to ferrous sulfate translated into better treatment acceptability in this population of infants and children. | iron polymaltose complex (ipc) offers similar efficacy with superior tolerability to ferrous sulfate in adults, but randomized trials in children are rare. in a prospective, open - label, 4-month study, 103 children aged > 6 months with iron deficiency anemia (ida) were randomized to ipc once daily or ferrous sulfate twice daily, (both 5 mg iron / kg / day). mean increases in hb to months 1 and 4 with ipc were 1.2 0.9 g / dl and 2.3 1.3 g / dl, respectively, (both p = 0.001 versus baseline) and 1.8 1.7 g / dl and 3.0 2.3 g / dl with ferrous sulfate (both p = 0.001 versus baseline) (n.s. between groups). gastrointestinal adverse events occurred in 26.9% and 50.9% of ipc and ferrous sulfate patients, respectively (p = 0.012). mean acceptability score at month 4 was superior with ipc versus ferrous sulfate (1.63 0.56 versus 2.14 0.75, p = 0.001). efficacy was comparable with ipc and ferrous sulfate over a four - month period in children with ida, but ipc was associated with fewer gastrointestinal adverse events and better treatment acceptability. |
a 51-year - old male patient with type 2 diabetes (bmi 50.2 kg / m, a1c 8.0%) treated with metformin, sulfonylurea, and insulin underwent a laparoscopic rygb for morbid obesity. on the second postoperative day, a leakage from the gastro - jejunostomy was suspected because of fever and abdominal pain. acute reoperation showed no firm signs of leakage, but nevertheless a percutaneous gastric tube was inserted into the bypassed gastric remnant. the tube served as the only route of nutrition during the following 3 weeks, after which the patient again was allowed peroral feeding through the gastric pouch according to a standard nutrition protocol (1,200 kcal / day). treatment with insulin and metformin was temporarily required after the reoperation but could be discontinued 3 weeks postoperatively. we examined the patient 5 weeks postoperatively, at which time the patient was fed perorally but still had the gastric tube. on 2 consecutive days at 8.30 a.m. after an overnight fast (8 h), a standard 200-ml liquid meal (nutridrink ; nutricia) containing 300 kcal, with 16% protein, 49% carbohydrate, and 36% fat, was given over a period of 10 min, on the first day through the gastric tube and on the second day perorally. blood samples were drawn from an antecubital vein at 15- to 30-min intervals (fig. plasma concentrations of glucose (a), insulin (b), c - peptide (c), glucagon (d), glp-1 (e), intact gip (f), pyy (g), and ffas (h) after peroral or gastroduodenal feeding in a rygb - operated patient.. triangles and dotted lines, peroral feeding ; circles and solid lines, gastroduodenal feeding. plasma glucose was measured by a glucose oxidase method (abl800flex ; radiometer, brnshj, denmark), peptide yy336 (pyy) with a radioimmunoassay kit (linco research), and free fatty acids (ffas) by an enzymatic colorimetric method (wako, dsseldorf, germany). plasma insulin, c - peptide, glucagon, and incretin hormone were quantified as earlier described (3). -cell function was evaluated by iaucinsulin, iauccpeptide, and insulinogenic index (igi) and calculated as (insulin30 insulinfasting)/(glu30 glufasting) and iauccpeptide / glu ratio. homeostasis model assessment of insulin resistance (homa - ir) was calculated as (insulinfasting glufasting)/22.5. plasma glucose was measured by a glucose oxidase method (abl800flex ; radiometer, brnshj, denmark), peptide yy336 (pyy) with a radioimmunoassay kit (linco research), and free fatty acids (ffas) by an enzymatic colorimetric method (wako, dsseldorf, germany). plasma insulin, c - peptide, glucagon, and incretin hormone were quantified as earlier described (3). -cell function was evaluated by iaucinsulin, iauccpeptide, and insulinogenic index (igi) and calculated as (insulin30 insulinfasting)/(glu30 glufasting) and iauccpeptide / glu ratio. homeostasis model assessment of insulin resistance (homa - ir) was calculated as (insulinfasting glufasting)/22.5. plasma concentrations and iauc for glucose, insulin, c - peptide, glucagon, total glucagon - like peptide-1 (glp-1), intact glucose - dependent insulinotropic polypeptide (gip), pyy, and ffas after peroral and gastroduodenal feeding are shown in fig. 1. plasma glucose concentration peaked earlier and returned more rapidly to fasting values after peroral than gastroduodenal feeding, as illustrated by a markedly reduced 2-h plasma glucose concentration (7.8 vs. 11.1 mmol / l). the peak values of plasma insulin and c - peptide were higher after peroral than gastroduodenal feeding (fourfold and twofold, respectively) and iaucinsulin and iauccpeptide were also clearly elevated. igi was improved after peroral feeding (115 vs. 72 pmol / mmol), and the iauccpeptide / glu ratio was more than twofold increased (0.90 vs. 0.40 nmol / mmol). homa - ir remained unchanged on the 2 examination days (3.3 vs. 3.5). glp-1 plasma concentration peaked simultaneously after peroral and gastroduodenal feeding, but the peak value was more than threefold increased (87 vs. 28 pmol / l), and iaucglp-1 was nearly fivefold increased after peroral feeding. 0.001) but not gastroduodenal (r = 0.55, p = 0.08) feeding. plasma concentrations of glucagon and intact gip were similar on both days. rapid improvement in glucose tolerance after rygb surgery is a clinical reality (2). here, we report important differences in -cell function and glucose metabolism after peroral compared with gastroduodenal feeding in a patient with rygb and a gastrostomy, where differences in insulin sensitivity, weight loss, and caloric restriction can be ruled out as explanations for the improved glucose tolerance. our results show marked improvement in glucose tolerance with near normalization of 2-h postprandial plasma glucose value and a 33% reduction in iaucglu after peroral feeding compared with gastroduodenal feeding. in contrast, during gastroduodenal feeding glucose tolerance was diabetic with a 2-h postprandial plasma glucose value 11 mmol / l. the improvement was accompanied by a twofold increase in -cell secretory response (auccpeptide / glu), which was associated with a fivefold increase in iaucglp-1. insulin and glp-1 concentrations during peroral feeding were strongly correlated, which is suggestive of a causal relationship. interestingly, the insulin and c - peptide response curves found after gastroduodenal feeding resemble the responses found in type 2 diabetic patients, whereas the response curves after peroral feeding are similar to those found in healthy control subjects (4). the emptying time is likely to be slower after feeding into the bypassed gastric remnant, which could explain the slower peak in plasma glucose observed after gastroduodenal feeding but would also, per se, be expected to result in decreased postprandial glucose excursions. the observed improvements in glucose tolerance and glp-1 secretion are in concordance with earlier findings from patients examined before and after rygb surgery (513). regarding gip, some studies have demonstrated increased (7,10) and others decreased (9,13) responses after rygb. in our patient, gip responses were similar on the 2 days, suggesting that changes in gip were not responsible for the differences in insulin secretion and glucose tolerance. also glucagon responses were similar. in conclusion, our results suggest that rygb has a direct beneficial effect on postprandial glucose metabolism, most likely due to an increased insulin secretion caused by the massive increase in glp-1 that is probably due to the rapid exposure of l - cells in the distal small intestine to nutrients (14). it has been suggested that duodenal exclusion inherent in the rygb somehow might be responsible for the improvement in glucose tolerance (15). in this respect, it is of interest that the secretion of the upper jejunal hormone, gip, was similar during peroral or gastroduodenal feeding. | objectiveto examine after gastric bypass the effect of peroral versus gastroduodenal feeding on glucose metabolism.research design and methodsa type 2 diabetic patient was examined on 2 consecutive days 5 weeks after gastric bypass. a standard liquid meal was given on the first day into the bypassed gastric remnant and on the second day perorally. plasma glucose, insulin, c - peptide, glucagon, incretin hormones, peptide yy, and free fatty acids were measured.resultsperoral feeding reduced 2-h postprandial plasma glucose (7.8 vs. 11.1 mmol / l) and incremental area under the glucose curve (iauc) (0.33 vs. 0.49 mmol l1 min1) compared with gastroduodenal feeding. -cell function (iauccpeptide / glu) was more than twofold improved during peroral feeding, and the glucagon - like peptide (glp)-1 response increased nearly fivefold.conclusionsimprovement in postprandial glucose metabolism after gastric bypass is an immediate and direct consequence of the gastrointestinal rearrangement, associated with exaggerated glp-1 release and independent of changes in insulin sensitivity, weight loss, and caloric restriction. |
approximately 400,000 coronary artery bypass graft (cabg) surgeries are performed in the united states each year. as these grafts age, patients are at high cumulative risk for developing new cardiovascular events due to graft occlusion. studies have suggested that saphenous vein graft (svg) occlusion occurs at a rate of approximately 10% in the first month and up to 20% in the first year, with a 2% annual risk for the first 5 years. recurrent infarction may occur in as many as 29% of patients in 10 years following cabg. in contrast to svgs, the left internal mammary artery (lima) graft has a 10% closure rate in 10 years. of note, at least 10% of percutaneous coronary interventions when patients with prior cabg present with chronically occluded svg, the options are often limited due to the suboptimal clinical results in recanalizing these grafts percutaneously. these patients usually have 3 options : (1) percutaneous coronary intervention of the severely diseased native vessels, (2) repeat cabg or (3) medical therapy alone. we present an unusual case of a patient presenting with a total occlusion of a svg which spontaneously recanalized over a 2 month period but with a persistent focal stenosis at the distal anastomosis. the distal anastomotic stenosis was treated with a drug eluting stent and the vessel remained patent angiographically at 1 year. the patient is a 76 year old gentleman with a history of known arteriosclerotic heart disease, status post cabg in june 1989 with a lima graft to the left anterior descending artery and separate svgs to the diagonal branch and right coronary arteries. he developed recurrent angina and subsequently had a bare metal stent placed in the native left circumflex coronary artery in 2002. in 2005, he developed atypical chest pain and a sestamibi scan showed anterolateral ischemia. four days prior to the january 2007 admission, he developed acute onset retrosternal chest pain that was associated with fatigue. he presented to medical care 4 days later with a blood pressure of 135/80, pulse of 80 pulse and normal chest, heart and vascular exam. an electrocardiogram revealed loss of anterior forces, new right axis deviation and borderline 1 mm of st elevation in leads i and avl. the troponin - i level was elevated at 7.9 ng / ml. lipid profile showed total cholesterol 171, triglyceride 103, hdl 48, ldl 102. he was maintained on aspirin 81 mg / day and ramipril that he was already taking and started on isosorbide mononitrate, metoprolol and ezetimibe / simvastatin. subsequent troponin levels decreased and the clinical diagnosis was acute coronary syndrome with recent myocardial infarction. coronary angiography showed complete thrombotic occlusion of the svg to the diagonal branch (fig. 1), with patent svg to the right coronary artery with jump to the posterolateral branch. the svg to the diagonal branch was large and completely filled with thrombus which was distinguished by multiple swirling filling defects. there was a faint trickle of flow in the graft body of the diagonal branch but the contrast did not reach the native diagonal branch. left ventriculography showed new evidence of anterolateral and apical akinesis and there was a 1 to 2 + mitral regurgitation. because this was felt to be a functionally occluded svg with a myocardial infarction approximately 5 days old, with no recurrent ischemia or other indication for revascularization, the eptifibatide was discontinued after the procedure after a total duration of approximately 3 h and the patient was treated medically and discharged on aspirin 81 mg / d as the only anti - platelet agent.fig. 1initial angiogram in january of 2007 shows complete occlusion of the svg to diagonal branch. the white arrow points to the area of occlusive thrombus seen near the ostium of the svg. two views are shown (lao cranial, rao cranial) initial angiogram in january of 2007 shows complete occlusion of the svg to diagonal branch. the white arrow points to the area of occlusive thrombus seen near the ostium of the svg. two views are shown (lao cranial, rao cranial) two months after the initial hospitalization the patient developed chest tightness lasting approximately 45 min associated with diaphoresis and shortness of breath. ecg revealed markedly inverted t - waves in leads i and avl as well as in leads v4v6 which were new compared to his ecg previously. he was treated with heparin and eptifibatide for approximately 4 h prior to being taken to the cardiac catheterization laboratory. repeat angiography showed complete patency of the previously occluded svg to the diagonal branch (fig. however, there was a focal 95% stenosis at the distal anastomosis, for which a drug eluting stent (3.0 18 mm cypher, cordis, miami, florida) was placed. the proximal segment of this stent that was in the bypass graft was treated with a 4.0 8 mm quantum balloon (boston scientific, maple grove, minnesota) and expanded to 16 atmospheres. he subsequently had complete resolution of angina and was treated with aspirin and clopidogrel indefinitely.fig. 2repeat angiogram performed 2 months later when the patient presented with more symptoms and negative cardiac markers. lao cranial and caudal views shown here with white arrow demonstrating stenotic occlusion at the anastomosis site repeat angiogram performed 2 months later when the patient presented with more symptoms and negative cardiac markers. lao cranial and caudal views shown here with white arrow demonstrating stenotic occlusion at the anastomosis site one year later the patient had another episode of resting but somewhat atypical chest pain and was admitted to the hospital. initial ecg was unchanged, however t wave inversions were noted in v2, v3, and patient was taken to the catheterization laboratory. angiography demonstrated no significant change from the prior cardiac catheterization, including a patent svg - to - diagonal graft with no in - stent restenosis (fig. the patient has no evidence of ischemia 24 months following the stent placement.fig. 3follow up angiography 1 year later demonstrating patent svg to diagonal - branch with patent cypher stent. angiography is performed in lao and rao cranial views with angulation similar to fig. 1 follow up angiography 1 year later demonstrating patent svg to diagonal - branch with patent cypher stent. angiography is performed in lao and rao cranial views with angulation similar to fig. 1 this report demonstrates the rare phenomenon of complete angiographic thrombus resolution and reperfusion of a thrombotically occluded svg following treatment with aspirin as the only outpatient anti - platelet therapy. furthermore, the svg had documented 1 year patency following drug eluting stent placement at the presumed site of thrombotic occlusion and a favorable clinical course for 2 years. this case suggests that a subset of patients may have the physiological capability for spontaneous fibrinolysis of even very large thrombi in completely occluded svgs. it also reflects the therapeutic efficacy of pci and drug eluting stents in maintaining graft patency, at least in the near term. their greater content of thrombus and other material such as necrotic debris predisposes them to a greater propensity for graft trauma, distal embolization and no - reflow phenomenon after an intervention. in the patient described in this report, repeat angiography 2 months following the initial angiogram demonstrated that the infarct related svg was patent. although the patient received four hours of eptifibatide prior to angiogram, it is unlikely that this had a significant thrombolytic effect as the svg was totally occluded during prior coronary angiography. prior studies in acs or stemi involving svgs have not generally shown any benefit from iib / iiia inhibitors or even thrombolytic therapy [4, 5 ]. for example, in the mayo clinic pci registry, results from 128 patients with prior cabg with primary pci in ami patients showed that when the treated vessel was a svg, there was a lower success rate, with increased inability to completely recanalize the svg along with increased risk of distal embolization, when compared to a native vessel intervention. similarly, in the pami-2 trial where the infarct related artery was an svg rather than a native coronary artery, both timi-3 flow grade (70.2% vs. 94.3%) and 6-month mortality (14.3% vs. 4.1%) were worse in patients with versus without previous cabg. similar outcomes were noted in the gusto - i trial in 1784 patients with stemi. nonetheless, in a subset of patients, such as the one presented here, they may enhance partial fibrinolysis as has been suggested by upfront iib / iiia inhibitor use in stemis showing enhanced timi iii flow on angiography prior to pci. it is intriguing to speculate that this process may be enhanced by novel anti - platelet agents that are being developed. in svg infarct cases, the decision to proceed to an intervention is made after taking into consideration infarct size and duration, patient condition, thrombus burden, and vessel anatomy. recanalization of the native coronary artery to which the svg was anastomosed may be a better option. in our case, no initial intervention was performed for the occluded svg due to the large thrombus burden in the length of the graft and the regression of symptoms. furthermore the native diagonal branch was totally occluded proximally and it was felt that it could not be recanalized. only two previous cases have been reported of spontaneous reperfusion of a svg related to an ami. the first is a patient with a nstemi due to svg thrombosis 1 month after discontinuation of antiplatelet therapy. the second case is a patient with a stemi due to an occlusion of one svg, who was treated with a stent to his native lad. our case represents the longest documented patency of a spontaneously recanalized graft, albeit it was also treated with a drug eluting stent at the distal anastomosis which likely has helped maintain patency. spontaneous reperfusion of an infarct related native coronary artery is not uncommon, particularly in this era of aggressive in - hospital anti - thrombotic therapy, and is associated with greater myocardial salvage and resolution of chest pain. this observation of spontaneous reperfusion is highly intriguing, particularly in an elderly individual, an age group where impaired fibrinolysis is common. it is possible that aspirin by itself potentiated a fibrinolytic effect, as demonstrated in the isis-2 thrombolytic trial where the aspirin alone was associated improved clinical outcomes. it has also been reported that the local profibrinolytic response in coronary arteries, by net coronary release of tissue type plasminogen activator (t - pa) following induced myocardial ischemia in pigs, could be a useful defense against coronary thromboembolic events. in fact, higher levels of t - pa are noted in st - segment elevation myocardial infarction, and are paradoxically associated with worse prognosis [16, 17 ]. possible mechanisms of spontaneous recanalization include the release of factors, activators and inhibitors of fibrinolysis, from the endothelium, neutrophils and monocytes. the role of thienopyridines such as clopidogrel in enhancing reperfusion has not been demonstrated, but one would predict it would be additive to aspirin, based on recent trials. in conclusion, we describe an unusual case of spontaneous reperfusion of an svg, subsequent treatment of a residual stenosis with a drug eluting stent and a favorable 2 year outcome. understanding the underlying mechanisms of enhanced thrombolysis may help discover new insights into sustained graft patency that may improve long term outcomes of patients undergoing cabg. although it has not been formally evaluated yet, defining the long - term efficacy of dual anti - platelet therapy in patients in preventing events in patients who have undergone cabg is a worthwhile clinical question that should be investigated. | acute myocardial infarction resulting from saphenous vein graft occlusion occurs not infrequently in patients who have undergone coronary artery bypass graft surgery. in this case report, we present a novel case of spontaneous recanalization of a thrombotic graft occlusion in a patient who presented with a subacute myocardial infarction. the patient was treated medically with aspirin as the only anti - platelet agent. interestingly, he presented 2 months later with new onset angina. coronary angiography demonstrated complete resolution of thrombus but a severe focal stenosis in the distal anastomoses. following drug eluting stent placement, a favorable clinical course has ensued and patency confirmed on follow up angiography at 1 year. |
a role for leukocytes in solid tumor development has long been suspected ; however, only recently have immune - competent spontaneous models of human cancer development enabled mechanistic evaluation of leukocytes to determine if their presence in solid tumors is coincidental or functional. with the advent of mouse models of multi - stage neoplastic progression, it is now clear that lymphoid and myeloid cells can either restrain or propel cancer development, depending on their maturation state, as well as the local microenvironment regulating their bioeffector phenotype [24 ]. a role for immune cells as mediators of therapeutic response in cancers has only recently been explored [5, 6 ]. chemotherapy (ctx) and radiation therapy (rt) remain as part of the standard therapeutic armament for patients with cancer, including breast cancer (bc). both ctx and rt impact growing cancers through their ability to induce cell death by disrupting various parameters of cell biology necessary for survival. however, recent data has emerged demonstrating that the type of cell death induced by cytotoxic therapy is significant with regards to the type of immune response elicited within a tissue. these studies have revealed that effectiveness of ctx and rt may in part depend on whether cell death (induced by cytotoxic therapy) is sensed by leukocytes [5, 9, 10 ]. leukocytes detect cell death through immune - based receptors for molecules released by dying cells (often termed danger signals), such as toll - like receptor (tlr)-4 and its ligands including the high - mobility group box protein (hmgb) 1. detection of danger signals in tissues by leukocytes activates an immune response involving cells of the innate (myeloid and natural killer cells) and adaptive (t and b cell) lineages. this review will focus on immunologic consequences of rt and discusses emerging data indicating that therapeutic reprogramming of immune responses in tumors may regulate efficacy and durability of rt. cancer research has primarily focused on the role of activating and/or inactivating mutations in genes regulating aspects of cell proliferation or cell death. much of that research has been geared towards understanding how these activating and/or inactivating mutations support the multi - step model of tumorigenesis where progressive accumulation of genetic changes in somatic cells act as drivers of cancer development. this neoplastic cell - intrinsic perspective of multi - stage tumorigenesis overlooks progressive alterations in the tumor microenvironment that also regulate cancer development. solid tumors contain neoplastic and non - neoplastic stromal cells embedded in a dynamic extracellular matrix (ecm) microenvironment. cellular components of tumor stroma include hematogenous and lymphatic vascular cells, infiltrating and resident leukocytes, various populations of fibroblasts and mesenchymal support cells unique to each tissue microenvironment. clinical and experimental studies have established that chronic infiltration of neoplastic tissue by leukocytes, i.e., chronic inflammation, promotes development and/or progression of solid tumors. however, the organ - specific cellular and molecular programs that favor pro - tumor, as opposed to anti - tumor immunity by leukocytes remain incompletely understood. retrospective clinical studies have revealed an increased presence of extra follicular b cells, t regulatory (treg) cells, high ratios of cd4/cd8 or th2/th1 t lymphocytes in primary tumors or in draining lymph nodes that correlate with tumor grade, stage and overall survival (os) [1422 ]. lymphocyte density in pretreatment biopsies has also been found to represent an independent predictor of complete pathologic response following anthracycline and taxane - based chemotherapy. on the other hand, high densities of macrophages in bc stroma, and some other solid tumors, correlates with increased vascular density and worse clinical outcome [2429 ]. infiltration of macrophages inside tumor nests however, particularly when cd8 cytotoxic lymphocytes (ctl) are also present, correlates with increased overall survival (os). these differences might be explained in part by the realization that macrophages exert either pro- or anti - tumor bioactivities depending on the types of cytokines to which they are exposed. macrophages exposed to th1 cytokines including interferon (ifn), tumor necrosis factor (tnf), and granulocyte monocyte - colony stimulating factor (gm - csf) exhibit enhanced cytotoxic activity, production of pro - inflammatory cytokines and antigen presentation [31, 32 ]. on the other hand, macrophages exposed to th2 cytokines such as interleukin (il)-4 and -13, immune complexes or immunosuppressive cytokines instead block ctl activity and promote angiogenesis and tissue remodeling [33, 34 ]. thus, the presence of leukocytes can exert either a positive or negative force depending on the functional properties they possess. historically, rt was thought to induce an immunosuppressive microenvironment largely based on experimental studies with whole body irradiation revealing lower levels of circulating lymphocytes resulting from increased radiation sensitivity of bone marrow as compared to other tissues. fas, a cell surface proapototic protein and member of the tumor necrosis factor receptor (tnf - r) family, contains an intracellular " death domain " that is activated following ligand binding that subsequently leads to apoptosis. fas is expressed on many cell types including lymphocytes and is upregulated in response to cell damage. activation of fas - mediated cell death is a mechanism by which immune cells eliminate damaged cells, including those damaged by rt. thus, while whole body radiation is immunosuppressive due to triggering widespread apoptosis of immune cells via fas, focal radiation such as that used for treatment of many types of solid tumors instead has limited immunosuppressive side effects, and may actually promote changes in the local tumor microenvironment that paradoxically enhance infiltration and activation of multiple immune cell types (fig. 1) that may either foster, and/or suppress tumor development. figure 1leukocyte infiltration following rt. representative tissue sections of mammary carcinomas stained with hematoxylin and eosin (a) following isolation from a murine mammary carcinoma 96 h after receiving 5 gy of localized gamma irradiation. cd45 staining (red) of adjacent tissue sections (b) demonstrates extent of leukocyte infiltration following rt. the percentage change in cd45 cell infiltration was assessed by flow cytometry of whole tumor cell suspensions revealing a significant increase in cd45 cells following rt. representative tissue sections of mammary carcinomas stained with hematoxylin and eosin (a) following isolation from a murine mammary carcinoma 96 h after receiving 5 gy of localized gamma irradiation. cd45 staining (red) of adjacent tissue sections (b) demonstrates extent of leukocyte infiltration following rt. the percentage change in cd45 cell infiltration was assessed by flow cytometry of whole tumor cell suspensions revealing a significant increase in cd45 cells following rt. at the most simplistic level, a main mechanism by which ionizing radiation mediates a biologic effect is via generation of free radicals that lead to genotoxic (dna) damage, and subsequent activation of stress - response pathways through activation of the dna damage pathway ataxia telangiectasia mutated (atm). activation of the atm protein pathway following rt involves activation of p53 and nuclear factor (nf)-b transcription factors [37, 38 ]. nf-b can also be activated independently of dna damage through radiation - induced activation of tnfr - associated factors (trafs) [39, 40 ]. pro - inflammatory immune response, including tnf-, interleukin (il)-1, chemokines such as ccl5 ; adhesion molecules including intracellular adhesion molecule (icam)-1 [44, 45 ], e - selectin and vascular cell adhesion molecule (vcam)-1, as well as major histocompatability complex (mhc) molecules (fig. 2), and expression of several anti - apoptotic genes including bax and bcl-2 [4952 ]. signaling cascades induced by radiation through atm / nf-b, in addition to the direct cell death resulting from radiation damage, stimulates influx and activation of leukocytes leading to a productive immune response. tumor cells respond to ionizing rt by upregulating cytokines (tnf, il-1/ and il-6), adhesion molecules (icam-1, vcam-1, e - selectin) and mhc class i. death of tumor cells also generates release of inflammatory molecules hmgb1 and atp. this response recruits macrophages and dcs to tumors where they then receive activation signals resulting in their migration to draining lymph nodes where apcs (macrophages and dendritic cells) present tumor - derived antigens and stimulate t cell responses. tumor - specific t cells then re - infiltrate tumors and induce death of damaged malignant cells. tumor cells respond to ionizing rt by upregulating cytokines (tnf, il-1/ and il-6), adhesion molecules (icam-1, vcam-1, e - selectin) and mhc class i. death of tumor cells also generates release of inflammatory molecules hmgb1 and atp. this response recruits macrophages and dcs to tumors where they then receive activation signals resulting in their migration to draining lymph nodes where apcs (macrophages and dendritic cells) present tumor - derived antigens and stimulate t cell responses. tumor - specific t cells then re - infiltrate tumors and induce death of damaged malignant cells. rt and cytokine expression cytokines are peptide - type regulatory proteins, such as the interleukins and lymphokines, released by immune cells leading to generation of an immune response. some cytokines act to inhibit immune responses, e.g. il-10 and transforming growth factor (tgf)-, or instead stimulate immune responses, e.g., tnf- or il-1. tnf- and il-1 are pro - inflammatory cytokines that also mediate leukocyte recruitment in tumors [53, 54 ]. in the 1980 s, hallahan and colleagues reported that tnf- mrna and protein levels were increased in human sarcoma cells following rt, an effect that sensitized tumor cells to radiation - induced cell death. macrophage - derived il-1 and il-1 have also been found increased in response to rt in vivo following sublethal total body irradiation [5658 ], as also have il- 6 and tgf-. consequences resulting from the release of these cytokines are recruitment and activation of leukocytes from peripheral blood and extravasation into tissue (tumor) parenchyma as is illustrated in fig. 1. adhesion molecules regulated by rt adhesion molecules are proteins located on the cell surface that mediate interaction with other cells or extracellular matrix. cell adhesion molecules such as icam-1, e - selectin and vcam-1 are upregulated on endothelial cells during inflammation and are critical for leukocyte trafficking across endothelial barriers. vascular endothelial cells within tumor vessels respond to rt by upregulation of icam-1 and e - selectin and thereby facilitate leukocyte arrest and adhesion prior to transmigration. blockade of cd11b, the ligand for icam-1, in a transplantable murine squamous carcinoma model significantly reduced tumor - infiltration by cd11b myeloid cells following rt resulting in diminished tumor growth. similarly, examination of tumor tissue removed from head and neck cancer patients following rt revealed marked increase in endothelial icam-1 expression, in concert with increased 2 integrin - positive myeloid cell infiltration. other adhesion molecules are also regulated by rt including vcam-1 in melanoma in an interferon (ifn)-dependent manner. chemokines and rt chemokines are a family of small chemotactic cytokines that regulate directional migration of cells expressing a cognate chemokine receptor. while some chemokines are important for homeostatic circulation of leukocytes, others are induced following tissue damage. two important chemokines regulated by rt are cxcl16 and sdf-1. using a murine model of mammary carcinogenesis, matsumura and colleagues reported that cxcl16, which is upregulated in tumors following rt, induced recruitment and activation of t cells expressing cxcr6, the ligand for cxcl16. mice deficient for cxcr6 exhibited decreased cd8 t cell recruitment in tumors and decreased rt responsiveness. murine melanoma, fibrosarcoma and colon carcinoma cell lines in vitro upregulate cxcl16 in response to rt indicating that cxcl16 expression may be a common response across many tumor types. thus, radiation - induced cxcl16 is an important mechanism by which rt promotes cd8 t cell infiltration leading to tumor suppression.stromal cell - derived factor (sdf)-1 is also upregulated following rt in bone marrow - derived cells and cell lines derived from brain tumors. using an in vivo model, kozin and colleagues observed that lung and breast xenograft tumors responded with increased cd11bf4/80 macrophage infiltration following rt that was dependent on expression of sdf-1. inhibition of the sdf-1 pathway with a small molecule inhibitor blocking the interaction of sdf-1 and cxcr4 prevented infiltration of macrophages and significantly delayed tumor regrowth following rt. studies such as these indicate that rt upregulates expression of some chemokines (cxcl16 and sdf-1) that can in turn regulate presence of either tumor suppressive lymphocytes (cd8 t cells), or tumor - promoting cells such as macrophages. rt and antigen presentation once leukocytes have migrated into sites of tissue damage in response to cytokines and chemokines, functional antigen - presenting cells (apc) are required for a productive anti - tumor t cell response to ensue. apcs capture antigens, and following processing, present them on their cell surface via mhc. t cells recognize antigens bound to mhc and respond by proliferating and generating anti - tumor t cells responses. lugade and colleagues, utilizing a transplantable murine model of melanoma, reported increased expression of mhc class i on tumor cells following rt, a response also observed on gl261 glioma tumor cells, indicating that rt enhances tumor cell recognition by t cells through upregulation of mhc class i on the surface of tumor cells, as well as on the surface of apcs [73, 74 ]. increased presence of radiation - specific peptides has also been identified as a mechanism whereby tumor - specific t cell responses are elicited by rt, a mechanism that also contributes to enhanced anti - tumor immunity. rt - induced immunogenic cell death radiation of tumor cells generally produces two responses : proliferative arrest (which in the case of senescence is indefinite) or cell death. tumor cell death can occur by several mechanisms including apoptosis, necrosis, autophagy or mitotic catastrophe. apoptosis is a stereotyped pattern of morphological changes involving chromatin condensation (pyknosis), nuclear fragmentation (karyorhexis), shrinkage of cytoplasm, blebbing of plasma membranes, and final disintegration of cells into membrane - surrounded apoptotic bodies [76, 77 ]. while often observed in vitro, apoptosis is rarely seen in vivo since dying cells are efficiently recognized, engulfed and eradicated by neighboring cells before they enter the late stages of the apoptotic process. necrosis is characterized by cell swelling followed by rupture of plasma membranes and subsequent spillage of cellular contents into intercellular spaces. autophagy is marked by sequestration of large parts of the cytoplasm in autophagic vacuoles typically before cells undergo apoptosis. finally, mitotic catastrophe is described by prolonged mitotic arrest with associated micro- and/or multinucleation prior to undergoing death. radiation - mediated cell death is generally thought to occur primarily through either apoptosis or mitotic catastrophe.the notion that immunogenic - mediated cell death is also an important aspect of rt response has been demonstrated by several groups. apetoh and colleagues immunized mice with tumor cells previously exposed to either chemotherapy or rt, and then re - challenged them with the tumor cells and monitored for tumor growth. immunization with tumor cells treated with either chemotherapy or rt prevented regrowth of tumors in ~30% of mice as compared to mice immunized with untreated tumor cells. when cells were harvested from draining lymph nodes in immunized mice, and rechallenged ex vivo, only lymph node cells from mice immunized with tumor cells treated with rt produced ifn- in response to re - challenge. protective immunization in this scenario was dependent on the presence of tlr-4 on dendritic cells (dcs) and its ligand hmgb1, both released by tumor cells following rt. two other factors, calreticulin and atp, also significantly contribute to immunogenic cell death, in a manner similar to hmgb1, where cell death triggers rapid translocation of calreticulin to the surface of cells thereby promoting antigen presentation by dying cells and dcs [79, 80 ]. mice previously vaccinated with irradiated tumor cells engineered to express an sirna against calreticulin exhibit a greatly reduced immune response to challenge as compared to irradiated cells alone.cytotoxic therapies (chemotherapy and rt) induce rapid release of atp from cells. atp acts on the p2x(7) purinergic receptor expressed by dcs, leading to activation of the nod - like receptor family, pyrin domain containing-3 protein (nlrp3)-dependent caspase-1 activation complex (also known as the inflammasome). inflammasome activation leads to release of pro - inflammatory cytokines such as il-1, which are important for priming t cells. when components of this pathway (nlrp3, caspase-1 or il-1r) are absent, reduced t cell responses towards cells killed by chemotherapy or rt are observed, thus indicating that release of atp from dying cells is a critical aspect of immunogenic cell death and anti - tumor immunity.further support for the importance of immunogenic cell death mediated by hmgb1 and tlr-4 bearing dcs has been provided by retrospective evaluation of a cohort of breast cancer patients treated with adjuvant anthracyclines following resection. women harboring an asp299gly tlr-4 polymorphism exhibited reduced response to hmgb1, and a significantly higher rate of metastatic disease. interestingly, similar to hmgb1, breast cancer patients treated with anthracycline who harbored a loss - of - function allele in p2rx7 (glu496ala) exhibited significantly worse metastasis - free survival as compared to patients with wildtype alleles. rt and activation of innate immune programs cells of the innate and adaptive lineages work in concert to provide rapid and effective responses to a wide variety of pathogens. while cells of the innate lineage provide an immediate and pre - programmed response, response by cells of the adaptive lineage are delayed but instead are antigen - specific and lead to prolonged memory. innate leukocytes, including dcs, macrophages, natural killer (nk) cells and mast cells, are referred to as first responders to inflammatory mediators, largely based on the fact that they are often prestationed in tissues.nave dcs continually sample antigens and migrate to draining lymph nodes for antigen presentation to t cells following their activation by inflammatory mediators. rt induces opposing responses in tumors with regards to dcs : directly - irradiated dcs are less effective apcs, however, the tumor microenvironment generated by rt enhances apc capabilities of dcs. in vitro examination of human dcs has revealed that rt induced a tolerogenic phenotype by decreasing the amount of il-12 produced by mature dcs, leading to decreased nave cd8 t cell priming. however, when dcs are adoptively transferred into tumors in combination with rt and chemotherapy in vivo, complete regression of tumors is enhanced. thus, while intratumoral dcs present at the time of chemotherapy or rt yield reduced immune responsiveness, the environment created by the rt fosters enhanced dc activation and enhanced anti - tumor immunity.tumor-infiltrating macrophages, derived from circulating monocytes, make up a substantial component of the leukocyte infiltrate in solid tumors. macrophages exhibit either anti- or pro- tumoral bioactivities dependent on the cytokines, chemokines and soluble mediators they are exposed to [87, 88 ]. given this duality, it is not surprising that the effect of radiation on macrophages is complex with evidence that radiation can support either their anti- or pro - tumor properties. using human macrophage - derived cell lines, lambert and colleagues observed that rt enhanced macrophage cytolytic activity. other groups have reported that low dose whole - body rt increased expression of tlr4/md2 and cd14 expression on murine peritoneal macrophages, leading to increased secretion of anti - tumor cytokines including il-12 and il-18, thus indicating that rt increases anti - tumor potential of macrophages. despite the evidence that rt can stimulate cytolytic activity and anti - tumor cytokine production in macrophages, there also exists extensive literature indicating that macrophages also promote resistance to rt. in orthotopically - transplanted sarcoma and carcinomas, presence of macrophages local rt of implanted tumors increased the number of apcs in draining lymph nodes and increased the number of cd11b cells in tumors. cd11b myeloid cells (a portion of which are macrophages) contribute growth factors such as vascular endothelial growth factor (vegf) and matrix metalloproteinase-9 (mmp-9) that supports angiogenic programs in growing tumors. preventing influx of cd11b myeloid cells following rt results in enhanced rt effects [63, 93 ] likely due to their increased expression of t cell suppressive molecules inos and arginase i [94, 95 ]. thus, while radiation can stimulate macrophage cytolytic activity and anti - tumor cytokine production, this may be insufficient to inhibit tumor growth if there is simultaneous recruitment or activation of macrophages harboring dominant pro - tumor properties.nk cells are lymphoid cells that, unlike b and t cells, do not possess specific antigen receptors, and thus are considered innate lineage cells. nk cells play an important role in tumors by targeting malignant cells by direct cytolysis and secretion of potent immune mediators including several cytokines and chemokines. examination of tumor cells exposed to ionizing radiation in vitro indicates that rt induces expression of nkg2d ligands, an activating receptor for nk cells [97, 98 ]. other pro - inflammatory stress molecules released by dying cells include heat shock protein 70 (hsp70), a stress response protein with a role in binding defective proteins and presenting them on the surface of cells. when exposed to rt, pancreatic and colon carcinoma cell release hsp70, thereby targeting them for lysis by nk cells. that nkg2d ligands and hsp 70 render cells more susceptible to nk - cell mediated cytolysis indicates that rt - stimulated nk activity may be an important component of rt - induced immune responsiveness.mast cells are pre - stationed in many tissues where they act as important sentinel cells capable of mounting rapid responses to tissue damage. mast cells also accumulate in tissues undergoing angiogenesis, wound healing and tissue repair. during these processes, they secrete angiogenic factors, such as vascular endothelial growth factor (vegf), and other inflammatory mediators such as histamine, heparin cytokines, chemokines, proteases and lipid mediators. heissig and colleagues reported that low - dose irradiation fostered mast cell - dependent vascular regeneration in a limb ischemia model where rt promoted vegf production by mast cells in a matrix metalloproteinase-9 (mmp-9)-dependent manner. rt, through mmp-9 up - regulated by vegf in stromal and endothelial cells, induced release of kit - ligand (kitl) and promoted migration of mast cells from bone marrow to the ischemic site similar to rt effects in the thoracic cavity where mast cell density increased in bronchoalveolar lavage fluid. influx of mast cells following rt is blocked by treatment with imatinib, a small molecule tyrosine kinase inhibitor with activity against kit, platelet derived growth factor receptor (pdgfr) and abelson murine leukemia viral oncogene homolog (abl). imatinib treatment inhibited proliferation and induced apoptosis of mast cells and increased efficacy of rt in several murine tumor models. given that low - dose rt fosters mast cell - dependent vascular regeneration during limb ischemia model, it seems reasonable to conclude that increased recruitment and activation of mast cells following rt and subsequent alterations in ischemic microenvironments and activation of angiogenic programs may paradoxically foster tumor growth. rt and adaptive immunity in experimental rodent models of cancer development, e.g. brain, sarcoma, lung and breast, rt alone or in combination with dc or immunostimulatory therapies enhanced generation of anti - tumor responses mediated by cytotoxic t cells [66, 72, 108 ]. rt alone can also stimulate anti - tumor t cell - based immunity when given at high - doses by increasing the number of activated cd8 t cells [109111 ]. in 4 t1 mammary tumors, recruitment of cytotoxic t cells is dependent on cxcr6, a receptor for cxcl16. rt in combination with anti - ctla-4 mab increases recruitment of cxcr6 cd8 t cells. given that patients undergoing rt for prostate cancer exhibited detectable anti - tumor cd4 and cd8 t cells responses following rt that were undetectable prior to therapy, it stands to reason that in addition to genotoxic damage, induction of anti - tumor immunity via t cell activation represents an important mechanisms mediating the efficacy of rt - based therapy. preclinical models since one consequence of rt in tissues is induction of various immune -mediated programs, several groups have explored the potential of augmenting rt responsiveness with immunotherapeutics (immunostimulatory cytokines, dc - based therapy and antibodies targeting t cell costimulatory pathways) engineered to bolster anti - tumor immunity.immunostimulatory cytokines including il-2, il-12 and tnf- have been used in combination with rt to stimulate anti - tumor t cell responses. addition of these pro - inflammatory cytokines enhances rt efficacy by bolstering cytotoxic t cell responses [113117 ]. interestingly, il-3, a cytokine that activates monocytes and mast cells, delays tumor growth in response to rt. the enhanced tumor inhibition observed by combining cytokines with rt provides evidence that effectiveness of rt depends on immune - mediated mechanisms that can be targeted effectively to enhance overall rt response. based on this postulate, several groups have attempted to increase the presence of antigen - presenting dcs in tumors. intratumoral injection of cpg oligodeoxynucleotides that activate tlr9 on macrophages and dcs resulted in increased rt response and resistance to a second challenge with the same tumor, thus indicating development of a durable immune response.antigen - presentation on the surface of dcs to t cells requires both mhc and costimulatory molecules, b7 molecules and ox40 [121, 122 ]. strategies to enhance costimulatory molecules in combination with rt have been employed in a transgenic model of colon carcinogenesis. carcinoma cells were engineered to express a human antigen (cea) when rt was given in combination with a viral vaccine expressing cea and t cell co - stimulatory molecules, complete tumor regression was observed accompanied by anti - tumor cd4 and cd8 t cell infiltration. inhibition of tumor growth and enhanced overall survival was also observed in a murine sarcoma model when rt was given in combination with an agonistic antibody for ox40, a costimulatory molecule found on activated t cells that stimulates t cell proliferation and differentiation. inhibition of ctla-4 costimulation also enhanced effectiveness of rt in 4 t1 mammary carcinomas carcinoma resulting in diminished metastasis and increased survival [124, 125 ] ; however, rt dose and timing were critical with regards to anti - ctla-4 therapy. despite these numerous successful preclinical trials demonstrating efficacy of immune - modulation therapy in combination with rt, only a few clinical studies have been initiated to date. clinical studies clinical evaluation of rt in combination with immunotherapeutic strategies are currently being conducted for prostate, melanoma and liver carcinomas. rt has been evaluated in conjunction with drugs that inhibit androgen production resulting in enhanced autoantibody responses in 1530% of prostate cancer patients and correlating with previous studies indicating that anti - androgen therapy also increases t cell activity due to thymic regrowth [128, 129 ]. vaccination of prostate cancer patients with recombinant viral - based vaccines expressing prostate - specific antigen (psa), in combination with the costimulatory molecule b7 - 1 and standard rt to the prostate (70 gy of rt in 1.8 to 2.0 gy fractions), resulted in a three - fold increase in psa - specific t cells and evidence of generating t cells against other prostate - specific antigens in 76% of patients. immunogenicity of irradiated tumor cells in patients with melanoma was examined in which autologous irradiated melanoma cells engineered to express gm - csf, a white blood cell growth factor, were injected into patients resulting in a significant anti - tumor immune response leading to tumor regression in 50% of patients. the synergy between rt and dcs has been further evaluated in a small study of patients with hepatoma where dcs were injected intratumorally following a single - dose of rt leading to development of tumor - specific immune responses in 30% of patients. though these clinical studies involve small numbers of non - randomized patients, they present compelling findings indicating that the durability of rt may be enhanced by combinatorial therapy with selective immune - based therapeutics. though many cancer patients will receive rt, it is clear that clinician - scientists are only just beginning to understand the full spectrum of biologic responses resultant from rt. rt clearly influences multiple immune - based programs in tissues, some of which lead to durable tumor regression, whereas others propel tumor development. it seems reasonable to conclude that identifying pathways mediating activation of myeloid - based protumor immunity induced by rt, will encourage development of novel therapeutics that suppress those activities to effectively bolster rt responses. moreover, blockade of these protumor immune - based pathways may also present the opportunity to then combine rt with anti - tumor immunotherapeutics to yield effective and durable suppression of tumors, resulting in improved outcomes for patients with cancer. | chemotherapy and radiation therapy (rt) are standard therapeutic modalities for patients with cancer, including breast cancer. historic studies examining tissue and cellular responses to rt have predominantly focused on damage caused to proliferating malignant cells leading to their death. however, there is increasing evidence that rt also leads to significant alterations in the tumor microenvironment, particularly with respect to effects on immune cells infiltrating tumors. this review focuses on tumor - associated immune cell responses following rt and discusses how immune responses may be modified to enhance durability and efficacy of rt. |
immunoglobulin g4 (igg4)-related sclerosing disease is a systemic disease characterized by extensive igg4-positive plasma cells and t - lymphocyte infiltration of various organs (1). aside from autoimmune pancreatitis, it is the most common expressed form of igg4-related sclerosing disease. numerous other manifestations of the disease has been noted : sclerosing cholangitis, sclerosing cholecystitis, sclerosing sialadenitis, retroperitoneal fibrosis, tubulointerstitial pneumonia, prostatitis, hypophysitis (1 - 3). in addition, it has been reported that the igg4-related sclerosing disease could also be represented as inflammatory pseudotumor (ipt) in various organs (1, 4, 5). to our knowledge, however, involvement of the urethra by the igg4-related sclerosing disease has not been reported in the previous literature. in this report, we present a case of igg4-related ipt in the urethra. about 17 years before the presentation, the patient had a past medical history of an eyelid mass, which was clinically diagnosed as ipt and was relieved by steroid therapy. about 15 years later, she also underwent a computed tomography (ct) scan for the acute abdominal pain, and it revealed diffuse swelling of the pancreas. by a percutaneous cutting needle biopsy of the pancreas, the lesion was pathologically diagnosed as igg4-related autoimmune pancreatitis, and the patient 's symptoms were dramatically relieved by the steroid therapy. to evaluate the patient 's dysuria at this visit, a urologist performed physical examinations and laboratory studies, which yielded no positive findings suggestive of an infection or a malignancy, except for hematuria of 30 to 49 red blood cells per high power field (hpf) on a random urine analysis. on a subsequent cystoscopy, the urinary bladder was free, but a firm mass was suspected in the posterior wall of the urethra. thus, ct and magnetic resonance (mr) imaging were performed for further characterization of the urethral mass. two phase (unenhanced, enhanced) ct images were acquired with an 8-channel multi - detector raw ct (lightspeed ultra ; ge medical systems, milwaukee, wi, usa). in addition, mr images were acquired with a 3.0 tesla mr scanner (magnetom trio tim ; siemens medical solutions, erlangen, germany). on unenhanced ct images, the urethral mass demonstrated similar attenuation compared to the adjacent muscles. on subsequent contrast enhanced images, routine pelvic mr images also revealed a well - defined mass in the urethra with isointensity to slight hyperintensity on both t1 and t2 weighted images (repetition time [tr]/echo time [te ], 790/14, 4800/95, respectively) (fig. 1b, c, respectively). on diffusion weighted images with a b value of 1000 s / mm, obtained by echo - planar trace sequence (tr / te, 4800/79 ; section thickness, 5 mm), the mass showed hyperintensity suggesting diffusion restriction (fig. concordantly, the mass showed a lower apparent diffusion coefficient value than that of the adjacent muscles (0.65 10 mm / s, 1.39 10 mm / s, respectively) (fig. on gadolinium - enhanced fat - saturation t1-weighted images (tr / te, 2.9/1.2 ; section thickness, 2.4 mm), the mass demonstrated rim - enhancement in the arterial phase and homogeneous enhancement in the 5-minute delayed phase (fig. 1f, g, respectively). to rule out a malignancy such as urethral carcinoma, the patient underwent a core needle biopsy of the urethral lesion under transvaginal ultrasonography (us) guidance. the mass was found to be encased in the urethra and showed heterogeneously low echogeneity (fig. the pathologic specimen showed linear spindle cell proliferation and inflammatory cell infiltration in lymphocytes and eosinophils (fig. on immunohistochemical staining, both smooth muscle actin and igg4 (more than 30/hpf) were positive (fig. all pathologic results were compatible with igg4-related sclerosing disease, which was presented as ipt (igg4-related ipt). following steroid therapy for a few months, the patient 's symptoms were relieved and the size of the mass was markedly decreased on follow - up mr images (fig. immunoglobulin g4-related sclerosing disease is an emerging disease entity that can involve the pancreas, bile duct, gallbladder, salivary gland, retroperitoneum, kidney, prostate, and so on. it is characterized by extensive igg4-positive plasma cells and t - lymphocyte infiltration of various organs associated with tissue fibrosis and obstructive phlebitis (1). on the other hand, ipt is a rare benign condition characterized by abundant spindle cells mixed with variable amounts of extracellular collagen, lymphocytes, and plasma cells (6). therefore, ipt can either be a manifestation of igg4-related sclerosing disease or a distinct simple ipt, according to the amount of igg4-positive plasma cells. pathophysiologically, the role of igg4-positive cell is still unclear in ipt (7, 8). nonetheless, identifying the ipt and clarifying its relationship with igg4-related sclerosing disease may be critical, because the ipt is a well - known mimicker of malignant tumors and it may show a different pathophysiology according to the presence of igg4-positive plasma cells (8, 9). in particular, yamamoto. (8) emphasized the evaluation of igg4-positive plasma cells and the presence of obstructive phlebitis as markers for the differential diagnosis between igg4-related ipt and an inflammatory myofibroblastic tumor (imt). as the imt is regarded as a neoplastic counterpart to the ipt, the evaluation of igg4-positive plasma cells is crucial to determining an optimal treatment plan. unfortunately, identifying igg4-related ipt, non igg4-related ipt, and imt is still an unexplored field from a radiologic point of view. park. (6) studied the imaging findings of ipts in the genitourinary tract without evaluating the association with igg4-related sclerosing disease. moreover, they reported that genitourinary ipt can be seen in variable patterns on us and ct, which may be attributed to varying degrees of fibrosis and inflammation (6). they also described the disease usually demonstrates delayed homogeneous enhancement, hypointensity on t2 weighted image, and diffusion restriction on mr imaging (6). these features can be regarded as projections of fibrotic change in the developing ipt (5, 6). however, the various degrees of fibrosis and the inflammatory process can result in a broad range of mr imaging manifestations (9). in our report, we were able to obtain ct, mr and us images of the igg4-related sclerosing disease manifested as a urethral ipt. ct and us features of our case were consistent with known imaging findings of ipt (6, 9, 10). on mr imaging of our case, although the presented case revealed isointense to slightly hyperintense t2 signal intensity, which is not typical of the fibrosis, this may be possibly understood as a result of the mixed inflammatory and fibrotic stages. meanwhile, a core needle biopsy under transvaginal us guidance was very useful in the diagnosis of our case. as a transvaginal us - guided biopsy is known as a safe and well - established method to obtain urethral and periurethral tissues (11), we believe this method should be considered to diagnose igg4-related sclerosing disease involving the urethra. although the ipt as a manifestation of the igg4-related sclerosing disease has been reported in various organs (1, 4, 5), most of the reports have provided limited information about imaging findings of the disease. most of all, to the best of our knowledge, no report handles urethral involvement of the igg4-related sclerosing disease. we described the imaging features of urethral involvement of the igg4-related ipt, which was developed metachronously after other organ involvement including the eyelid and the pancreas. in patients with a past history or suspicion of igg4-related sclerosing disease, urethral mass should be carefully considered as a potential manifestation of the igg4-related sclerosing disease. furthermore, acknowledging that the igg4-related sclerosing disease may present as a urethral mass is essential to avoid unnecessary surgery or anti - cancer treatment. in some cases, a needle biopsy under transvaginal us guidance can be helpful to diagnose urethral involvement of igg4-related sclerosing disease. | immunoglobulin g4 (igg4)-related sclerosing disease is a systemic disease characterized by extensive igg4-positive plasma cells and t - lymphocyte infiltration in various organs. we described the imaging findings of an igg4-related inflammatory pseudotumor in the urethra. the urethral mass showed isoattenuation on unenhanced ct images, delayed enhancement on enhanced ct images, iso- to slight hyper - intensity on t1 and t2 weighted magnetic resonance images, diffusion restriction on diffusion weighted images, and heterogeneously low echogeneity on ultrasonography. |
paraquat (pq) is a nonselective contact herbicide, which is used world - widely for its high efficiency and low residues in the crops. it has been extensively demonstrated that it is highly toxic to multiorgans when absorbed through ingestion, skin contact, or inhalation. the primary target organ for pq toxicity is the lung as a consequence of its accumulation, against a concentration gradient, through the highly developed polyamine uptake system [13 ]. the toxicity mechanism of pq is mainly due to a sustained redox - cycling effect, resulting in oxidative stress - related insults such as lipid peroxidation. pq - induced lung injury results in alveolar epithelial cells (type i and ii pneumocytes) and clara cell disruption, impairments of the surfactant system, hemorrhage, edema, hypoxemia, infiltration of inflammatory cells into the interstitial and alveolar spaces, proliferation of fibroblasts, and excessive collagen deposition [3, 4 ], which ultimately leads to pulmonary fibrosis and respiratory failure. survivors of pq poisoning may be left with a restrictive type of long - term pulmonary dysfunction. so far, there are no known pharmacological antidotes for pq poisoning [68 ] and therapeutics have been disappointing and the mortality has still remained high. thus agent with both antioxidant and regulating fibrosis properties would have favorable value in the treatment of paraquat - induced lung injury. pyrrolidine dithiocarbamate (pdtc) is a low - molecular - weight thiol compound, which has a variety of biochemical activities, such as redox state alternation [10, 11 ], heavy metal chelation, and enzyme inhibition. pdtc was initially regarded as a potent inhibitor of nf-b [10, 14 ], and it has been used as an antioxidant compound to counteract the toxic effects of free radicals and to interfere with the generation of proinflammatory cytokines. it has the potential to activate gene expression of endogenous antioxidants such as superoxide dismutase, independent of any effects on nf-b. it induces the genes encoding the two subunits of the enzyme gcs and increased de novo synthesis of the cellular protectant gsh. pdtc reduces oxidant - mediated cellular injury, as demonstrated by a reduction in the accumulation of malondialdehyde. antioxidants such as pdtc and their modulatory effects on nf-b activation suggest that these agents may offer therapeutic benefits in acute lung injury caused by pq. therefore, the present study was designed to evaluate the effects of pdtc in a rodent model of acute lung injury induced by pq and observe its potential therapeutic effect in order to provide scientific basis for the treatment strategy of paraquat - induced lung damage. pyrrolidine dithiocarbamate (99%) was purchased from sigma - aldrich (st. louis, mo, usa). fifty - four male sprague - dawley (sd) rats which initially weighed between 180 and 220 g were purchased from the laboratory animal research center of fudan university. the animals had free access to the pellet diet and water ad libitum always, and were maintained on 12-hour diurnal cycles and in a controlled environment with a temperature of 20 ~ 22c and humidity of 50 5% for a period of 1 week before usage. animals were randomly divided into the control group, n = 6 : animals treated with the saline solution ; the pq group, n = 24 : animals were orally given aqueous solution of paraquat (40 mg / kg) by gastric gavage and sacrificed at 3rd day (n = 6, pq 3d group), 7th day (n = 6, pq 7d group), 14th day (n = 6, pq 14d group) and 21st day (n = 6, pq 21d group) ; pq+pdtc group, n = 24 : animals intoxicated with 40 mg / kg pq followed by immediate injection 120 mg / kg pdtc (ip), and sacrificed at 3rd day (n = 6, pq+pdtc 3d group), 7th day (n = 6, pq+pdtc 7d group), 14th day (n = 6, pq+pdtc 14d group), and 21st day (n = 6, pq+pdtc 21d group). on days 3, 7, 14, and 21 after treatments, one group rats of each treatment were sacrificed with an ip injection of 10% chloralum hydratum (3 ml / kg body weight). a total of 6 ml of venous whole blood was collected in heparin - containing vacutainer, and centrifuged at 400 g for 10 minutes collecting supernatant for measurement of the activities of glutathione peroxidase (gsh - px), superoxide dismutase (sod), the level of maleic dialdehyde (mda), and tgf-1 protein. after collected blood sample, immediately thoracotomy was sterilely performed to obtain bronchoalveolar lavage fluid (balf). once ligating the left major bronchial beneath the tracheal crotch, bronchoalveolar lavage (bal) was performed by flushing right lung with 5 ml of saline through the tracheal cannula three times. the supernatant was harvested for the activities of sod, gsh - px, and mda analysis. right lung was harvested and frozen at 80c for rna extraction and the content of hydroxyproline (hyp) in lung homogenate measurement, a small piece of left lung was fixed in 3% glutaraldehyde and immediately sent for ultra - morphological examination, and the remaining lungs were fixed in 10% formaldehyde solution before histological analysis. he and masson 's trichrome staining of lung section were undertaken, the latter was used observing collagen fibers. according to the methodology described by szapiel the activities of gsh - px, sod, and the level of mda both in plasma and balf of rats were measured using qualified kits. the hydroxyproline contents of lung tissues were determined and the data were expressed as ng / g wet lung tissue. the 100 mg frozen lung tissue from control, pq - treated and, pq+pdtc - treated rats was thoroughly homogenized in distilled water and measured using qualified kits. total rna was extracted using the trizol reagent (life technologies, grand island, ny, usa) according to the manufacturer 's instructions. yield and purity of the isolated rna solution were determined by a260 and a280 readings on a spectrophotometer. reverse transcription was performed on 3g of rna with oligo - dt primers and avian myeloblastosis virus reverse transcriptase (mbi fermentas, st. the primers for mrna analysis were upper 5 gctcgctttgtacaacagca 3 and lower 5 gagttctacgtgttgctcca 3 yielding a 280-bp product for tgf-1. the primers for mrna analysis were upper 5 cctctatgccaacacagtgc 3 and lower 5 gtactcctgcttgctgatcc 3 yielding a 210-bp product for housekeeping gene -actin. the pcr products were analyzed by electrophoresis on an agarose gel, stained with ethidium bromide, and photographed. to determine the linear range of the pcr, the intensity of the amplified products was plotted against the cycle number. the plasm sample was used for measurements tgf-1 levels with enzyme - linked immunosorbent assay rat tgf-1 kit (bender medsystems, lot : 20280013) according to the manufacturer 's protocol. protein quantification was performed according to the method of lowry., using bovine serum albumin as standard. comparisons between control, pq - treated, and pq+pdtc - treated groups at each time point were made using unpaired student 's t - test. polypnea, blausucht, crouch, diarrhea, anorexia were present especially in animals exposed to pq during the first 24 hours. one animal died on the third day after treatment in the pq group and pq+pdtc group, respectively. the results of our experiment using pq - treated rats (40 mg / kg pq) indicated that lipid peroxidation marker mda levels in plasma and balf were significantly increased and the activities of gsh - px and sod were significantly decreased (p <.01) compared with that in the control group. when the rats were cotreated with pq and pdtc, we found the activities of gsh - px and sod in the pdtc treatment group (120 mg / kg) was markedly higher than that of pq - treated group (p <.05 or p <.01), and the mda levels was lower correspondingly (table 1). the content of hyp in lung tissue was increased significantly (p <.05) compared with the control group at 7th day after the treatment of pq 40 mg / kg, while the content of hyp in lung tissue was slightly decreased in cotreated with pq and pdtc group (figure 1). pq - induced lung structural, ultrastructural alterations, and alleviative effects of pdtc on pq - damages are depicted in figures 2 and 3. histologic changes were assessed with h&e and correlated with lung fibrosis, which was identified by using masson 's trichrome stain for collagen. according to the methodology described by szapiel, animals from control group (saline solution) presented a normal pulmonary structure at light microscopy (lm) and electron microscopy (em), without evidences of alveolar collapse, cellular infiltrations, or collagen accumulation. pq administration induced marked alterations compared to the control pattern, mainly characterized by a diffuse alveoli collapse with an increased thickness of its walls. it was noticed that an intense vascular congestion with numerous activated platelets and polymorphonuclear cells inside the capillaries. the majority of pneumocytes showed, at least, one ultrastructural abnormality, mitochondrial swelling being the most frequent alteration, abundant rough endoplasmic reticulum (rer), and rich ribosome in the fibroblast. in comparison with the pq group, the occurrence of the above, referred alterations were drastically attenuated in the pq+pdtc groups, particularly inflammation, hemorrhage, and the amount of accumulation of collagenous fiber. despite the existence of several pneumocytes with mitochondrial swelling and evidences of interstitial edema, the exuberance of those signals and the ratio of affected cells were drastically attenuated in pq+pdtc animals. furthermore, comparing to the pq group, the vascular congestion and the alveolar collapse were not as noticeable in pq+pdtc animals. in order to analyze the effects of pq on the tgf-1, we tested the mrna and protein levels of tgf-1 in lung tissues from rats treated with pq. as expected, the levels of both mrna and protein of tgf-1 were significantly drastically increased by the pq (p <.05 or p <.01). cotreatment of pdtc with pq significantly decreased the levels of both mrna and protein in comparison with pq treatment alone (p <.05 or p < paraquat is a highly toxic compound for humans and animals. over the past few decades, many cases of acute poisoning and death have been reported [22, 23 ]. the major cause of death in paraquat poisoning is respiratory failure due to an oxidative insult to the alveolar epithelium with subsequent obliterating fibrosis. the cellular damage mediated by pq is essentially due to its redox - cycle leading to continuous superoxide radicals (o2) production. this then sets off the well - known cascade leading to generation of the hydroxyl radical (ho), which has been implicated in the initiation of membrane injury by lipid peroxidation during the exposure to pq [2426 ]. in addition, researchers have proposed the hypothesis of cytotoxicity via mitochondrial dysfunction caused by pq [24, 25 ]. the data presented in table 2 showed significant changes in endogenous antioxidant system and lipid peroxidation during the treatment of rats with pq, where the sod and gsh - px activities were decreased in blood plasma and balf, and the level of mda increased which is indicative for excessive lipid peroxidation. the aforementioned observation confirms the consequence of the intracellular accumulation of reactive oxygen species (ros) with subsequent development of lungs injury [24, 26 ]. in the present study, we performed qualitative and semiquantitative analysis of the morphological injury of lung by pq (table 2 and figures 2 and 3). we have observed the characteristic pq - induced pathological alterations including alveolar edema, hemorrhage, inflammatory cell infiltration, the swollen - type ii alveolar epithelial cells, and deformed mitochondria by electro - microscopy in less than 7 days after pq exposure. the abundant rough endoplasmic reticulum (rer) and rich ribosome in the fibroblast and the development of an extensive fibrosis in lung during 1421 days after pq exposure, are probably a compensatory repair mechanism to damaged alveolar epithelial. the alveolar walls were thickened, predominantly with collagen ; mild mononuclear cell infiltration in the alveolar walls and alveolar collapse were observed in necropsy specimens of two patients 8 and 10 days after paraquat poisoning. in this study, cotreatment with pdtc was very effective in the preventing oxidative damage induced by pq, which are characterized by the reversal of pq - induced tissue damages. in addition, the pq - induced biochemical changes as indicated by significant decrease of sod and gsh - px activities, along with an increase mda level in blood plasm and balf, were also alleviated by pdtc (table 1). the mechanism of pdtc 's antioxidant effect could be explained in part by activating gene expression of endogenous antioxidants such as superoxide dismutase and a reduction in the accumulation of malondialdehyde. these results showed that the pdtc as an antioxidant compound to counteract the toxic effects of free radicals and to interfere with the generation of proinflammatory cytokines efficiently protect lung from pq - induced oxidative damage. moreover, these morphological evidences of cellular aggression were attenuated by pdtc treatment (table 2 and figure 2), and the reduced accumulation of collagenous fiber observed in pq+pdtc - treated animals during 1421 days may be interpreted as a consequence of regulated collagen gene expression, which pq+pdtc - treated groups could attenuate paraquat - induced upregulation of tgf-1 mrna expression levels. tgf-1 is a key growth factor that initiates tissue repair and its sustained production underlies the development of tissue fibrosis. in experimental models of lung fibrosis, tgf-1 has been shown to be an important upstream effector of collagen gene expression [30, 31 ]. the attenuation of pq - induced damages by pdtc suggests that the alleviation of pq - induced fibrosis may be due to the inhibitory effects of pdtc on nf-b activation which then led to reduced tgf-1 gene expression. in conclusion, our results in the present study clearly demonstrated that pdtc significantly increased sod, gsh - px activities, decreased mda, hyp levels, and reduced accumulation of collagenous fiber in paraquat - treated rat. these findings suggested that pdtc may exert its protective effects on paraquat - induced pulmonary damage by alleviating the earlier inflammation damage via paraquat - induced oxidative stress and the later fibrosis in rat lung and by regulating the mrna expression of tgf-1. future studies are warranted to further investigate the underlying mechanisms involved in this complicated process. | paraquat (pq) has been demonstrated that the main target organ for the toxicity is the lung. this study aimed to investigate the potential protective effect of pdtc on the pq - induced pulmonary damage. fifty - four rats were divided into control, pq - treated and pq+pdtc - treated groups. rats in the pq group were administrated 40 mg / kg pq by gastric gavage, and pdtc group with 40 mg / kg pq followed by injection of 120 mg / kg pdtc (ip). on the days 3, 7, 14 and 21 after treatments, the activities of gsh - px, sod, mda level and the content of hyp were measured. tgf-1 mrna and protein were assayed by rt - pcr and elisa. mda level in plasma and balf was increased and the activities of gsh - px and sod were decreased significantly in the pq - treated groups (p <.05) compared with control group. while the activities of gsh - px and sod in the pq+pdtc - treated groups was markedly higher than that of pq - treated groups (p <.05), and in contrast, mda level was lower. tgf-1 mrna and protein were significantly lower in the pq+pdtc - treated groups than that of pq - treated groups (p <.05). the histopathological changes in the pq+pdtc - treated groups were milder than those of pq groups. our results suggested that pdtc treatment significantly attenuated paraquat - induced pulmonary damage. |
o - methylguanine - dna methyltransferase (mgmt) is a dna repair protein that protects cells against the carcinogenic and cytotoxic effects of alkylating agents.1 mgmt activity has been determined in various types of tumors, and was found to be relatively high in colon, ovary, breast and brain cancers.2,3 the lack of mgmt expression may be related to the development of gliomas,4 non - small cell lung cancers,5 and colon cancers.6,7 in the absence of mgmt activity, o - alkylguanine mispairs with thymine during dna replication and results in guanine - cytosine to adenine - thymine transitions.6,8 other studies that showed loss of mgmt protein expression associated with methylation in diffuse large b cell lymphoma and colorectal and brain tumors.6,7 aberrant methylation of 5 cytosine residues of a guanine residue in cpg islands in the promoter regions of tumor suppressor genes is an important mechanism of gene transcriptional inactivation and has been associated with tumorigenesis. several studies have provided evidence of the linkage between epigenetic inactivation of mgmt and the appearance of g to a transition mutations in genes in human primary tumors.6 tumor suppressor gene p53 plays an important role in the cell cycle, dna damage, cell death and cell differentiation, and it is commonly mutated in human tumors.9 several studies have suggested the involvement of p53 protein in the expression of the mgmt gene. there are studies reported that mgmt gene expression was significantly lower in p53 altered tumors,4 and mgmt promoter methylation may increase the occurrence of p53 mutation in lung cancer.10 other studies reported that wild - type p53 acts as an inhibitor of mgmt gene expression.11 to the best of our knowledge, the relationship between mgmt and p53 expression in tissues of endometrial cancer has not yet been studied in korea. so we investigated the expression patterns of mgmt and p53 using immunohistochemistry to elucidate the tissues - specific relationship between mgmt and p53 expressions in endometrial cancers. a retrospective study was carried out on 36 cases with well differentiated endometrial adenocarcinomas admitted in local hospital from 2008 to 2010 in busan. the ages of the 36 patients ranged from 34 to 68 years (median age : 52 years). all cancer cases were obtained endometrial curettage, and histopathologically confirmed and had no preoperative chemotherapy or radiotherapy. the he stained were reviewed in each case to confirm the original diagnosis, which was based on the figo classification. immunohistochemical study for mgmt and p53 was performed on the formalin - fixed, paraffin - embedded, 4 m thick tissue section using the avidin - biotin - peroxidase complex method. deparaffinization of all the sections was performed through a series of xylene baths, and rehydration was performed with a series of graded alcohol solutions. to enhance immunoreactivity, microwave antigen retrieval was performed at 750 w for 30 min in tris - edta buffer (ph 9.0). after blocking endogenous peroxidase activity with 5% hydrogen peroxidase for 10 min, the primary antibody was a mouse monoclonal antibody directed against mgmt (lab vision, fremont ca, usa) and p53 (dakocytomation, denmark) used in a 1:100 dilution. an envision chem kit (dakocytomation, carpinteria, ca, usa) was used for the secondary antibody at room temperature for 30 min. after washing the tissue samples in tris - buffered saline for 10 min, 3, immunoreactivity for mgmt expression was defined by presence of nuclear and cytoplasmic staining and that for p53 expression was defined by presence of nuclear staining. the percentage scoring of the immunoreactive tumor cells was categorized into four groups : 0 (0%), 1 (110%), 2 (1150%), and 3 (> 50%). the staining intensity was also categorized into four groups : 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). a final score was obtained for each case by multiplying the percentage and the intensity score. finally, tumors with multiplied score exceeding 3 (i.e., tumors with a moderate and strong intensity of > 10% of the tumor cells) was recorded as positive immunoreactivity to mgmt and p53 ; all the other scores were considered negative. statistical analysis was performed using spss for windows standard version 19.0 (spss, chicago, il, usa). fisher exact test was performed to assess the relationship between the expression patterns of mgmt and p53. a p value less than 0.05 was considered to be statistically significant. a retrospective study was carried out on 36 cases with well differentiated endometrial adenocarcinomas admitted in local hospital from 2008 to 2010 in busan. the ages of the 36 patients ranged from 34 to 68 years (median age : 52 years). all cancer cases were obtained endometrial curettage, and histopathologically confirmed and had no preoperative chemotherapy or radiotherapy. the he stained were reviewed in each case to confirm the original diagnosis, which was based on the figo classification. immunohistochemical study for mgmt and p53 was performed on the formalin - fixed, paraffin - embedded, 4 m thick tissue section using the avidin - biotin - peroxidase complex method. deparaffinization of all the sections was performed through a series of xylene baths, and rehydration was performed with a series of graded alcohol solutions. to enhance immunoreactivity, microwave antigen retrieval was performed at 750 w for 30 min in tris - edta buffer (ph 9.0). after blocking endogenous peroxidase activity with 5% hydrogen peroxidase for 10 min, the primary antibody was a mouse monoclonal antibody directed against mgmt (lab vision, fremont ca, usa) and p53 (dakocytomation, denmark) used in a 1:100 dilution. an envision chem kit (dakocytomation, carpinteria, ca, usa) was used for the secondary antibody at room temperature for 30 min. after washing the tissue samples in tris - buffered saline for 10 min, 3, immunoreactivity for mgmt expression was defined by presence of nuclear and cytoplasmic staining and that for p53 expression was defined by presence of nuclear staining. the percentage scoring of the immunoreactive tumor cells was categorized into four groups : 0 (0%), 1 (110%), 2 (1150%), and 3 (> 50%). the staining intensity was also categorized into four groups : 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). a final score was obtained for each case by multiplying the percentage and the intensity score. finally, tumors with multiplied score exceeding 3 (i.e., tumors with a moderate and strong intensity of > 10% of the tumor cells) was recorded as positive immunoreactivity to mgmt and p53 ; all the other scores were considered negative. statistical analysis was performed using spss for windows standard version 19.0 (spss, chicago, il, usa). fisher exact test was performed to assess the relationship between the expression patterns of mgmt and p53. a p value less than 0.05 was considered to be statistically significant. the loss of mgmt expression was detected in 11 (30.6%) out of the 36 endometrial cancers, and p53 immunoreactivity was detected in 23 (63.9%) out of the 36 endometrial cancers. ten (90.9%) of the 11 cases with negative mgmt immunoreactivity showed positive p53 expression, whereas 1 (9.1%) of the 11 cases with negative mgmt immunoreactivity showed negative p53 expression. so the loss of mgmt expression was significantly associated with the p53 overexpression (p=0.03) (table 1, fig. loss of mgmt expression has been reported to have a significantly role in carcinogenesis in various organs.4 in this study, the loss of mgmt expression was detected in 11 (30.6%) out of the 36 endometrial cancers. there are studies that a lack of mgmt expression may be related to guanine to adenine mutation, and may be related to the development of gliomas,4 non - small cell lung cancers,5 and colonic cancers.6,7 however, other studies have been reported that the lack of mgmt expression in not commonly due to mutation, deletion, or rearrangement of the mgtm gene.12 hypermethylation of cpg islands in its promoter region is the most important mechanism.13 esteller. reported that the mgmt gene is epigenetically inactivated by promoter hypermethylation in many primary tumor types, and a direct relationship between mgmt aberrant methylation and g : c to a : t transition mutations of p53 in colorectal tumors.6,7 methylated mgmt gene promoter has been associated with loss or decrease of mgmt expression in tumor tissues of various organs, including lung tumors.5,7 in lung carcinoma, 25% of non - small cell lung carcinoma and 37% of adenocarcinoma have been reported to show loss of mgmt expression.5 p53 is the most commonly mutated gene in human cancer with transition mutations being the main type of p53 mutation observed.9 several studies that mgmt inactivation by hypermethylation has been reported to be associated with a shift from the g : c to a : t mutation in the p53 gene,10 which is known to be one of the most important tumor suppressor genes in human. myong reported that mgmt loss associated with p53 overexpression in lung cancers, especially adenocarcinomas.14 also, osanai. reported that expression of p53 may be associated with the regulation of mgmt expression in breast tumors, and that mgmt immunonegativity and p53 immunopositivity may be strong predictors of breast cancer survival.15 however, other studies that wild - type p53 is accompanied by lower mgmt protein expression.11 in this studies that 10 (90.9%) of the 11 cases with negative mgmt immunoreactivity showed positive p53 expression, whereas 1 (9.1%) of the 11 cases with negative mgmt immunoreactivity showed negative p53 expression. so the loss of mgmt expression was significantly associated with the p53 overexpression (p=0.03). reported that mgmt gene expression was significantly lower in p53 mutated tumors, because mutations of p53 in human cancer are common and occur in all malignant cell types, the relationship observed between mgmt expression and p53 is highly relevant.4 recently, the genetic and epigenetic pathways are not isolated, but rather a complex network of cross - talk between genetic and epigenetic factors may exist. the results of this study because it is limited to immunohistochemical test, further studies are needed to promoter methylation. these findings suggest that the loss of mgmt expression may be one of factors capable of p53 overexpression in endometrial cancer, but some follow - up is needed since this study was limited to 36 cases of well differentiated endometrial adenocarcinoma. further studies are needed to define the relation between mgmt and p53 for examining the mechanisms of tissue - specific mgmt expression. | o6-methylguanine - dna methyltransferase (mgmt) is a dna repair protein, the loss of mgmt expression was commonly known due to hypermethylation of cpg islands in its promoter region. overexpression of p53 protein may be associated with downregulated mgmt expression in brain tumors. the aims of this study were to investigate the role of mgmt expression loss and its correlation with p53 overexpression in endometrial cancers. mgmt and p53 expression was examined in formalin - fixed, paraffin - embedded tissues from 36 endometrial cancer cases using immnunohistochemical staining. the loss of mgmt expression was detected in 11 (30.6%) out of the 36 endometrial cancers and p53 immunoreactivity was detected in 23 (63.9%) out of the 36 endometrial cancers. ten (90.9%) of the 11 cases with negative mgmt immunoreactivity showed positive p53 expression, so the loss of mgmt expression was significantly associated with the p53 overexpression (p=0.03). these findings suggest that the loss of mgmt expression may be one of factors capable of p53 overexpression in endometrial cancer. further studies are needed to define the relation between mgmt and p53 for examining the mechanisms of tissue - specific mgmt expression. |
stroke is a known manifestation of human varicella zoster virus (vzv) infection seen in children with chicken pox and in adults with zoster. this vzv associated vasculopathy affects either large (granulomatous arteritis) or small vessels in both immunocompetent and compromised patients. it is thought that the virus travels from the ipsilateral trigeminal ganglion through its branches to the vessel wall and cause inflammation. there are also few reports that raise a possibility of contiguous vascular, cerebrospinal fluid (csf), or hematogenous spread of the virus from the ganglion (trigeminal / thoracic) to the cerebral blood vessels. in this case report, we describe an immunocompromised patient secondary to human immunodeficiency virus infection (hiv) with left herpes zoster ophthalmicus who subsequently developed bilateral cerebral infarctions. a 43-year - old male was brought to our hospital by his elder sister with history of sudden onset of inability to understand the speech and bumping on to objects on the right side of his way and later on progressively becoming drowsy for duration of 15 days. he was a known hiv seropositive patient not on any treatment and his cd4 count and viral load were not available. three months prior to this illness, he had developed skin lesions suggestive of herpes zoster involving his left forehead and periorbital area 's with redness of the eye. he was treated by an ophthalmologist with oral acyclovir tablets, analgesics, and eye drops for 14 days. the lesions had healed during next 15 days with mild scarring of the left cornea with impaired vision. on examination he had the scar of herpes zoster with keratitis on the left side. he was speaking some words unrelated to context of the conversation. reading, writing, and repetition could not be tested. there was spasticity of all four limbs (right more than left) with mild right sided weakness requiring one - person support to walk. investigations revealed normal hemoglobin, erythrocyte sedimentation rate, vasculitis profile, renal and hepatic parameters, electrolytes, lipid profile, cardiac evaluation, and serum venereal disease research laboratory (vdrl) test. magnetic resonance imaging (mri) of the brain plain and contrast [figure 1 ] showed left temporal, left parietal, left basal frontal and periventricular and right basal ganglionic infarcts with hemorrhagic transformation. the distal left internal carotid, bilateral middle cerebral arteries were irregular with nonvisualization of the left anterior cerebral artery [figure 2 ]. india ink, cryptococcal antigen, antitoxoplasma, antimycobacterial, and anticysticercal antibodies were negative. very high titers of csf igg antibodies to vzv (5000 iu / ml) by enzyme linked immuno absorbent assay (elisa) were seen while igm antibodies were negative. magnetic resonance imaging of the brain t1 (a), t2 (b), fluid attenuated inversion recovery (c and d), diffusion weighted image (e) and apparent diffusion weighted image (f) sequences showing bilateral infarcts with hemorrhagic transformation magnetic resonance angiography of the brain (a and b) showing irregularity and dilation of intracranial vessels (arrows) he was diagnosed as a case of left herpes zoster ophthalmicus with vasculopathy with acquired immuno deficiency syndrome (aids) with bilateral ischemic infarcts. he was treated with intravenous acyclovir 500 mg 8 hourly for 14 days (weight : 48 kg), methylprednisolone 1 g / day for 5 days, injection heparin followed by oral acenocoumarol with prothrombin time (international normalization ratio) maintained at around 1.5 for 3 months. he also received other supportive therapy during hospitalization and tab clopidogrel 75 mg / day after 3 months. over the next 15 days, the patient gradually improved in the sensorium and language function, in the form that he could understand simple commands, indicate his toilet needs, and walk independently. repeat csf examination done after 6 months was normal (cells -3/l, protein -0.25 g / l repeat antibodies not done). he was also started on antiretroviral therapy and 2 years in to his follow - up he is maintaining good health with normalization of the cd4 counts (700 cells / mm). our patient who was a known case of hiv seropositive state presented with bilateral cerebral infarcts 3 months after left zoster ophthalmicus. the internal, middle and anterior cerebral arteries ipsilateral to the zoster and branches of the contralateral middle cerebral arteries were involved in the form of irregularity and beading of the vessels. other causes of stroke were excluded by appropriate history, examination, and investigations and it was attributed to varicella vasculopathy with positive csf igg anti vzv antibodies. following treatment with acyclovir, short course steroid therapy, anticoagulants, and antiplatelets and later on with antiretroviral therapy he made significant recovery and is on follow - up. the salient and interesting features about the present patient are (a) bilateral involvement of the cerebral vessels with unilateral zoster ophthalmicus with immunocompromised state and (b) favorable response to the treatment with acyclovir, short course of steroid, anticoagulants, and later antiplatelets. this case report also supports the possibility of contiguous vascular, or csf or hematogenous spread of the virus in the pathogenesis of zoster - related vasculopathy in addition to the classical theory of neurogenic spread. infection with vzv is associated with spectrum of neurological manifestations in humans like zoster, preherpetic and postherpetic neuralgia, myelitis, large vessel granulomatous arteritis, and small vessel encephalitis. large vessel infarcts following zoster infection of the trigeminal ganglion are thought to be from axonal transport of the virus from trigeminal afferent fibers that innervate the vessels of the anterior cerebral circulation with subsequent vasculitis. there are few reports of cerebral infarcts following sacral and thoracic ganglion involvement, suggesting csf / hematogenous spread of the virus or simultaneous reactivation in the trigeminal ganglion. recently there have been reports of hiv seropositive and negative patients with bilateral cerebral infarcts with unilateral zoster suggesting vascular spread of the virus, which is similar to our case. they also demonstrated, like in our case, the vzv antibodies in csf and improvement in clinical condition with acyclovir. the best recognized are the infectious complications, which include vzv, cytomegalovirus, tuberculosis, cryptococcosis, syphilis, and toxoplasmosis. we have ruled out conventional causes of stroke and above etiologies including tuberculosis by negative csf antibodies, other blood and imaging studies. the temporal association of stroke with zoster ophthalmicus, positive csf antibodies, response to treatment, and absence of other demonstrable etiologies suggest that vzv is the reason for stroke in our patient. five modes of vzv infection of the central nervous system in aids patients are described : (i) multifocal encephalitis predominantly involving the white matter likely to be due to hematogenous spread, (ii) ventriculitis due to necrosis of the ventricular wall, (iii) acute hemorrhagic meningo - myeloradiculitis with necrotizing vasculitis due to secondary seeding of the csf, (iv) focal necrotizing myelitis due to neural spread from the diseased dorsal root ganglion, and (v) vasculopathy involving leptomeningeal arteries and causing cerebral infarcts associated with meningitis. vasculopathy involving large vessels present with acute stroke with in a mean period of 7 weeks between the onset of neurological disease and zoster, but intervals as long as 6 months have been recorded. mental symptoms are common and up to 25% of patients may succumb due to their illness. cerebral angiography usually shows the focal constriction and segmental narrowing of the affected vessels and commonly middle, anterior, and internal carotid arteries are involved. other vasculopathies can produce the same neurologic symptoms, signs, csf changes, and imaging abnormalities ; hence detection of anti - vzv igg antibody in csf, which is the virologic test of choice is required for the diagnosis. our patient had presented 3 months after the zoster and had involvement of vessels bilateral to the rash with hemiparesis and encephalopathic symptoms. these features probably suggest that the vzv virus had spread through cerebral arteries or csf or hematogenous route as compared with classical the trigeminal nerve spread. steroids, intravenous acyclovir and anticoagulation (to prevent progression of thrombosis) have been advocated as the treatment modalities. because the virus is present in the arteries and it is associated with inflammation there is a role for acyclovir and steroids. we treated our patient with the above modalities and he made a good recovery and was continued on antiplatelet medicine because we could not repeat imaging of the blood vessels. when a diagnosis (clinically and radiologically) of vzv with stroke is suspected, virological confirmation is required it can be either from detecting vzv dna by polymerase chain reaction (pcr) or by anti - vzv igg antibody in csf. in a study, 93% of the 30 patients with vzv vasculopathy had anti - vzv igg in the csf compared with only 30% with vzv dna in csf by pcr. this may be due to protracted clinical course of vzv vasculopathy, usually lasting for weeks to months ; hence antigen may disappear and only antibodies might persist. positive csf pcr for vzv is helpful ; however a negative pcr does not exclude the diagnosis. the hiv itself is a neurotropic virus and has predilection for the endothelial cells and may present with granulomatous angitis, eosinophilic vasculitis, and nonspecific small vessel angitis. the causes of infarctions in aids patients include vasculitis, coagulation abnormalities due to virus, infections, neoplasms and cardiac. viral load has no correlation with vasculopathy and it is a diagnosis of exclusion and it is important that treatable causes are excluded. in our patient it is difficult to exclude the hiv associated primary vasculopathy but clinical circumstances, csf vzv antibody positivity and response to treatment favor vzv vasculopathy. in conclusion, this case report of unilateral zoster ophthalmicus in an immunocompromised patient presenting with bilateral stroke highlights the csf / hematogenous / vascular spread of the virus as opposed to the conventional neurogenic spread. patient made a good recovery with a choice of modalities of treatment, which act on the various aspects of the pathogenic mechanism of the stroke in a patient with zoster. | ischemic stroke is a recognized complication of herpes zoster ophthalmicus. arterial involvement is usually seen on the side of the rash. it is thought to be due to vessel inflammation by the virus, which travels from the trigeminal ganglion. few case reports of bilateral and distant site of zoster lesions with stroke in the brain have been described. these reports suggest possibility of contiguous vascular, cerebrospinal fluid (csf) or hematogenous spread of the virus from the ganglion to the cerebral blood vessels. therapeutically acyclovir, anticoagulation, and steroids have been used in the treatment of the zoster associated with stroke. we describe a case of immunocompromised patient with ipsilateral zoster ophthalmicus with bilateral anterior circulation strokes, who was treated with above measures and made successful recovery. this report also raises / supports possible csf / vascular / hematogenous spread of the virus from the ganglion to involve cerebral blood vessels leading to the stroke. |
convergence insufficiency (ci) is a neuromuscular deficiency of binocular eye alignment (grisham, 1988) characterized by a reduction in the degree of convergence or the inability to maintain the necessary convergence of the eyes on a near target while keeping this target single, clear, and comfortable to look at. ci is associated with a wide range of symptoms, such as eyestrain, headaches, blurred vision, fatigue, sleepiness, difficulty concentrating while reading, and double vision (lavrich, 2010). two earlier studies have reported that a clinical diagnosis of ci was more frequent in patients with parkinson s disease (pd ; lepore, 2006 ; repka, claro, loupe, & reich, 1996). more recently, it was reported that both ci - related symptomatology and a clinical diagnosis of ci were more prevalent in individuals with versus without pd (irving., 2016). the therapy of choice for ci consists of orthoptic treatment (ot ; scheiman & wick, 2014). its success rate is high (grisham, 1988) and has been documented in pediatric (scheiman, mitchell, cotter, kulp, rouse,., 2005), young adult (scheiman, mitchell, cotter, kulp, cooper,., 2005), and older adult populations (birnbaum, soden, cohen, 1999 ; wick, 1977). unfortunately, ot for ci has never been evaluated in pd, even if it has been suggested that the effectiveness of vergence exercises to improve convergence in these patients seemed warranted (almer, klein, marsh, gerstenhaber, & repka, 2012). we present a case series of two older adults with pd in whom ot decreased the signs and symptoms of ci. in an earlier study investigating ci in pd, 25 (31.3%) of 80 pd participants received a diagnosis of symptomatic ci and were offered ot. seven participants accepted therapy, three abandoned therapy, and two were lost to follow - up. this report thus presents a case series of the two participants who completed ot for their symptomatic ci. the protocol was approved by the research ethics committee of our institutions and participants signed an informed consent form. positive symptomatology was based on a global score of 21 on the convergence insufficiency symptom survey (ciss-15) questionnaire (rouse., 2004 ; the ciss-15 measures the frequency and severity of ci - type symptoms and has been shown to be a valid and reliable instrument for ci treatment outcomes (rouse., 2004). a positive diagnosis of ci was based on the following criteria (rouse., 2004) : (a) exophoria at near at least 4 prism diopters () greater than at far, (b) receded near point of convergence (npc) break of 6 cm, and (c) insufficient positive fusional vergences (pfv) at near (see figure 1 for explanations). glossary of terms and explanation of criteria used to define convergence insufficiency in this report. the ci - specific ot program used in the participants had a duration of 8 weeks. the treatment consisted of three home - based exercises : pencil pushups, brock string, and variable vectograms. the exercises were to be done 5 days / week, 3 times / day for 10 min each time (i.e., 2 min : pencil pushups ; 3 min : brock string ; 5 min : variable vectograms). participants were instructed to spread the exercise sessions throughout the day, at times when they felt less fatigued. participants were given a daily log / diary to record progress and time spent doing exercises. a phone appointment was conducted every week to ensure that all was going well and to answer any questions participants may have had. a follow - up appointment was conducted 1 and 2 months after the beginning of therapy. during these visits, diagnostic tests for ci were performed, that is, horizontal phorias at 6 m and 40 cm, pfv at 40 cm, and npc, 3 times each, and the average represented the value for each test. the ciss-15 was also administered, the exercises and log / diary reviewed and corrected if required, and the next phase of exercises provided and explained. finally, a 6-month period of reinforcement therapy (rt) was offered, during which training sessions were reduced to once per day 5 days per week for the first month and then one session twice per week for the remainder of the reinforcement period. the first participant was a 68 years old married man with pd diagnosed 6 years ago, having multimorbidity (n = 6) and polymedication (n = 17). he was therefore highly motivated to undergo treatment, with the help of his wife who was very supportive. he was very diligent in doing his exercises, and it was only at the end of the 2-month period that he felt the benefits. the clinical findings indicated that he improved by 8.3 cm on the npc, 9.3 on the pfv, and 14 points on the ciss-15 (table 1). he was satisfied with the results obtained and, for that reason, declined rt. a phone call 3 months later indicated that things remained stable for him, and he was still happy to read and even bought a digital tablet. although the ciss-15 is not validated specifically for phone - administration, it was performed that way to get an idea of the participant s level of symptomatology post - treatment. key clinical findings showing the improvement in symptomatic ci pre- versus post - treatment for the case series. note. ci = convergence insufficiency ; npc = near point of convergence ; pfv = positive fusional vergences ; ciss = convergence insufficiency symptom survey. the second participant was an 80 years old married man with pd diagnosed 5 years ago, with comorbidities (n = 3) and several medications (n = 7). he was very motivated to do ot, knowing that he would receive support from his wife and from the ot team. after 1 month of therapy, he felt his two eyes were working well together, and on completion of ot, he was able to read the newspaper. the clinical findings indicated that he improved by 6.6 cm on the npc, 11.3 on the pfv, and 10 points on the ciss-15 (table 1). this participant called 3 months after his last visit to indicate that he had performed rt for 2 months after which he abandoned because of health problems. at that time, he was still doing well from a vision standpoint, and a phone - administered ciss-15 gave a score of 22. the first participant was a 68 years old married man with pd diagnosed 6 years ago, having multimorbidity (n = 6) and polymedication (n = 17). he was therefore highly motivated to undergo treatment, with the help of his wife who was very supportive. he was very diligent in doing his exercises, and it was only at the end of the 2-month period that he felt the benefits. the clinical findings indicated that he improved by 8.3 cm on the npc, 9.3 on the pfv, and 14 points on the ciss-15 (table 1). he was satisfied with the results obtained and, for that reason, declined rt. a phone call 3 months later indicated that things remained stable for him, and he was still happy to read and even bought a digital tablet. although the ciss-15 is not validated specifically for phone - administration, it was performed that way to get an idea of the participant s level of symptomatology post - treatment. key clinical findings showing the improvement in symptomatic ci pre- versus post - treatment for the case series. note. ci = convergence insufficiency ; npc = near point of convergence ; pfv = positive fusional vergences ; ciss = convergence insufficiency symptom survey. the second participant was an 80 years old married man with pd diagnosed 5 years ago, with comorbidities (n = 3) and several medications (n = 7). he was very motivated to do ot, knowing that he would receive support from his wife and from the ot team. after 1 month of therapy, he felt his two eyes were working well together, and on completion of ot, he was able to read the newspaper. the clinical findings indicated that he improved by 6.6 cm on the npc, 11.3 on the pfv, and 10 points on the ciss-15 (table 1). this participant called 3 months after his last visit to indicate that he had performed rt for 2 months after which he abandoned because of health problems. at that time, he was still doing well from a vision standpoint, and a phone - administered ciss-15 gave a score of 22. the first participant was a 68 years old married man with pd diagnosed 6 years ago, having multimorbidity (n = 6) and polymedication (n = 17). he was therefore highly motivated to undergo treatment, with the help of his wife who was very supportive. he was very diligent in doing his exercises, and it was only at the end of the 2-month period that he felt the benefits. the clinical findings indicated that he improved by 8.3 cm on the npc, 9.3 on the pfv, and 14 points on the ciss-15 (table 1). he was satisfied with the results obtained and, for that reason, declined rt. a phone call 3 months later indicated that things remained stable for him, and he was still happy to read and even bought a digital tablet. although the ciss-15 is not validated specifically for phone - administration, it was performed that way to get an idea of the participant s level of symptomatology post - treatment. key clinical findings showing the improvement in symptomatic ci pre- versus post - treatment for the case series. note. ci = convergence insufficiency ; npc = near point of convergence ; pfv = positive fusional vergences ; ciss = convergence insufficiency symptom survey. the second participant was an 80 years old married man with pd diagnosed 5 years ago, with comorbidities (n = 3) and several medications (n = 7). he was very motivated to do ot, knowing that he would receive support from his wife and from the ot team. after 1 month of therapy, he felt his two eyes were working well together, and on completion of ot, he was able to read the newspaper. the clinical findings indicated that he improved by 6.6 cm on the npc, 11.3 on the pfv, and 10 points on the ciss-15 (table 1). this participant called 3 months after his last visit to indicate that he had performed rt for 2 months after which he abandoned because of health problems. at that time, he was still doing well from a vision standpoint, and a phone - administered ciss-15 gave a score of 22. to our knowledge, this is the first report of ot for pd individuals having symptomatic ci. the clinical findings and subjective report of the participants presented here indicate that ot was successful in decreasing the signs and symptoms of ci. these results thus indicate that pd is not a contraindication to ot for ci and that it can be offered to those having symptomatic ci. however, in spite of a 31% prevalence of symptomatic ci in pd (irving., 2016), we also found that the uptake and compliance for treatment is rather poor, with only 7 out of 25 patients agreeing to undergo treatment, and 2 out of 7 completing treatment. those who abandoned the treatment generally reported that manifestations of pd rendered them too fragile and too tired to want to pursue ot on a regular basis. although limited, the positive outcomes obtained in our two participants provide evidence for the need of further research in the area. our data suggest that it would be worthwhile to screen patients for ci as soon as a diagnosis of pd is made and offer ot to those who have symptomatic ci. it might be that at an early stage of pd, with less burden from the disease, compliance to treatment could improve. it is clear also that patients need to be highly motivated and to have strong support at home and from the treating team. it has been suggested that pd may play a role in the oculomotor control of convergence. an earlier report indicated that one patient with pd had ci during his off period and that it cleared up under levodopa therapy during his on period (racette, gokden, tychsen, & perlmutter, 1999). a more recent study investigating dopaminergic medication on ocular function in pd patients indicated that although the degree and amplitude of convergence were better in the on versus off states of the disease, convergence ability remained poorer in pd patients than in controls even in the on state (almer., 2012). this study further indicated that if dopaminergic treatment helps, the convergence ability fluctuates, therefore complicating the clinical management of ci. although ci in pd may be exacerbated by dopamine deficiency, the fact that dopaminergic therapy could help restore the exodeviation at near in the single case, and provided a better degree and amplitude of convergence during the on versus off states in the other pd patients, indicates that the anatomical structures responsible for convergence are intact. furthermore, in a large majority of pd patients with ci, neuroimaging showed no structural brainstem changes (lepore, 2006). the present report thus complements these studies, showing that it is possible to improve convergence with ot in symptomatic pd individuals. this was demonstrated by a greater ability to converge, an increase in the fusional range of convergence and a decrease in symptomatology. furthermore, subjectively, these participants still benefited from ot for at least 3 months post - therapy, that is, the time of our last communication. in future research attempts to confirm our results in a wider pd population, efforts should be devoted into finding the most effective ot modality for these individuals. this could be to carefully select patients for whom symptoms have a large impact on quality of life, decrease the daily session time, do group - training within movement disorder clinics, or make it part of another overall exercise program, for example. the results presented here are novel as this case series show that ot for ci in pd is possible. careful attention on proper patient selection and measures to improve compliance will be necessary. in the meantime, the positive results obtained in these two cases should encourage clinicians to consider ot (a therapy with no / minimal risk) for ci in patients with pd whose quality of life is affected by this binocular dysfunction. | introduction : this study reports a case series of orthoptic treatment (ot) for convergence insufficiency (ci) in individuals with parkinson s disease (pd). method : we are reporting two cases of individuals with pd who completed ot for ci. both had a confirmed diagnosis of ci, accompanied by ci - type symptomatology. they each underwent an ot program consisting of three office - based visits and 8 weeks of home - based exercises. treatment outcome was based on the changes measured pre- versus post - ot on the near point of convergence, positive fusional vergences, and symptomatology score. results : the two participants successfully completed therapy, gained ability to converge, had fewer symptoms, and were satisfied with the ot - induced changes they felt in their day - to - day lives. conclusion : this case series show that ot for ci in pd is possible. further research is required as these results demonstrate that ot has the potential to improve symptomatic ci in these patients. in the meantime, the positive results obtained in these two cases should encourage clinicians to consider ot (a therapy with no / minimal risk) for ci in patients with pd whose quality of life is affected by this binocular dysfunction. |
the advent of computed tomography (ct) has started a revolution of information in health studies and has contributed to planning, diagnosis, treatment, and prognosis analysis of several diseases. cone - beam computed tomography (cbct) is a recently developed technology with potential for applications in different areas of research and clinical dentistry. in endodontics, conventional radiographic images provide a two - dimensional (2d) rendition of a three - dimensional (3d) structure, which may result in interpretation errors. periapical lesions of endodontic origin may be present but not visible on conventional 2d radiographs. these lesions are visible more often on cbct images, and new methods using cbct scans to investigate apical periodontitis and root resorption have been developed, as new imaging tools are now used in several endodontic research areas artifacts can cause low image quality and poor image contrast leading to limited interpretation of the 3d volumes. there is a concern with artifacts and the search for beam hardening corrections have been the focus of several studies. according to ketcham and carlson (2001) beam hardening beam hardening causes the edges of an object to appear as cupping, streaks, dark bands, or flare artifacts, and is caused by preferential absorption of low - energy photons by absorbing materials with higher atomic numbers (e.g. metals). (2006) related that the cbct image defects often appeared in images of solid non anatomical objects placed on the dental arch, such as diagnostic stents (guide splint) for the accurate positioning of dental implants and rectangular radiopaque reference markers for the assessment of periodontal disease. this was more frequent when radiopaque materials, such as hydroxyapatite containing resin and aluminum, were used. it is essential to understand, however, that image artifacts are likely to occur because of the density of several materials used in root canal treatment. alterations from true dimension of rcf may offer false interpretation on real dentin remnants. thus, evaluating the dimension of endodontic materials on cbct images may warn the clinicians about the potential risks of misdiagnosis. this study evaluated the discrepancy of rcf measurements obtained from original root specimens and cbct images. seventy - two human maxillary anterior teeth, extracted for different reasons, were obtained from the dental urgency service of the federal university of gois - school of dentistry, goinia, go, brazil. this study was approved by the ethics committee of the federal university of gois, brazil. preoperative radiographs of each tooth were taken to confirm the absence of calcified root canal, internal or external resorption, and the presence of a fully formed apex. the teeth were removed from storage in 0.2% thymol solution and immersed in 5% sodium hypochlorite (fitofarma, goinia, go, brazil) for 30 min to remove external organic tissues. the crowns were removed to set the remaining tooth length to a standardized 13 mm from the root apex. after taking the initial radiographs, standard access cavities were prepared, and the cervical third of each root canal was enlarged using iso # 50 up to # 90 gates - glidden drills (dentsply / maillefer, ballaigues, switzerland). teeth were prepared up to an iso#50k - file (dentsply / maillefer) 1 mm short of the apical foramen. during instrumentation, the root canals were irrigated with 3 ml of 1% naocl (fitofarma) at each change of files. root canals were dried and filled with 17% edta (ph 7.2) (biodinmica, ibipor, pr, brazil) for 3 min to remove the smear layer. after that, the root canals were irrigated again with 3 ml of 1% naocl, and dried with paper points (dentsply / maillefer). the teeth were randomly divided into 8 experimental groups with 9 specimens each, according to different sealers : group 1 - sealapex (sybron endo, glendora, ca, usa) ; group 2 - sealapex + gutta - percha points (dentsply / maillefer) ; group 3 - sealer 26 (dentsply ind., petrpolis, rj, brazil) ; group 4 - sealer 26+gutta - percha points ; group 5 - ah plus (dentsply / maillefer) ; group 6 - ah plus+gutta - percha points ; group 7 - grossman sealer (endofill, dentsply) ; group 8 - grossman sealer+gutta - percha points. the chemical composition of sealers used are : sealapex (calcium oxide, bismuth trioxide, zinc oxide, sub - micron silica, titanium dioxide, zinc stearate, tricalcium phosphate, and blend - ethyl toluene sulfonamide, polymethylenemethylsalicylate resin, isobutyl salicylate and a pigment) ; sealer 26 (bismuth trioxide, calcium hydroxide, tetraminehexamethilene, titanium dioxide and bisphenol epoxy resin) ; ah plus (paste a - epoxy resins, calcium tungstate, zirconium oxide, silica, iron oxide pigments, and paste b - amines, calcium tungstate, zirconium oxide, silica, silicone oil) ; grossman sealer (zinc oxide, hydrogenated resin, bismuth subcarbonate, barium sulfate, sodium borate, eugenol and almond oil). after root canal preparation, groups 1, 3, 5, and 7 were filled with the corresponding sealers prepared according to the manufacturer 's directions and taken using a lentulo spiral. in groups 2, 4, 6, and 8, teeth were filled with the corresponding sealer and gutta - percha points using the conventional lateral condensation technique. the teeth were wrapped in wet gauze and placed in an incubator at 37c for 72 h to allow complete set of the root canal filling materials. the coronal and the apical portion of the teeth were rendered waterproof with a layer of cyanoacrylate adhesive (super bonder, itapevi, sp, brazil) to protect against the influence of the water on materials. teeth were positioned in the center of a bucket filled with water to simulate soft tissue and supported by a plastic platform, based on the model used in previous studies. cbct images were acquired with the first generation i - cat cone beam 3d imaging system (imaging sciences international, hatfield, pa, usa). exposure time was 40 s. after obtaining the cbct scans, the roots of all teeth were carefully sectioned in axial, sagittal or coronal planes using a high - speed endo z bur (dentsply / maillefer) under water spray cooling. the axial cuts were obtained at 6.5 mm from the root apex ; and for sagittal and coronal planes, the roots were sectioned longitudinally, in the center of the root canal (figure 1). schematic representation of sectioning root method and post length, showing the sagittal, axial, and coronal views all measurements of the sectioned roots were made by two endodontists using a digital caliper accurate to 0.01 mm (fowler / sylvac ultra - cal mark iv electronic caliper, crissier, switzerland). the calibrated examiners measured all the specimens and cbct images, and assessed rcf dimensions in the directions described below. all measurements made on the cbct images were acquired by two dental radiology specialists with the scanner 's proprietary software (xoran version 3.1.62 ; xoran technologies, ann arbor, mi, usa) in a pc workstation running microsoft windows xp professional sp-2 (microsoft corp, redmond, wa, usa), with processor intelcore 2 duo-6300 1.86 ghz (intel corporation, usa), nvidia geforce 6200 turbo cache videocard (nvidia corporation, usa) and monitor eizo - flexscan s2000, resolution 1600x1200 pixels (eizo nanao corporation hakusan, japan). all cbct scans were reformatted using 0.2 mm, 0.6 mm, 1.0 mm, 3.0 mm and 5.0 mm slice thickness. to determine the discrepancy between the rcf measurements made on the original root specimens and those made on the cbct images, the same sites were evaluated on the cbct scans using the same orientation (axial, sagittal and coronal). in all planes, on axial images, the rcf measurement was made in the buccal / palatal direction ; on sagittal images, in the mesial / distal direction ; and on coronal images, in the buccal / palatal direction. one - way analysis of variance (anova) and tukey tests were used for statistical analyses. seventy - two human maxillary anterior teeth, extracted for different reasons, were obtained from the dental urgency service of the federal university of gois - school of dentistry, goinia, go, brazil. this study was approved by the ethics committee of the federal university of gois, brazil. preoperative radiographs of each tooth were taken to confirm the absence of calcified root canal, internal or external resorption, and the presence of a fully formed apex. the teeth were removed from storage in 0.2% thymol solution and immersed in 5% sodium hypochlorite (fitofarma, goinia, go, brazil) for 30 min to remove external organic tissues. the crowns were removed to set the remaining tooth length to a standardized 13 mm from the root apex. after taking the initial radiographs, standard access cavities were prepared, and the cervical third of each root canal was enlarged using iso # 50 up to # 90 gates - glidden drills (dentsply / maillefer, ballaigues, switzerland). teeth were prepared up to an iso#50k - file (dentsply / maillefer) 1 mm short of the apical foramen. during instrumentation, the root canals were irrigated with 3 ml of 1% naocl (fitofarma) at each change of files. root canals were dried and filled with 17% edta (ph 7.2) (biodinmica, ibipor, pr, brazil) for 3 min to remove the smear layer. after that, the root canals were irrigated again with 3 ml of 1% naocl, and dried with paper points (dentsply / maillefer). the teeth were randomly divided into 8 experimental groups with 9 specimens each, according to different sealers : group 1 - sealapex (sybron endo, glendora, ca, usa) ; group 2 - sealapex + gutta - percha points (dentsply / maillefer) ; group 3 - sealer 26 (dentsply ind., petrpolis, rj, brazil) ; group 4 - sealer 26+gutta - percha points ; group 5 - ah plus (dentsply / maillefer) ; group 6 - ah plus+gutta - percha points ; group 7 - grossman sealer (endofill, dentsply) ; group 8 - grossman sealer+gutta - percha points. the chemical composition of sealers used are : sealapex (calcium oxide, bismuth trioxide, zinc oxide, sub - micron silica, titanium dioxide, zinc stearate, tricalcium phosphate, and blend - ethyl toluene sulfonamide, polymethylenemethylsalicylate resin, isobutyl salicylate and a pigment) ; sealer 26 (bismuth trioxide, calcium hydroxide, tetraminehexamethilene, titanium dioxide and bisphenol epoxy resin) ; ah plus (paste a - epoxy resins, calcium tungstate, zirconium oxide, silica, iron oxide pigments, and paste b - amines, calcium tungstate, zirconium oxide, silica, silicone oil) ; grossman sealer (zinc oxide, hydrogenated resin, bismuth subcarbonate, barium sulfate, sodium borate, eugenol and almond oil). after root canal preparation, groups 1, 3, 5, and 7 were filled with the corresponding sealers prepared according to the manufacturer 's directions and taken using a lentulo spiral. in groups 2, 4, 6, and 8, teeth were filled with the corresponding sealer and gutta - percha points using the conventional lateral condensation technique. the teeth were wrapped in wet gauze and placed in an incubator at 37c for 72 h to allow complete set of the root canal filling materials. the coronal and the apical portion of the teeth were rendered waterproof with a layer of cyanoacrylate adhesive (super bonder, itapevi, sp, brazil) to protect against the influence of the water on materials. teeth were positioned in the center of a bucket filled with water to simulate soft tissue and supported by a plastic platform, based on the model used in previous studies. cbct images were acquired with the first generation i - cat cone beam 3d imaging system (imaging sciences international, hatfield, pa, usa). after obtaining the cbct scans, the roots of all teeth were carefully sectioned in axial, sagittal or coronal planes using a high - speed endo z bur (dentsply / maillefer) under water spray cooling. the axial cuts were obtained at 6.5 mm from the root apex ; and for sagittal and coronal planes, the roots were sectioned longitudinally, in the center of the root canal (figure 1). schematic representation of sectioning root method and post length, showing the sagittal, axial, and coronal views all measurements of the sectioned roots were made by two endodontists using a digital caliper accurate to 0.01 mm (fowler / sylvac ultra - cal mark iv electronic caliper, crissier, switzerland). the calibrated examiners measured all the specimens and cbct images, and assessed rcf dimensions in the directions described below. all measurements made on the cbct images were acquired by two dental radiology specialists with the scanner 's proprietary software (xoran version 3.1.62 ; xoran technologies, ann arbor, mi, usa) in a pc workstation running microsoft windows xp professional sp-2 (microsoft corp, redmond, wa, usa), with processor intelcore 2 duo-6300 1.86 ghz (intel corporation, usa), nvidia geforce 6200 turbo cache videocard (nvidia corporation, usa) and monitor eizo - flexscan s2000, resolution 1600x1200 pixels (eizo nanao corporation hakusan, japan). all cbct scans were reformatted using 0.2 mm, 0.6 mm, 1.0 mm, 3.0 mm and 5.0 mm slice thickness. to determine the discrepancy between the rcf measurements made on the original root specimens and those made on the cbct images, the same sites were evaluated on the cbct scans using the same orientation (axial, sagittal and coronal). in all planes, on axial images, the rcf measurement was made in the buccal / palatal direction ; on sagittal images, in the mesial / distal direction ; and on coronal images, in the buccal / palatal direction. one - way analysis of variance (anova) and tukey tests were used for statistical analyses. the variation of rcf dimensions on cbct images ranged from 9% to 100% greater than those measured on the original root specimens. the lowest rcf dimensions (percentage values) corresponded to sealer 26 and sealer 26 plus gutta - percha points. groups that were filled with sealers alone showed the greatest dimensional values in cbct images when compared with groups filled with sealer and gutta - percha points, with statistically significant difference among the groups. when slice thicknesses varying from 0.2 mm to 5.0 mm were measured, an increase of 46.16% to 50.53% in rcf dimensions the different visualization planes analysis revealed an increase in rcf dimensions ranging from 35.48% (sagittal slice) to 59.28% (axial slice) (with statistically significant difference). figures 2 - 3 illustrate the sagittal, axial and coronal views of the rcf using cbct. percentage (%) of root canal filling dimension increase from original specimens to cone - beam computed tomography images according to slice thickness and planes for each endodontic material (=5%) interaction between type of cut and slice thickness significantly by tukey test. gpp - gutta - percha points percentage (%) of root canal filling dimension increase from original specimens to cone - beam computed tomography images for each group according to sealers, slice thickness and planes, and statistical analysis (=5%) different letters in horizontal demonstrate statistically significant difference with p<0.05. p=0.0001 by anova test and p=0.0001 by tukey test ; p=0.647 by anova test and p=0.272 by tukey test ; p=0.0001 by anova test and p=0.0001 by tukey test. gpp - gutta - percha points. cone - beam computed tomography images of root canal filling with sealapex (a), sealapex+gutta - percha (b), sealer 26 (c), and sealer 26+gutta - percha (d) in different slice thickness and planes (sagittal, axial and coronal) cone - beam computed tomography images of root canal filling with grossman sealer (a), grossman sealer 26+gutta - percha (b), ah plus (c), and ah plus+gutta - percha (d) in different slice thickness and planes (sagittal, axial and coronal) a new standard of contemporary endodontics has been created with the advent of cbct. for several years, rcf quality was evaluated in clinical practice according to a 2d image of 3d structures. the radiographic appearance of the filled root canal space is used to evaluate its sealing quality and to indicate the presence of apical periodontitis. however, the limitations of a radiographic assessment as a reference and study method have been demonstrated in several studies. there is a possibility of interference from artifact caused by different densities from endodontic materials, which can cause errors of interpretation. our main purpose was to determine the discrepancy of rcf measurements between original root specimens and cbct images, and our findings showed that measurements obtained from cbct images of rcf with sealers and sealers plus gutta - percha were greater than those obtained on the original root specimens (tables 1 and 2). these results bring important implications for the clinical evaluation of rcf and anatomic dental structures. special attention should be paid depending on the density of the endodontic material and slice thickness / orientation which can lead to misdiagnosis. (2008) compared the accuracy of cbct and multislice ct for linear jaw bone measurements, and found that both methods were accurate when used to evaluate an ex vivo specimen. (2008) determined the geometric accuracy of cbct scans in comparison with a multidetector computed tomography (mdct) scanner. their results showed that the cbct devices provide satisfactory information about linear distances and volumes. mdct scans proved slightly more accurate in both measurement categories, but this difference may be irrelevant for most clinical applications. recent studies using cbct images detected voids in root filling and incomplete removal of filling material during endodontic retreatment. (2007) showed that image quality of storage phosphor images was subjectively as good as conventional film images and superior to limited - volume cbct images for the evaluation of both homogeneity and length of root fillings in single - rooted teeth. (2009) analyzed voids in root fillings using intraoral analogue, intraoral digital and cbct images. voids larger than 30 m were detected by all imaging techniques. for small void detection, all digital intraoral techniques performed better than intraoral analogue and cbct images. the difference in density of rcf materials may have affected the results of several studies, and was also found in our specimens. the endodontic sealers and gutta - percha used in our study have different physical and chemical properties because of different radiopaque substances (bismuth oxide, barium sulfate, zinc oxide). (2011) evaluated the effect caused by intracanal posts (glass - fiber post, carbon fiber root canal, pre - fabricated post - metal screws, silver alloy post and gold alloy post) on the dimensions of cbct images of endodontically treated teeth. gold alloy and silver alloy post dimensions were greater on cbct scans than on the original root specimens. cbct reconstructions may show higher rcf dimensional values, as well as lack of image homogeneity and definition. (2007) conducted an in vitro study about the effect of projection data discontinuity - related artifacts in limited - volume cbct imaging of jaws. the effects of artifacts were scored as the difference in relative density between the lingual and buccal soft tissue. the intensity of artifacts increased when more objects were outside the area being imaged. fewer artifacts were noted in images produced by the particular flat cbct panel detector used in this investigation. (2009) analyzed the relationship between density values and cbct volume size using an alphard cbct system capable of providing different - size imaging volumes. the authors found that the data discontinuity - related effect was different in limited - volume cbct scanning. considering that the higher density of rcf materials may produce image artifacts, special attention should be paid to the rcf evaluation of endodontically treated teeth. any change from true dimension of rct may favor false interpretation on real dentin remnants, and constitute potential risks of misdiagnosis. the implication of density artifacts on diagnostic procedures seems to be obvious, and different correction methods have been investigated. (2008) studied the effect of incident radiation energy on the amount and extent of image artifacts over adjacent anatomic structures. the use of a harder energy beam during scanning appears to result in less extensive artifact formation. a strong correlation between gray scale values on cbct images and bone densities has been found by haristoy,. this has implications for potential quantitative radiological approaches to determine bone density from cbct images. however, given the variation of gray scale values despite normalization, it may be necessary to use calibration phantoms scanned simultaneously with the patient to ensure an accurate determination of bone density. hunter and mcdavid (2009) showed that additional copper filtration suppresses beam hardening artifacts. in micro - computed tomography scanning based on bone mineral density measurements, the effects of beam hardening - induced cupping artifacts may be also minimized by beam filtration. cbct provides a three - dimensional image, in which a new plane has been added : depth. its clinical application results in greater accuracy and may be used in nearly all areas of dentistry : surgery, implants, dentistry, orthodontics, endodontics, periodontics, temporomandibular dysfunction, image diagnosis, and so on. few studies investigated the dimensional alterations of endodontic materials observed on cbct images, and further studies should include other variables, such as the effect of artifacts on intracanal post alloys. the evolution of the new softwares certainly can reduce metallic artifact and dimensional alterations in future reconstructions of cbct images. periapical radiographs should be used as a reference standard, together with cbct image interpretation, when making endodontic diagnoses. the results of this study showed that rcf dimensions were greater on cbct images than on the original root specimens, especially when only sealer was used. | objective to evaluate the discrepancy of root canal filling (rcf) measurements obtained from original root specimens and cone - beam computed tomography (cbct) images. material and methods seventy - two human maxillary anterior teeth were prepared up to an iso # 50 k - file 1 mm short of the apical foramen. thus, the teeth were randomly divided into 8 groups, according to the root canal filling material : sealapex, sealapex+gutta - percha points, sealer 26, sealer 26+gutta - percha points, ah plustm, ah plustm+gutta - percha points, grossman sealer, and grossman sealer+gutta - percha points. after root canal preparation and rcf, cbct scans were acquired and the specimens were sectioned in axial, sagittal and coronal planes. the rcf measurements were obtained in different planes and thicknesses to determine the discrepancy between the original root specimens (using a digital caliper) and the cbct images (using the scanner 's proprietary software). one - way analysis of variance and tukey tests were used for statistical analyses. the significance level was set at =5%. results measurements of the different endodontic filling materials were 9% to 100% greater on the cbct images than on the original root specimens. greater rcf dimensions were found when only sealers were used, with statistically significant difference among the groups. conclusions rcf dimensions were greater on cbct images than on the original root specimens, especially when only sealer was used. |
on january 14, 2016, a woman with behavioral changes and hydrophobia visited a regional hospital in cap - hatien, nord department, haiti. a local clinic treated the wound on the day the bite occurred but did not offer rabies vaccination. healthcare workers at the regional hospital made a presumptive diagnosis of rabies but were unable to offer palliative care. the hospital administrator reported the suspected rabies case to the national department of epidemiology and laboratory research (delr), as required by haiti s national surveillance system. without contact information, delr was unable to investigate further. haiti s ministry of health (ministre de la sant publique et de la population [mspp ]), with assistance from the us centers for disease control and prevention (cdc), has developed a robust surveillance system for 44 conditions, 13 of which are immediately reportable, including suspected human rabies. under this surveillance system, health alerts for suspected human rabies cases are investigated to confirm clinical cases of rabies, identify persons or animals exposed to a rabid animal, and identify healthcare and community contacts of the person suspected of having rabies. since february 2015, cdc has assisted delr in 3 human rabies investigations, which identified 27 rabies - exposed persons in addition to patients. the person with suspected rabies in this case report was not admitted to the hospital, and no contact information was obtained. therefore, public health investigators could not determine her health outcome, gather potential human and animal exposures, or complete classification of this case on the basis of the world health organization s clinical case definition for rabies (2). on march 14, 2016, three months after the woman with suspected rabies had visited the regional hospital, a cdc - trained veterinarian who was conducting a rabies survey among mongooses was alerted by community members to a potential human rabies death. initial reports led the veterinarian to believe that the person who died was the same woman who sought care at the cap - hatien hospital. a team of healthcare workers from cdc and the pan american health organization (paho) had already planned training on integrated bite case management (ibcm) in this area. in addition to the training, during march 30april 10, cdc and paho assisted haiti s ministry of agriculture, natural resources and rural development (marndr) and delr in investigating the suspected human rabies case. on april 5, 2016, the investigation team conducted a verbal autopsy with the decedent s husband. the investigation confirmed that the person who died was a 54-year - old woman who was bitten on the left hand on november 30, 2015, while fending off a dog that was acting aggressively toward her goats. the woman visited a local healer, who administered 1 shot of an unknown substance. except for residual pain in the hand, the woman remained healthy until january 10, 2016, when her husband recognized signs of confusion ; notably, she had placed common household items in unusual locations. during january 1113, fevers, hypersalivation, agitation, and incoherent speech developed. on january 14, the woman accused her husband of trying to kill her when he offered her water (presumed hydrophobia). on that day, the husband and wife traveled to a health clinic and were immediately referred to the regional referral hospital in cap - hatien. the husband reported that palliative care was denied, and they left the hospital without providing contact information. the wife died later that night. according to who clinical rabies case definitions, the woman s illness was a probable rabies case. the investigators verified that neighbors had killed an abnormally aggressive dog on approximately november 30, 2015. neighbors reported that the dog had attempted to bite several persons, but it was killed without further human exposures. the dog had bitten 1 pig, which could not be located because of the delay between the bite event and the case investigation. few cases of rabies in haiti are reported to health authorities ; in 2015, only 7 cases were documented, 5 of which were detected through the veterinary sector. lack of recognition of rabies has been attributed to low awareness, unique cultural beliefs, and a high incidence of numerous conditions (i.e., cerebral malaria, meningitis, viral encephalitis, and tetanus) that may confound rabies diagnosis (3,4). furthermore, diagnosis of human rabies is not performed in haiti due to limitations in diagnostic capacity and cultural aversion to collection of postmortem samples. the healthcare team identified no other human rabies exposures in this investigation. given the delay in investigating this case, if any persons had been exposed, they likely would have already succumbed to rabies, underscoring the importance of timely reporting and investigation. in 2015, in an effort to improve healthcare provider recognition of rabies cases and surveillance reporting, paho and mspp developed a rabies training course for healthcare providers. although animal rabies is a reportable condition to marndr and bite events are reportable to mspp, neither the rabid dog nor the bite event were reported in this situation. a 2014 survey estimated that 95,000 animal bites occur annually in haiti (1% bite rate) (5). however, only 6,500 bites (6.8% of estimated bites) were reported through the national surveillance program that year. to improve bite detection and healthcare - seeking behaviors, cdc and paho collaborated with marndr, delr, and mspp to develop an ibcm system to assist in reporting bites to marndr for animal investigation. results are reported to bite victims and to the responsible healthcare sector. since its inception, the ibcm system has increased detection of animal rabies cases 18-fold and improved patient healthcare - seeking behavior. the ibcm program is now operational in 3 of haiti s 10 departments but is not yet available in cap - hatien. therefore, even if the bite had been reported through appropriate surveillance channels, follow - up likely would not have occurred. to improve reporting in nord department (cap - hatien), the investigation team has trained 11 veterinary professionals to use the ibcm program (figure). a team consisting of workers from the us centers of disease control and prevention ; the pan american health organization ; haiti s ministry of agriculture, natural resources and rural development ; and christian veterinary mission trained 11 veterinary professionals on principles of animal rabies surveillance. here, trainees gain experience drawing up sedative medications into a pole syringe, which is used to sedate suspected rabid animals from a safe distance. haiti has made considerable strides in controlling dog - mediated human rabies deaths through efforts such as dog vaccination, the implementation of the ibcm system, and medical provider training. these advances have been made through collaborative work with haiti s government institutions and international partners. through the continued support and expansion of these programs, this case is an unfortunate reminder that dog - mediated human rabies deaths continue to occur in some western hemisphere countries ; however, this death provided stimulus for training local health officials and served as a reminder of why haitians and international partners seek elimination of this disease. | haiti has experienced numerous barriers to rabies control over the past decades and is one of the remaining western hemisphere countries to report dog - mediated human rabies deaths. we describe the circumstances surrounding a reported human rabies death in 2016 as well as barriers to treatment and surveillance reporting. |
optic neuritis related to ms is an acute optic neuropathy occurring in young people, especially in women. the clinical picture shows a decline in visual acuity, a pain which increases with movements of the eye, a decline in contrast sensitivity, dyschromatopsia and changes in the visual field (1). it occurs more often unilaterally in young people, predominantly in women with an incidence of 1 - 5 per 100,000 annually (2). the main aim of this study was to determine the onset of optic neuritis as the first ms clinical presentation. we also wanted to analyze the role and significance of morphometric changes in the optic nerve and visual field by analyzing parameters of optic coherent tomography (oct) and computer perimetry in these patients. one of the aims was to determine the efficiency of pulse corticosteroid therapy in those patients. a clinical, retrospective study was conducted at the eye clinic and the neurological clinic, sarajevo university clinical centre (succ) at the time of the march 20092011. after the first clinical signs follower is a detailed examination of patients by neurologists and ophthalmologists. here we did not analyzed cases of ms relapses. in all analyzed patients we found a unilateral optic neuritis. we approached ms verification according to paty or brakhof modern clinically defined multiple sclerosis (cdms) criteria (3, 4). first criterion : at least two separate clinical episodes of the disease that occurred at different times and at least two fields of demyelination. of radiological tests, mri of the brain and spinal cord were analyzed. second criterion : two ms clinical episodes, one mri manifest lesion and paraclinical symptoms. laboratory - supported ms diagnosis: two episodes, one clinically and paraclinically confirmed lesion and immunoglobulin abnormalities of cerebrospinal fluid. one episode, two clinically separate lesions and cerebrospinal abnormalities. first criterion : at least two separate clinical episodes of the disease that occurred at different times and at least two fields of demyelination. of radiological tests, second criterion : two ms clinical episodes, one mri manifest lesion and paraclinical symptoms. laboratory - supported ms diagnosis : two episodes, one clinically and paraclinically confirmed lesion and immunoglobulin abnormalities of cerebrospinal fluid. one episode, two clinically separate lesions and cerebrospinal abnormalities. of neurological parameters we analyzed clinical manifestations of motor, sensory and sensitive disturbances, relevant to the confirmation of ms diagnosis. of ophthalmological parameters, we analyzed : visual acuity (va) by snellen charts, visual field (vf) by octopus 100, and thickness of the peripapillary rim nerve fibres by stratus zeiss optic coherent tomography (oct).. age structure of ms patients with verified optic neuritis gender structure of ms patients with verified optic neuritis the age structure analysis of the sample shows that optic neuritis was most often present in the patients aged 18 to 30 years. in the entire sample, onset of optic neuritis was more frequent in the female patients (70%) compared to the male patients. the first clinical manifestation of ms in our sample statistically significant tests (x=9,7 p=0,01) we have confirmed that the optic neuritis is one of the first clinical manifestation of ms, compared to the other motor, sensory and sensory events in our sample. ophthalmological parameters we followed after 7 and 15 days, after 1, 3, 6 and 12 months. analyzing disturbances in the computerised visual field : centrocecal and paracentral scotoma and diffuse sensitivity, we find most frequent disturbances in the centrocecal region in 50% of the cases. different kinds of scotoma in visual field in ms patients with optic neuritis by analysis of oct results in part of the nerve fibre thickness of papillary and parapapillary layers (of the neuroretinal rim), we had, after three months, results showing the thinning of the nerve fibres in the whole circumference, most often in the upper quadrant. retinal nerve fibre layer (rnfl) by optic disc visual acuity on first examination and one month after corticosteroid therapy visual acuity in all patients with optic neuritis at the first examination ranged from 0.1 to 0.3 with correction. after the administration of pulse corticosteroid therapy, all patients with optic neuritis had a significant improvement of va, and a month upon administration it varied from 0.6 to 1.0. in our sample of 89 patients with the verified ms diagnosis, 10 patients had a clinical picture of optic neuritis, as a first clinical sing of disease, it was statistically significant (x= 9,7 p=0,01). the optic neuritis study group describes the changes of visual functions in ms patients, which usually present as a decline in vision, blurring, decline in contrast sensitivity, color vision disturbances, as well as disturbances in the visual field (1 - 5). in their studies, allanore y. and deretzi g.confirm the genetic impact on ms development as an autoimmune disease in some families (6). studies were performed to analyze the influence of stress, viral and bacterial infections of respiratory tract, urinary and gastrointestinal systems on the appearance and occurrence of ms exacerbations (7, 8). in our study, the disease appeared most often in the patients aged 18 - 30 years. population get results which show that the ratio of the incidence of occurrence of ms ranged from 1.9 : 1 to 3.6:1 in favor of women (9). a large number of studies confirm the ms and on occurrence at a younger age and more often in women (1, 2, 5, 10). balashov ke. finds a higher on incidence in the spring months in the patients without verified demyelisating lesions, which has been observed in our past practice too, and which could be an aim of our future studies (11). cigarette smoking is also a risk factor in the development of ms and on (12). in 50% of our subjects we had centrocecal scotoma in the visual field, and sensitivity depression in 30%. disturbances in the central areas of the visual field were verified in other studies as well (1, 13). visual acuity in all patients with optic neuritis at the first examination ranged from 0.1 to 0.3 with correction. all patients with an on and verified ms diagnosis monitored in this study were treated with pulse corticosteroid therapy in a dosage of 1,000 mg for three days, followed by 8 days of 1mg / kg b.w. a month after pulse corticosteroid therapy, visual acuity in all patients with on ranged from 0.6 to 1.0. corticosteroids administered intravenously by the pulse therapy scheme prevent on relapse, but they have the same effect on definite va as corticosteroids orally administered. ontt finds that the on patients treated with prednisolone orally administered in a dosage of 1mg / kg b.w. for 14 days ontt shows that intravenously administered corticosteroids inhibit ms development for a 2-year period, bur after three years that effect disappears (14, 15). three months after the first on presentations, the oct analysis of retinal nerve fibre thickness and neuroretinal rim confirmed the nerve fibre thinning in all subjects. ontt shows that 50% of the patients with on develop ms over a 15-year period. ontt also finds that in on patients without mri changes, ms develops in 25% of the cases, and in the cases with one or more cns lesions that is 75%. all patients had oct - verified nerve fibre atrophy after on (1, 14). according to other researchers, by oct analysis it can discover in vivo atrophy of nerve fibres in patients with ms as structural damages of axons of afferent fibres of the visual pathway (15, 16). oct rnfl thickness average analysis studies show, by morphometric analysis of the structure of nerve fibres by oct, that there is fibre atrophy in the ms patients with and without on. however, retinal nerve fibre atrophy is more significant in the patients with on (16). in our sample with the predominantly female gender ranging from 18 to 30 years of age, on occurred as the first clinical presentation of ms. the analysis of the computerised visual field confirmed centrocecal disturbances as the most common, and optic coherent tomography registered the thinning of the nerve fibres of the neuroretinal rim in all subjects. a month after pulse corticosteroid therapy, | aim : to analyze the clinical signs of multiple sclerosis (ms) and show that optic neuritis is one of the first event, which indicates the development of disease.patients and methods : the study involved 89 cases in which it confirmed ms at the time of the march 20092011. since ophthalmological parameters were analyzed visual acuity (va), visual field (vf), and retinal nerve fibre layer (rnfl) thickness of peripapillary rim by optic coherent tomography (oct).results : ten(10) patients had on as the first clinical manifestation of the disease which was statistically significant (x2 = 9,7 p=0,01) compared to the manifestation of other clinical signs of disease. in vf, centrocecal scotomas were predominant in 50% of the subjects ; the rnfl thinning of the neuroretinal rim was verified in all patients, most often in the upper quadrant. a month after pulse corticosteroid therapy, visual acuity in all patients with on ranged from 0.6 to 1.0.conclusion:on is one of the first ms clinical manifestation. in vf, the most common disturbances are in the centrocecal area. the rnfl thinning was verified in all patients with oct. |
retroperitoneal hemorrhage or retroperitoneal hematoma (rh) refers to an accumulation of blood found in the retroperitoneal. the retroperitoneum is a large space bounded anteriorly by the posterior parietal peritoneum, posteriorly be the transversalis fascia, and superiorly by the diaphragm. retroperitoneum contains some vascular structures in the gastrointestinal, genitourinary, vascular, and musculoskeletal system. a mortality rate of traumatic rh is reported as high as 1860% in english literature. it is actually possible that rh mortality from resource - poor countries such as nigeria is actually higher because death from trauma ranked high as a cause of mortality in such setting, previous work showed that death from trauma ranked second over a three decade period in a tertiary hospital setting in nigeria. other authors have also shown that trauma from road traffic accidents from nigeria is often very fatal with poor outcome. however, there is a virtual paucity of literature to appraise this. despite all advances in the field of technology and surgical techniques, rh resulting from blunt injuries remains a challenge for the surgeon. because of low pressure of bleeding due to venous injuries, hemostasis may be achieved spontaneously. thus, rh caused by venous bleeding are usually restricted and located at the right side of the linea alba, i.e., midline. on the other hand, rh originating from arterial bleedings appear as a bright red mass, expand rapidly, and often locate on the left side of the midline, rh may occur after blunt and penetrating traumas. several classifications of rh have been made based on the localization of hematomas. in this study, we used kudsk and sheldon 's classification described in 1982. in this classification, centromedial localization was described as zone 1, flank localization as zone 2, and pelvic localization as zone 3 rh. accurate characterization of rh and associated injury is best done with computerized axial tomography scan (ct - scan) can affect clinical management and can help minimize unnecessary laparotomies. equivocal findings at initial abdominal ct should prompt close clinical follow - up with possible imaging follow - up, particularly for suspected occult duodenal and pancreatic injuries. many at times, diagnosis is often delayed most especially in a poor resource country such as nigeria where full armamentaria of radiological diagnostic tools such as ct - scan are not readily available at the accident and emergency department, the usual first point of call for the patient with traumatic condition like rh. despite all these challenges, we present the pattern and outcome of patients with rh in resource - poor setting such as the tertiary hospital in nigeria. we highlight the severity and various challenges faced while managing patients with this condition in our peculiar setting. this was a retrospective study of all patients with blunt or penetrating abdominal trauma needing emergency surgical exploration. since it was almost impossible to make diagnosis of rh preoperatively because of the limited diagnostic armamentaria available in our center, all cases of abdominal trauma who had surgical exploration were sieved to find out the cases of rh. the study was carried out at a tertiary hospital southwest, nigeria, after strict compliance with the ethical standard. the medical record of all 247 patients who had exploratory laparotomy on account of blunt or penetrating abdominal injury in the last decade between 2005 and 2015 were called for but only 161 complete medical records were found. out of this, 43 patients had an operative diagnosis of rh. usually, indication for surgery would be ultrasound findings suggestive of hemoperitoneum, positive abdominal paracentesis, or positive diagnostic peritoneal lavage. data such as mechanism of injury, clinical features at presentation, time interval between trauma and arrival in emergency room, time interval between arrival and surgery, intraoperative classification of the zone of rh, operative procedure done, postoperative hospital stay, mortality, and morbidity were extracted from the patient record, the data were analyzed using the statistical program for social sciences (spss 12.0.1 for windows ; spss inc. in the last one decade spanning 20052015, our operation database record showed that 247 patients had exploratory laparotomy for blunt and penetrating abdominal trauma, out of this, only 115 complete records were found, 15 patient had wrongly quoted case note record number and 71 case record were missing. out of the 115 complete record, 43 patients had rh. table 1 showed patient characteristics with a median age of 30 years and the most common age group for rh occurrence to be 2029 years, female : male ratio occurrence of 1:13, and student being the most susceptible group. rh is far more common in blunt abdominal trauma 26 (60.5%) compared to penetrating injury 17 (39.5%). mechanism of injury showed that motor vehicular accident is the most common cause of injury 10 (23.3%). other features such as etiology, mechanism of injury, and clinical features at presentation in hospital are shown in table 2. patient characteristics etiology / mechanism of injury / clinical features challenges encounter during treatment of patients with rh in our setting are well illustrated in table 3. only eight patient (18.6%) reached the hospital from the site of accident within the first golden hour (gh) of accident, and only two patients (4.7%) got into the operating theater within 1 h of reaching the hospital. preoperative ultrasound findings, intraoperative findings, zones of hematoma, overall outcome, and postoperative morbidity were outlined in table 4. details of intra - abdominal organ injury associated with rh are shown in table 5. intervention challenges intervention findings and associated abdominal injury associated intra - abdominal organ injury two mortality were recorded during the study period. the first case was a 32-year - old man who presented with close range gunshot injury to the abdomen. intraoperative findings included nonexpanding zone i and ii rh, multiple jejunal perforation, pancreatic avulsion, left renal contusion, and 2.5 l hemoperitoneum. he had repair of bowel injury, packing of retroperitoneal hemorrhage, and peritoneal lavage. however, the patient developed pancreatic fistula and roaring peritonitis and died 48 h postsurgery. the second patient was a 35-year - old driver involved in a vehicular road traffic accident. he sustained blunt abdominal injury, findings at surgery were 3.0 l hemoperitoneum, zone 2 rh, avulsion of splenic pedicle, hepatic laceration, and rupture transverse colon. he had splenectomy, peritoneal lavage, and colostomy, there was difficulty securing hemostasis of retroperitoneal bleeding. traumatic rh is a life - threatening complication of abdominal and pelvic injuries, early diagnosis and urgent surgical intervention are of utmost importance to give any surviving chance to the patient involved in this type of accident. in this series, only eight patient (18.6%) of the total number of 43 patients the importance of trauma patient accessing definitive surgical intervention in the early hour of trauma after which morbidity and mortality increases significantly has been emphasized by many workers in the past. there is the possibility that the poor and inefficient prehospital transport and lack of adequate emergency medical service that is often prevalent in many low - income and resource - poor countries such as nigeria, is responsible for the delay transport of trauma patients to the hospital. this probably suggests that the 43 cases (17.4%) of posttraumatic rh, who had exploratory laparotomy for blunt and penetrating abdominal injury is a tip of the iceberg. quite a proportionate of patients with severe injury including more extensive rh would have died at the site of the accident and are not able to make it to the hospital. this is even more so considering the fact that trauma from road traffic accident is one of the leading cause of death in nigeria, this has been established by the previous work. in this study, rh arising from blunt or penetrating abdominal injury sustained in road traffic accident accounted for 24 cases (55%) of all the cases.. also found to be more challenging and troubling in this cohort of the patient is the delay in getting to the operating theater room even after reaching the hospital. in this study, only eight patients (18.6%) of the total number of patient with rh were operated on within 1 h of presentation in the hospital [table 4 ]. the reason for delayed surgery in this study include lack of sterile operation bundles, gown and outfit as at when need, limited and busy operating theater space, delay in getting blood for transfusion, and also delay in processing laboratory result. these causes of delay has been documented in other studies to be prevalent in low - resources country such as nigeria. patients were operated on based on main clinical findings of intra - abdominal organ injury as manifested by sign of peritonitis / peritonism, progressive abdominal distension, abdominal paracentesis, or positive diagnostic peritoneal lavage that suggest hemoperitoneum. the majority of the patient also had abdominopelvic ultrasound. in 32 patients (74.4%) who had abdominal ultrasound none of them had a preoperative diagnosis of rh, in fact, the most common diagnosis on ultrasound was hemoperitoneum and splenic injury 25 cases (58.1%). this is not surprising because ultrasound of the abdomen is known to be poor in making diagnosis of blood collection in a deeply situated space such as retroperitoneum with multilayers of overlying gas bearing bowel, instead ct scan has been found to be more accurate in detecting retroperitoneal injuries and collection. limitation of resources in developing countries means that high - end radiological investigation gadget like ct scan are not readily available in most hospitals. in situation where there is only one ct scan machine like in this center, such machine is deployed in central radiology suite to serve general diagnostic purpose rather than deploying it in accident and emergency centre for trauma patient. the implication for this is that many patients with an occult retroperitoneal injury can remain undiagnosed and may not be surgically explored thus dying from their injury. zone 2 rh is the most common type encountered in this study group, 18 patients (41.9%) present with zone 2 rh alone and zone 2 plus hematoma in other zones is seen in 21 patients (48.8%). meanwhile, zone 3 rh alone is seen in 12 patients (27.9%) while zone 3 rh in combination with rh in other zones is seen in 17 patients (39.5%). the zone 1 rh involvement is the least common in this series, 6 patients (13.9%). only one patient had extensive rh in three zones 1, 2, and 3. this pattern is in contrast with what is reported in other parts of the world. in a study of extensive rh by abdullah and al - salamah, zone 3 rh with extension to the lateral or central zone was the most common type of rh accounting for 65.2% of the patients. ishikawa. found that rh extended out of the pelvis involving zone 3 and 2 in 66 of his studied patients (39.1%), extension through the three zones in 41 of his patient (24.3%) and these have the worst prognosis. the reason for the low incidence of zone 1 rh and also low occurrence of extensive rh involving zone 1, 2, and 3 in our study patient can be due to the fact that these extensive rh which usually has poor prognosis probably never made it to the hospital, in view of the poor state of prehospital care and poor patient evacuation from the site of accident that is commonly seen in resource - poor countries like nigeria. it is most likely that some of the patients even died while in the hospital due to delay in having surgery done. as previously stated above, the classification of traumatic rh into three zones proposed by kudsk and sheldon was used in this study. other workers like feliciano has proposed sub - classification and location - based treatment protocol. zone 1 rh includes the midline area between the aortic hiatus and sacral promontory major vessels of the abdomen lies in this zone and most of the time it is recommended that hematoma in this zone be surgically explored to repair bleeding major vessels. zone 2 encompasses the lateral retroperitoneum, including the right and left perirenal spaces, management depends on severity many perirenal, and peri colonic hematomas are self - limiting, and patients can be treated with observation alone if they remain hemodynamically stable, even in these group of patient it is imperative to establish accurate diagnosis with ct scan of the abdomen. follow - up imaging can be used to assess the stability of retroperitoneal hemorrhage when observation is chosen. zone 3 encompasses the pelvic retroperitoneum, surgical intervention is avoided in most cases of blunt pelvic trauma with external fixation and angiographic embolization being the preferred for large bleeding. in our study, none of the patients with zone 1 hematoma who had surgical intervention had surgical repair of an injured major vessels in zone1. in fact, the common surgical procedure carried out in most of the patients are those targeted toward repairing associated injury. thus, in the whole cohort of patients, the most common surgical procedure is splenectomy and peritoneal lavage carried out in 9 patients (20.9%). followed by repair of bowel injury and peritoneal drainage in 8 patients (18.6%). many other researchers have also reported a wide - ranging mortality rate of between 12.9% and 26% in their studies. the reason for the deceptively low mortality rate in our series can be deductively inferred from the fact that most of our patients had stopped bleeding actively by the time they had surgical intervention. this became clear because most of them had non expanding hematoma and the majority surgical procedure carried out was lavage and drainage in most instances. the probability is that those patient with active massive ongoing bleeding did not make it to the operating table due delay from various reasons already alluded to above. logistical infrastructural inadequacies such as lack of sterile theater bundle and drapes / nonavailability or busy theater space caused delay for patients between presentation in accident and emergency center and operating theater. none of our patients had a preoperative diagnosis of rh because of lack of access to ct scan dedicated to trauma in accident and emergency center. the overall mortality of 4.7% in this study, which is on the low side, tends to suggest that mostly mild and stable cases which can make it to the operating table despite all the delay were eventually operated on. allocation and distribution of resources in tertiary hospitals in low - income countries should be in such a way as to equip the accident and emergency centre with modern diagnostic tools such as ct scan dedicated to trauma patients. | background : retroperitoneal hematoma (rh) can present as an acute life - threatening condition, report on rh in low - income countries are lacking.objective:we present the severity, pattern, challenges, and outcome of rh in a low - resource country such as nigeria.methods:this was a retrospective observational study of all patients with blunt or penetrating abdominal injury needing surgery, patients with rh among them were analyzed.results:in the last one decade spanning 2005 - 2015, our operation database record showed that 247 patients had exploratory laparotomy for blunt and penetrating abdominal trauma. out of the 115 patients with complete record available, only 43 had rh. the median age of the patients was 30 years, and the most affected age group was 2029 years. female to male ratio was 1:13. only eight patients (18.6%) reached the hospital from the accident site within the first golden hour of accident, which is the first 1 h postrauma during which treatment intervention believed to have the best outcome. only two patients (4.7%) got to operating theater within 1 h of reaching hospital. none of our patients had preoperative diagnosis of rh ; overall, mortality was two patients (4.7%).conclusion : logistical infrastructural inadequacies such as lack of sterile theater bundle and drapes / nonavailability or busy theater space caused delay for patients between presentation in the accident and emergency center and operating theater. none of our patients had a preoperative diagnosis of rh because of lack of access to computerized tomography scan dedicated to trauma in accident and emergency center. the overall mortality of 4.7% in this study, which is on the low side, tends to suggest that mostly mild and stable cases which can make it to the operating table were eventually operated upon. |
taenia crassiceps is a cestode that, when adult, lives in the intestinal lumen of some carnivore species (i.e., fox) and in the subcutaneous connective tissue and pleural and peritoneal cavities of rodents (i.e., mice) in its metacestode (cysticercus) stage. for experimental conditions, the infection with t. crassiceps once in the peritoneal cavity, the cysticerci reproduce asexually by budding, until reaching massive parasite loads in a matter of 36 months that weigh as much as the host. measuring parasite intensity in such conditions is an easy task requiring no more than a magnifying glass to count the number of parasites installed in the host at the time they are harvested by way of thoroughly washing the infected peritoneal cavity. thus, experimental murine intraperitoneal (ip) cysticercosis by t. crassiceps orf strain (expmuriptcrascistiorf) has been extensively used for genetical, immunological, endocrinological, and behavioral studies of host - parasite relationships [37 ]. notwithstanding its usefulness, expmuriptcrascistiorf is plagued by unexplained great individual mouse variability in parasite loads and in igg antibody responses, even within the same genetic strain and sex of the murine host and time of infection. a number of factors from the individual host, the parasite, and the environment have been invoked as being involved in such variability. the possible role of inherent variation in the putatively identical parasites composing the infecting inoculums has received less attention. there are two major sources of possible parasite variation between inoculums, one technical and the other biological. the technical sources are the number of infecting cysticerci in the inoculums, the time of infection studied, and the degree of injury suffered by the cysticerci upon their passage through the syringes ' very tight caliber needles when squirted into the peritoneal cavities of the infected mice. the biological sources of unexplained variation are also plenty and involve both host and parasite genetic and epigenetic physiological factors at the individual level. to test the hypothesis of inherent budding variability among infecting cysticerci, without the participation of the host 's responses, we counted the number of buds they produced in in vitro cultures in 1, 5, or 10 ml of rpmi medium 1640 without fetal serum or added supplements, in isolated conditions (1 cyst / culture well), and in crowded conditions (5 and 10 cysts / well) or with various supplements during 10 days in the different culture conditions. the cysticerci employed came from two different balbc / ann female mice that had been infected ip 2 months before to develop a massive parasite load. harvesting the cysticerci implies killing the donor mice by etherization (in accordance with our institute 's ethical procedure in dealing with experimental animals (at http://www.biomedicas.unam.mx/codetico_archivos/reglamento_bioterio.pdf) and immediately afterwards slitting its peritoneal cavity to release hundreds of cysticerci into a petri dish containing phosphate - buffered saline (pbs) and 100 g / ml antibiotic (penicillin / streptomycin) at room temperature. typically, the collected cysticerci are presented in three phases : initial (no buds and transparent vesicle), larval (filled with buds and transparent vesicle) and final (no buds and opaque vesicle),. a significant fraction of the harvested cysticerci (~10%20%) is the subpopulation of tiny (0.10.3 mm) nonbudded motile and transparent cysticerci, from which 10 cysticerci are selected to constitute each of the inoculums with which to infect experimental mice. such selection of cysticerci expected would reduce variability in the resulting parasite loads between infected mice ; and it does so to some extent, but significant individual variation in parasite loads usually subsists and not rarely, depending on strain and sex of infected recipient mice, some of the challenged mice are totally spared from infection [49 ]. it is from this subpopulation of tiny nonbudded cysticerci that the cysticerci employed in this in vitro study of their budding process were selected. the independent variables were the initial density of cysts cultured in each well (density = 1, 5, 10 cysts / well with 1 ml of medium / well ; in a dish with 6 wells), the nature of supplements to the culture medium, and the days of culture (0 to 10) at 37 or 42c with 5% co2. the dependent variables were the number of buds found under light microscopy attached to each cyst (buds / cyst) in each well and the sum of all buds in each well (buds). the cysticerci came from two different donor mice and were cultured in the three density conditions without supplements in experiment number 1 (from donor number 1) or were subjected to various treatments in experiment number 2 (from donor number 2), such as heating (42c) and peroxide (30 mm) to induce stress in the cultured parasites, or supplemented with 17 - estradiol (30 nm), insulin (1.5 u / ml), glucose (56 mm), or insulin+glucose (same concentrations as when by themselves only) to provide with energy resources and restore putatively energy limiting conditions. the culture medium in the wells was changed by fresh medium every 24 hours for the first two days and every 36 hours thereafter. statistical analysis was performed with spss a student 's t - test to study the significance of contrasts between the different densities. figure 1 shows that in experiment number 1 the sum of buds produced in each well (budding) progressively increases with increasing parasite densities in a wave - like fashion more clearly visible at density = 10. it took 8 days for density = 1 to initiate budding and by only 1 of the 6 cysts, while it took 3 and 2 days for densities = 5 and 10, respectively, for most or all cysts to bud and 7 days for the higher densities to start a second wave of budding. the first line of table 1 shows that in experiment number 1 the final budding efficiency (final buds/cysticerci) at each density increased from 0.17 to 3.00 to 10.3 for densities 1, 5, and 10, respectively. from these results it is clear that there is considerable initial variation in budding among cysts and that increasing parasite density increases the production of buds and reduces the proportion of nonbudding cysts. thus, initial differences in the distribution of readiness to bud among the cysts (as defined by the time it takes a resting cysticercus to start budding plus the time taken for a bud to become a cysticercus capable of budding) may well explain the variation of parasite loads in mice infected with apparently similar inoculums. to minimize variation in parasite loads users of expmuriptcrascistiorf may try to presynchronize in vitro the cysts meant to be inoculated at 10 cysts / ml until most (80%) are already well into budding (> 1 buds / cyst) before their selection and inoculation through a procedure nondisruptive of the cysts. are the differences in budding related to and/or result from different responses to negative or positive pressures to bud existing in the over - crowded conditions in the peritoneal cavities of the donor mice and/or in the culture tubes ? [1014 ]. experiment number 2 was designed to address those questions, bearing in mind that increasing density may decrease resource availability and lead the parasites to enter into stress. accordingly, the cultured cysticerci from donor number 2 were submitted to standard stress (heat and peroxide) or favorable conditions (addition of 17 - estradiol, insulin, glucose, insulin+glucose) and cultured in vitro as done in experiment number 1. figure 2 shows the budding process at the different conditions from day 0 to day 10. table i most clearly shows that the total number of buds / cyst produced in vitro are increased about twofold with respect to unsupplemented control values at densities 1 and 5 but not at density 10, which is in fact reduced by the supplementations. the great difference between the control values of buds / cyst in experiments number 1 and number 2 (0.17 and 3.0, resp.) speaks of there being such differences between the harvested cysticerci from the two donor mice in the cysts ' readiness to bud, possibly depending on the state and terms of each host - parasite relationship established with the donor mice. it is not surprising that the supplements stimulated budding at low parasite densities because the synthesis and the role of estradiol in stimulating cysts reproduction in vivo and in vitro have been well established [7, 15 ] and so is also the role of heat. likewise, the insulin pathway has been shown to be present in a large variety of invertebrates, including the most primitive metazoan phyla (cnidaria and sponges), and to play a central role in cell division and differentiation. as other possible chemical mediators, we suspect cytokine - like substances which influence reproduction and apoptosis of heterologous cell lines, which cysticerci seem capable of producing and secreting in vivo and in vitro [1820 ]. tgf -, egf, and insulin pathways are conserved in helminth parasites with receptor functions probably similar to those of invertebrate and vertebrate orthologs. indeed, host - derived signals still present in the harvested cysts could have activated parasite receptors and modulated parasite development and differentiation [16, 21 ]. that crowding cysts at density = 10 per se promotes the highest budding efficiency is a novel finding which indicates that crowding is a powerful factor controlling the population of cysts. possibly, crowding may act by the release of growth factors [19, 20 ] by the cysts most differentiated and ready to begin budding when placed in vitro, which then recruit those most laggard. additionally, crowding may be mediated by adhesive molecules or membrane sensors sensitive to contact, as it has been previously reported by haas. and loverde. that the supplements did not improve, but rather lowered, the budding efficiency at density = 10 suggests that the capacity to bud has an upper limit. such hysteresis in the system controlling budding could also explain the wave - like form in the dynamics of budding. overall, the results are congruent with the hypothesis that parasite inoculums composed of 110 apparently identical small nonbudded cysts would likely include a variety of cysts differing in their initial readiness to bud and thereby induce variation in parasite loads in infected mice at early times after infection when parasite loads are relatively low, followed by a progressive tendency towards uniformity at later times. | taenia crassiceps cysticerci (cysts) reproduce by budding. the cysts ' production of buds was measured in vitro to explore parasite and environmental - related factors involved in the extreme individual variation in parasite loads of inbred mice. cysts were placed in in vitro culture for 10 days at initial parasite densities of 1, 5, 10 cysts / well in 1 ml of rpmi medium 1640 without serum. results showed that there is considerable intrinsic initial variation among inoculated cysts in their production of buds and that increasing parasite density (crowding) stimulates the overall production of buds and recruit into budding most of the cysts. identical cultures were then subjected to various treatments such as heating and exposure to peroxide to induce stress, or to 17 - estradiol, insulin, glucose, or insulin+glucose to supplement putatively limiting hormonal and energy resources. all treatments increased budding but the parasites ' strong budding response to crowding alone overshadows the other treatments. |
nguyen. (1) reported an increasing trend in the prevalence of knee pain in the united states after they evaluated data from the national health and nutrition examination surveys between 1971 and 2004 and from the framingham osteoarthritis study between 1983 and 2005. kim. (2) investigated 504 community residents of chuncheon, korea, aged 50 yr, and reported that the prevalence of knee pain was 46.2% (32.2% in men, 58.0% in women) and increased with age in women. the prevalence of knee osteoarthritis (oa) in elderly koreans has been reported by several researchers. cho. (3) reported that the prevalence of radiographic oa was 38.1% in residents aged 65 yr in seongnam, korea. kim. (4) reported that prevalences of radiographic oa and symptomatic oa were 37.3% and 24.2%, respectively, in residents aged 50 yr in chuncheon. although oa is the most common cause of knee pain in elderly people (5), disorders other than oa may also cause knee pain in this population. it has also been reported that radiographic changes correlate poorly with pain and that knee pain is a better predictor of disability than radiographic changes (6). previous studies that investigated the epidemiology of knee pain or oa in elderly koreans were limited to some local areas in korea. thus, arguments could be raised against the representativeness of these results. additionally, there is no reported study presenting severity profiles of knee pain in koreans. we investigated epidemiological indices of knee pain and its severity - prevalence, risk factors, and impact on quality of life - in a representative sample of elderly koreans. the korea center for disease control and prevention has conducted the korea national health and nutrition examination survey (knhanes) on a random sample of the general korean population to assess health and nutritional status through interviews and health examinations. the fifth knhanes was conducted from 2010 to 2012, assessing the whole nation each year without overlapping of survey areas or participants. a multistage stratified probability sampling was used according to geographical area, gender, and age groups, based on the statistics korea registries. 1 is a flowchart that shows the inclusion of subjects in this study. of the 8,958 participants in the fifth knhanes conducted in 2010, we excluded 203 persons who did not respond to the question about the presence of knee pain and 16 persons who did not report the severity of knee pain or reported a pain score of 0 even though they reported that they had knee pain. a questionnaire was distributed to the participants to evaluate demographic, socioeconomic, lifestyle, and health status information, including presence of knee pain and its severity. subjects were divided into three age groups : 50 - 59, 60 - 69, and 70 yr. for smoking status, subjects were classified into former or current smokers vs non - smokers. alcohol consumption was classified according to frequency by modifying the definitions used by anttila. those who drank alcohol at least once per month in the past year were defined as frequent drinkers. those who drank alcohol less than once per month or who did not drink in the past year were defined as infrequent or non - drinkers. regular exercise was defined as practicing high - intensity physical activities (strenuous or gasping activities such as running, high - speed cycling, and swimming) for at least 20 min at one session and at least 3 days per week, or practicing moderately intense physical activities (slightly strenuous or gasping activities such as slow swimming, badminton, and table tennis) for at least 30 min at one session and at least 5 days per week, or walking for at least 30 min at one session and at least 5 days per week. the presence of hypertension was defined as a systolic blood pressure 140 mmhg, a diastolic pressure 90 mmhg, or taking anti - hypertensive medication(s). the presence of diabetes mellitus was defined as a fasting glucose level 126 mg / dl, a history of taking oral hypoglycemic agent(s) or insulin injection, or physician - diagnosed diabetes mellitus. the presence of obesity was defined as a body mass index (bmi) 25 kg / m, calculated by dividing weight (kg) by the square of height (in meters). participants aged 50 yr underwent knee radiography ; the radiographs were evaluated using the kellgren - lawrence grading scale : grade 1, doubtful narrowing of joint space and possible osteophytic lipping ; grade 2, definite osteophytes and possible narrowing of joint space ; grade 3, moderate multiple osteophytes, definite narrowing of joint space, some sclerosis, and possible deformity of bone contour ; and grade 4, large osteophytes, marked narrowing of joint space, severe sclerosis, and definite deformity of bone contours (8). to maintain quality control of the radiographic examinations, the radiology technicians were taught how to use the digital x - ray machine and the standard radiography procedures before they participated in the survey. two independent radiologists examined the radiographs ; if the two reported different kellgren - lawrence grading scales for a radiograph and the difference was greater than 2, then the radiograph was transferred to a third radiologist for final determination of the scale value (9). radiographic oa was defined by a kellgren - lawrence grade 2 or higher radiographic change. knee pain in the fifth knhanes was defined as the presence of pain in the knee joint lasting 30 or more days during the most recent 3 months. participants with knee pain were asked to rate their pain severity on a 10-point numerical rating scale (nrs), where 0 was the absence of pain and 10 was the most severe pain. more than half of the subjects reported a score of 0 (i.e., no knee pain). the extreme skew creates a potential problem with treating this variable as an interval level variable and modeling it with linear regression. thus, we transformed the severity score into an ordered variable with four levels : none, mild, moderate, and severe. the cutoff points between mild, moderate, and severe pain levels were based on the study by jones. they assigned a nrs to a four - level metric after extensively reviewing literature, consultations with several pain experts, and consideration of pain guidelines and recommendations. the assignments were as follows : 0=no pain, 1 - 3=mild, 4 - 6=moderate, and 7 - 10=severe. in the fifth knhanes, it consists of five questions on mobility, self - care, pain, usual activities, and psychological status, with three possible answers for each item (1=no problem, 2=moderate problem, 3=severe problem) (11). a summary index with a maximum score of 1 (eq-5d index) the maximum score of 1 indicates the best health state, in contrast to the scores of individual questions, where higher scores indicate more severe or frequent problems (12). the prevalence of knee pain and profiles of its severity in elderly koreans were calculated and represented diagrammatically. logistic regression analysis was used to estimate crude and adjusted odds ratio (or) for knee pain with regard to risk factors. the proc logistic procedure in the sas statistical package (ver. 9.3 ; sas institute, inc., cary, nc, usa) was used to conduct logistic regression analysis for knee pain. the proportional odds model is an extension of logistic modeling and is used for assessing proportionality where the response variable takes on values in a set of ordered categories. it was used to estimate crude and adjusted proportional odds ratio (por) for severity of knee pain with regard to risk factors. if por for a specific risk factor is 2.0, the subjects are 2.0 times more likely to report a greater severity of knee pain. the proc genmod procedure was used to calculate pors for severity of knee pain, with a multinomial distribution and cumulative logit link function (13). the eq-5d index was analyzed and compared between subjects with knee pain and without knee pain, and between subjects with individual severity profiles and without knee pain using student 's t - test. crude and adjusted ors for belonging to the worst group of eq-5d indices with regard to knee pain or severity profiles were calculated. the proc logistic procedure was used to conduct logistic regression analysis for belonging to the worst group of eq-5d indices. the study protocol was reviewed and approved by the institutional review board of jeju national university hospital (jejunuh 2013 - 05 - 036). as this study used open - source data not containing personal information, the korea center for disease control and prevention has conducted the korea national health and nutrition examination survey (knhanes) on a random sample of the general korean population to assess health and nutritional status through interviews and health examinations. the fifth knhanes was conducted from 2010 to 2012, assessing the whole nation each year without overlapping of survey areas or participants. a multistage stratified probability sampling was used according to geographical area, gender, and age groups, based on the statistics korea registries. 1 is a flowchart that shows the inclusion of subjects in this study. of the 8,958 participants in the fifth knhanes conducted in 2010, we excluded 203 persons who did not respond to the question about the presence of knee pain and 16 persons who did not report the severity of knee pain or reported a pain score of 0 even though they reported that they had knee pain. a questionnaire was distributed to the participants to evaluate demographic, socioeconomic, lifestyle, and health status information, including presence of knee pain and its severity. subjects were divided into three age groups : 50 - 59, 60 - 69, and 70 yr. for smoking status, subjects were classified into former or current smokers vs non - smokers. alcohol consumption was classified according to frequency by modifying the definitions used by anttila. those who drank alcohol at least once per month in the past year were defined as frequent drinkers. those who drank alcohol less than once per month or who did not drink in the past year were defined as infrequent or non - drinkers. regular exercise was defined as practicing high - intensity physical activities (strenuous or gasping activities such as running, high - speed cycling, and swimming) for at least 20 min at one session and at least 3 days per week, or practicing moderately intense physical activities (slightly strenuous or gasping activities such as slow swimming, badminton, and table tennis) for at least 30 min at one session and at least 5 days per week, or walking for at least 30 min at one session and at least 5 days per week. the presence of hypertension was defined as a systolic blood pressure 140 mmhg, a diastolic pressure 90 mmhg, or taking anti - hypertensive medication(s). the presence of diabetes mellitus was defined as a fasting glucose level 126 mg / dl, a history of taking oral hypoglycemic agent(s) or insulin injection, or physician - diagnosed diabetes mellitus. the presence of obesity was defined as a body mass index (bmi) 25 kg / m, calculated by dividing weight (kg) by the square of height (in meters). participants aged 50 yr underwent knee radiography ; the radiographs were evaluated using the kellgren - lawrence grading scale : grade 1, doubtful narrowing of joint space and possible osteophytic lipping ; grade 2, definite osteophytes and possible narrowing of joint space ; grade 3, moderate multiple osteophytes, definite narrowing of joint space, some sclerosis, and possible deformity of bone contour ; and grade 4, large osteophytes, marked narrowing of joint space, severe sclerosis, and definite deformity of bone contours (8). to maintain quality control of the radiographic examinations, the radiology technicians were taught how to use the digital x - ray machine and the standard radiography procedures before they participated in the survey. two independent radiologists examined the radiographs ; if the two reported different kellgren - lawrence grading scales for a radiograph and the difference was greater than 2, then the radiograph was transferred to a third radiologist for final determination of the scale value (9). radiographic oa was defined by a kellgren - lawrence grade 2 or higher radiographic change. knee pain in the fifth knhanes was defined as the presence of pain in the knee joint lasting 30 or more days during the most recent 3 months. participants with knee pain were asked to rate their pain severity on a 10-point numerical rating scale (nrs), where 0 was the absence of pain and 10 was the most severe pain. although the pain severity score was a numeric, the distribution was extremely skewed. more than half of the subjects reported a score of 0 (i.e., no knee pain). the extreme skew creates a potential problem with treating this variable as an interval level variable and modeling it with linear regression. thus, we transformed the severity score into an ordered variable with four levels : none, mild, moderate, and severe. the cutoff points between mild, moderate, and severe pain levels were based on the study by jones. they assigned a nrs to a four - level metric after extensively reviewing literature, consultations with several pain experts, and consideration of pain guidelines and recommendations. the assignments were as follows : 0=no pain, 1 - 3=mild, 4 - 6=moderate, and 7 - 10=severe. in the fifth knhanes, it consists of five questions on mobility, self - care, pain, usual activities, and psychological status, with three possible answers for each item (1=no problem, 2=moderate problem, 3=severe problem) (11). a summary index with a maximum score of 1 (eq-5d index) the maximum score of 1 indicates the best health state, in contrast to the scores of individual questions, where higher scores indicate more severe or frequent problems (12). the prevalence of knee pain and profiles of its severity in elderly koreans were calculated and represented diagrammatically. logistic regression analysis was used to estimate crude and adjusted odds ratio (or) for knee pain with regard to risk factors. the proc logistic procedure in the sas statistical package (ver. 9.3 ; sas institute, inc., cary, nc, usa) was used to conduct logistic regression analysis for knee pain. the proportional odds model is an extension of logistic modeling and is used for assessing proportionality where the response variable takes on values in a set of ordered categories. it was used to estimate crude and adjusted proportional odds ratio (por) for severity of knee pain with regard to risk factors. if por for a specific risk factor is 2.0, the subjects are 2.0 times more likely to report a greater severity of knee pain. the proc genmod procedure was used to calculate pors for severity of knee pain, with a multinomial distribution and cumulative logit link function (13). the eq-5d index was analyzed and compared between subjects with knee pain and without knee pain, and between subjects with individual severity profiles and without knee pain using student 's t - test. crude and adjusted ors for belonging to the worst group of eq-5d indices with regard to knee pain or severity profiles were calculated. the proc logistic procedure was used to conduct logistic regression analysis for belonging to the worst group of eq-5d indices. the study protocol was reviewed and approved by the institutional review board of jeju national university hospital (jejunuh 2013 - 05 - 036). as this study used open - source data not containing personal information, the average age of the subjects was 63.69.1 yr. of the subjects, 56.3% were women ; 10.2% were college graduates ; 34.2% had manual occupations ; 15.9% were current smokers ; 43.5% consumed alcohol ; 50.7% performed regular exercise ; 48.8% had hypertension ; 16.9% had diabetes mellitus ; 34.6% had obesity (bmi25 kg / m) ; 31.1% had hyperlipidemia ; 39.0% had radiographic oa, defined as the presence of kellgren - lawrence grade 2 or more radiographic change (table 1). the prevalence of knee pain in all subjects was 23.1% (11.7% in men, 31.9% in women). as age increased, the prevalence of knee pain increased. in every age group, the prevalences of mild, moderate, and severe knee pain in all subjects were 4.3%, 9.1%, and 9.7%, respectively. those in men were 2.8%, 5.4%, and 3.5%, respectively ; those in women were 5.4%, 12.0%, and 14.4%, respectively (fig. crude ors showed significantly higher risk of knee pain in those 60 yr old, women, those with a low level of education, hypertension, obesity, or radiographic oa. current and former smokers or those who frequently consumed alcohol had significantly lower risks for knee pain. the adjusted ors revealed that those 60 yr old, women, and those who had a low level of education (less than college), a manual occupation, obesity, or radiographic oa had significantly higher risks for knee pain, compared with those without (table 2). based on the crude pors, those 60 yr old, women, those with a low level of education, hypertension, diabetes mellitus, obesity, or radiographic oa reported greater severity of knee pain. former and current smokers or those who frequently consumed alcohol were more likely to report lower severity of knee pain. the adjusted pors revealed that those 60 yr old, women, and those who had a low level of education (less than college), a manual occupation, obesity, or radiographic oa had a risk for reporting greater severity of knee pain, compared with those without (table 3). all subjects, all men, or all women with knee pain had significantly lower eq-5d index scores than all subjects, all men, or all women without knee pain. all subjects or all women with mild knee pain had significantly lower eq-5d index scores than all subjects or all women without knee pain. men with mild knee pain had lower eq-5d index scores than men without knee pain, but the difference was not significant. all subjects, all men, or all women with moderate knee pain or severe knee pain had significantly lower eq-5d index scores than all subjects, all men, or all women without knee pain (table 4). all subjects, all men, or all women with knee pain had significantly higher risks for belonging to the worst eq-5d index quartile compared with all subjects, all men, or all women without knee pain, unadjusted or after adjusting for other risk factors. all subjects or all women with mild knee pain had significantly higher risks for belonging to the worst eq-5d index quartile compared with all subjects or all women without knee pain. men with mild knee pain had a higher risk for belonging to the worst eq-5d index quartile than men without knee pain, but the difference was not significant. all subjects, all men, or all women with moderate or severe knee pain had significantly higher risks for belonging to the worst eq-5d index quartile compared with all subjects, all men, or all women without knee pain (table 5). the knhanes is a nationwide survey, and data are obtained from subjects who are randomly sampled from the general korean population. thus, we consider that our study is representative of the prevalence of knee pain and its severity profile in the general korean elderly population. the prevalence of knee pain in the present study (23.1% in all subjects, 11.7% in men, and 31.9% in women) was lower than that reported by kim. (2) (46.2% in all subjects, 32.2% in men, and 58.0% in women), who investigated the prevalence of knee pain in community residents of chuncheon, aged 50 yr. the difference in prevalence between the two studies may be attributable to the definition of knee pain and the residence distribution (community residents vs nationwide sample). the mean age of subjects in the present study was significantly lower than that reported by kim. old age, female gender, a low level of education, manual occupation, obesity, and radiographic oa were significant risk factors for knee pain. a prospective study of knee pain and its risk factors revealed that age, previous knee injuries, being overweight, and knee - straining work were risk factors for knee pain (14). no association with age, education, manual occupation, or obesity was reported by kim. differences in study design, subjects, and adjustment of variables may explain the discrepancies between studies. further studies to identify risk factors for knee pain are required. according to the crude ors, smoking and alcohol consumption significantly reduced the risk of knee pain. however, the adjusted ors indicated that their influence on the study variables was insignificant. the prevalence of musculoskeletal disorders is lower in men than in women (15). thus, the apparently lower risk for knee pain associated with smoking and alcohol consumption, based on the crude ors, probably resulted from the confounding effects of gender. the association of smoking and alcohol consumption with joint pain in previous reports is inconsistent. (16) found a protective association between smoking and oa when studying knee oa in the first u.s. they suggested that smoking, or some unidentified factor associated with smoking, modestly protects against development of knee oa. however, amin. (17) reported that men with knee oa who smoke have greater cartilage loss and more severe knee pain than men who do not smoke. (18) reported that alcohol consumption is inversely associated with the risk and severity of rheumatoid arthritis. however, adamson. (19) reported an inconsistent association between alcohol consumption and joint pain among men and women in the west of scotland twenty-07 study. to our knowledge, this is the first reported study to present a severity profile of knee pain in elderly koreans, risk factors that worsen the severity of knee pain, and the influence of the severity of knee pain on quality of life. this study revealed that a considerable number of elderly koreans suffer from mild, moderate, or severe knee pain. this study showed that the risk factors for knee pain were also risk factors for worse pain severity. creamer. (20) reported that helplessness, education, and bmi were important factors in determining pain severity in knee oa. people with a lower level of education generally work in manual occupations. compared with office work, manual work usually requires high physical loads, resulting in various musculoskeletal disorders, including knee pain, and worsening severity of musculoskeletal disorders. obesity is an emerging epidemic, and weight loss has been shown to improve pain and function in knee joints with osteoarthritis (21). among the risk factors for knee pain and its severity, manual occupation and obesity are modifiable factors. thus, workplace environment modifications, and weight loss programs for patients with knee pain, focusing especially on those with manual workers and those with high bmis, may reduce the prevalence of knee pain or at least the severity of knee pain. excluding men with mild knee pain, participants with knee pain had significantly lower quality of life than participants without knee pain. thus, the management of knee pain, even mild knee pain, is important for improving quality of life in the elderly population. considering the trend toward an ageing population in korea, early interventional approaches are needed to reduce the medical, social, and economic burdens of knee pain. | this study investigated the epidemiology (prevalence, risk factors, and impact on quality of life) of knee pain and its severity in elderly koreans. the subjects (n=3,054) were participants aged 50 yr from the fifth korea national health and nutrition examination survey, conducted in 2010. knee pain was defined as pain in the knee lasting 30 days during the most recent 3 months ; severity was categorized as mild, moderate, or severe. eq-5d was used to measure quality of life. the prevalence of knee pain was 23.1% (11.7% in men, 31.9% in women). the prevalences of mild, moderate, and severe knee pain were 4.3%, 9.1%, and 9.7%, respectively (2.8%, 5.4%, and 3.5% in men and 5.4%, 12.0%, and 14.4% in women). old age, female gender, a low level of education, a manual occupation, obesity, and radiographic osteoarthritis were risk factors for knee pain, and were associated with increased severity of knee pain. excluding men with mild knee pain, people with knee pain had significantly lower quality of life than those without knee pain. early interventional approaches are needed to reduce the medical, social, and economic burden of knee pain in elderly koreans. |
anti - neutrophil cytoplasm antibody (anca)-associated vasculitis (aav) is the term used to describe a group of systemic diseases characterized by small vessel vasculitis. there are three clinical syndromes : granulomatosis with polyangitis (gpa) ; microscopic polyangitis (mpa) ; and eosinophilic granulomatosis with polyangitis (egpa). within a caucasian population, gpa is usually associated with antibodies against proteinase-3 (pr3), mpa is usually associated with antibodies against myeloperoxidase (mpo) and egpa is often associated with anti - mpo antibodies but is sometimes anca negative. these are rare conditions with an annual incidence of around 20 cases per million within the uk. they are often associated with significant morbidity and mortality despite progress in immunosuppressive regimens designed to maximize efficiency and limit toxicity. the aetiology of aav remains uncertain ; however, it is likely that both genetic predisposition and environmental triggers are important. there is also mounting evidence for the pathogenicity of anca. we report the very unusual occurrence of two brothers with aav, but with differing clinical syndromes and anca types. a 49-year - old caucasian man presented as an emergency with a 1 day history of severe epistaxis. he had a 3-month history of nasal congestion, left ear and supraorbital pain, and lethargy. on examination he had bilateral uveitis and episcleritis and a faint purpuric rash over his groin. investigations showed preserved renal function, creatinine 91 mol / l and mild anaemia, haemoglobin 12 g / dl. inflammatory markers were raised, c - reactive protein 368 mg / l and erythrocyte sedimentation rate 119 mm / h. computed tomography (ct) chest demonstrated diffuse ground glass infiltrates throughout both lung fields (figure 1). a diagnosis of probable vasculitis was made and the patient was started on 30 mg / day of oral prednisolone. he was found to be c - anca positive with anti - pr3 titre of 431 iu (normal range < 25). he developed haemoptysis with a fall in his haemoglobin to 10.5 g / dl, and acute kidney injury with peak creatinine 185 mol / l. nasal swabs were negative for methicillin - resistant staphylococcal aureus but were not tested for methicillin - sensitive staphylococcal aureus. the diagnosis of gpa was made and he was transferred to our centre and treated by our current protocol for vasculitis with pulmonary haemorrhage. he received 60 mg / day oral prednisolone, 1 g iv cyclophosphamide and ten 4 l plasma exchanges. following discharge he received five further doses of iv cyclophosphamide (total 4 g) and 2 1 g iv rituximab (again in keeping with our current protocol). he had some minor infective complications with recurrent dental infections due to underlying dental caries which resolved following tooth extraction. two and a half years later he is in complete remission with a creatinine of 82 mol / l, although he remains c - anca positive with anti - pr3 titre of 70 iu. six months later the patient 's brother presented at the age of 53 years with 6 weeks of worsening lethargy, myalgia and arthralgia. he had a dry cough but no haemoptysis, and weight loss of 10 kg. investigations showed acute kidney injury, creatinine 338 mol / l (no baseline available but 213 mol / l when checked by his general practitioner 2 days previously). he was p - anca positive with anti - mpo titre of 357 iu (normal range < 25). ct chest was unremarkable except for a single pulmonary nodule which did not meet criteria for further investigation. renal biopsy showed pauci - immune crescentic glomerulonephritis and necrotizing vasculitis (figure 2). silver stain showing glomerulus with cellular crescent formation (courtesy of professor terry cook). silver stain showing glomerulus with cellular crescent formation (courtesy of professor terry cook). the diagnosis of mpa was made, and he was treated with 60 mg / day oral prednisolone, 1 g iv rituximab and 750 mg iv cyclophosphamide. following discharge he received a further 1 g iv rituximab and five further doses of iv cyclophosphamide (total 3.5 g) and was maintained on low - dose oral prednisolone (5 mg / day) and azathioprine 75 mg / day. his treatment was by our current protocol for treatment of patients with vasculitis but not requiring dialysis and without pulmonary haemorrhage. he remains in remission 2 years later with stable renal function (creatinine 140 mol / l) although still p - anca positive with anti - mpo titre of 40 iu. a 49-year - old caucasian man presented as an emergency with a 1 day history of severe epistaxis. he had a 3-month history of nasal congestion, left ear and supraorbital pain, and lethargy. on examination he had bilateral uveitis and episcleritis and a faint purpuric rash over his groin. investigations showed preserved renal function, creatinine 91 mol / l and mild anaemia, haemoglobin 12 g / dl. inflammatory markers were raised, c - reactive protein 368 mg / l and erythrocyte sedimentation rate 119 mm / h. computed tomography (ct) chest demonstrated diffuse ground glass infiltrates throughout both lung fields (figure 1). a diagnosis of probable vasculitis was made and the patient was started on 30 mg / day of oral prednisolone. he was found to be c - anca positive with anti - pr3 titre of 431 iu (normal range < 25). he developed haemoptysis with a fall in his haemoglobin to 10.5 g / dl, and acute kidney injury with peak creatinine 185 mol / l. nasal swabs were negative for methicillin - resistant staphylococcal aureus but were not tested for methicillin - sensitive staphylococcal aureus. the diagnosis of gpa was made and he was transferred to our centre and treated by our current protocol for vasculitis with pulmonary haemorrhage. he received 60 mg / day oral prednisolone, 1 g iv cyclophosphamide and ten 4 l plasma exchanges. following discharge he received five further doses of iv cyclophosphamide (total 4 g) and 2 1 g iv rituximab (again in keeping with our current protocol). he had some minor infective complications with recurrent dental infections due to underlying dental caries which resolved following tooth extraction. two and a half years later he is in complete remission with a creatinine of 82 mol / l, although he remains c - anca positive with anti - pr3 titre of 70 iu. six months later the patient 's brother presented at the age of 53 years with 6 weeks of worsening lethargy, myalgia and arthralgia. he had a dry cough but no haemoptysis, and weight loss of 10 kg. investigations showed acute kidney injury, creatinine 338 mol / l (no baseline available but 213 mol / l when checked by his general practitioner 2 days previously). he was p - anca positive with anti - mpo titre of 357 iu (normal range < 25). ct chest was unremarkable except for a single pulmonary nodule which did not meet criteria for further investigation. renal biopsy showed pauci - immune crescentic glomerulonephritis and necrotizing vasculitis (figure 2). silver stain showing glomerulus with cellular crescent formation (courtesy of professor terry cook). silver stain showing glomerulus with cellular crescent formation (courtesy of professor terry cook). the diagnosis of mpa was made, and he was treated with 60 mg / day oral prednisolone, 1 g iv rituximab and 750 mg iv cyclophosphamide. following discharge he received a further 1 g iv rituximab and five further doses of iv cyclophosphamide (total 3.5 g) and was maintained on low - dose oral prednisolone (5 mg / day) and azathioprine 75 mg / day. his treatment was by our current protocol for treatment of patients with vasculitis but not requiring dialysis and without pulmonary haemorrhage. he remains in remission 2 years later with stable renal function (creatinine 140 mol / l) although still p - anca positive with anti - mpo titre of 40 iu. we describe the occurrence of two different anca - associated vasculitides, gpa and mpa, in two brothers who were brought up together in west london. they presented within 6 months of each other with distinct clinical pictures and differing anca specificity. one brother had renal, pulmonary, and ear, nose and throat (ent) involvement with positive pr3-anca typical of gpa, and the other brother had positive mpo - anca and clinical features in keeping with mpa. they had no significant family history, in particular no history of vasculitis or autoimmune disease. the brothers have a shared hla haplotype of a3, b44, cw7, dr11, dq7, dqb1 03/05, which to our knowledge has not been reported to be associated with aav. the majority describe familial clusters of patients presenting separated in time, but with similar clinical phenotypes and the same anca type. we previously reported three members of an indo - asian family with gpa who all presented with ent and renal involvement. reported on two siblings with gpa, both with renal and pulmonary involvement, and sewell and hamilton reported a mother and daughter with gpa, both with ent and renal involvement. there are even fewer reported cases of first - degree relatives presenting with small vessel vasculitis with different anca types. a family reported by gomes. includes a father and daughter who both presented with renal disease ; one was anti - pr3 positive and the other anti - mpo positive. reported two brothers with egpa (anca negative) and gpa (anti - pr3 positive). aav is known to have important genetic associations. a genome - wide association study identified four single nucleotide polymorphisms (snps) that associated with aav. gpa / pr3-anca associated with snps within hla - dp, serpina1 (which encodes 1-antitrypsin) and prtn3 (which encodes proteinase 3), whereas a snp within hla - dq associated with mpa / mpo - anca. further analysis showed that these snps were more strongly associated with anca type than with clinical phenotype, suggesting that these diseases should perhaps be re - classified as pr3-anca or mpo - anca vasculitis, rather than gpa or mpa. environmental factors are also likely to be important in the pathogenesis of aav and a variety of toxins, infections and drugs have been implicated. silica is the environmental toxin for which there is the greatest evidence, with a population case brener. reported the case of two brothers with pulmonary and renal vasculitis (anti - mpo positive) ; both brothers had been exposed to silica but four other unexposed siblings did not have vasculitis. neither of our patients reported exposure to any environmental factor which could have predisposed them to developing aav. given the identification of a difference in genetic risk factors associated with different aav phenotypes and anca specificity it is perhaps surprising that first - degree relatives develop aav with differing clinical and serological features. it may be that there was some unrecognized environmental exposure, such as a toxin or infection, within their shared upbringing. our report illustrates the complex aetiology of aav and suggests that further research on the interaction of genetic and environmental factors is needed. | anti - neutrophil cytoplasm antibody (anca)-associated vasculitis (aav) is a group of rare autoimmune diseases. although the aetiology of aav is uncertain, it is likely that genetic and environmental factors contribute. we report the unusual case of two brothers presenting with aav with differing clinical pictures and differing anca specificity. there is a recently identified difference in genetic risk factors associated with anca specificity, making it surprising that first - degree relatives develop aav with differing clinical and serological features. our report illustrates the complex aetiology of aav and suggests that further research on the interaction of genetic and environmental factors is needed. |
hypernatremia (serum sodium concentration > 146 millimoles per liter [mmol / l ]) is a common electrolyte disorder that can occur in very young or elderly patients.1,2 severe hypernatremia is defined as a serum sodium concentration > 160 mmol / l.3 hypernatremia may be caused by simple excess of sodium, often resulting from dehydration, due to excessive water loss or the ingestion of hypertonic fluids. large losses of low - salt body fluids can occur during episodes of diarrhea or after the administration of diuretics or may result from renal tubular damage in patients with nephrogenic diabetes insipidus.1 hypernatremia may occur with the use of certain drugs, such as lithium and valproate, which may lead to renal impairment.4,5 hypernatremia can also occur in patients treated with excess sodium bicarbonate (nahco3) to correct metabolic acidosis, which may increase the concentration of sodium in the serum.6 particular attention must be paid to hypernatremic episodes occurring in elderly patients, which are usually caused by high - salt loads due to poor oral fluid intake. multiple myeloma is one of the common hematological diseases in elderly patients. in the past, reports are available on igg - kappa - type multiple myeloma and proximal renal tubular acidosis (fanconi syndrome),615 but the occurrence of hypernatremia in such cases has rarely been described.16 here, we report the case of an elderly patient presenting with multiple myeloma and underlying renal tubular damage, who developed extreme hypernatremia (peak serum sodium concentration, 183 mmol / l) and hyperchloremia (peak serum chloride concentration, 153 mmol / l) after intravenous treatment with bortezomib / dexamethasone / elcatonin and nahco3 in normal saline. a 77-year - old man (height, 170 cm ; body weight, 55 kg ; blood pressure, 172/66 mmhg ; and heart rate, 84 beats per minute) was transferred to our care after being injured in a road traffic accident. on admission a computed tomography scan revealed a fracture of the left pelvic bone (figure 1). the laboratory findings were : white blood count, 5600/l ; hemoglobin, 9.9 g / dl ; platelet count, 142,000/l ; aspartate transaminase, 42 (normal range ; 1337) units / l ; alanine aminotransferase, 21 (845) units / l ; lactate dehydrogenase, 402 (122228) units / l ; ammonium, 61 (90) ml / min / l ; ca, 11.9 (8.710.3) mg / dl ; inorganic phosphate (ip), 2.6 (2.94.9) mg / dl ; na, 140 (138146) mmol / l ; k, 2.8 (3.65.1) mmol / l ; cl, 102 (99108) mmol / l ; and serum beta 2-microglobulin, 3.1 (0.91.9) mg / l. urinalysis revealed a ph of 5.0 with occult blood (2 +) and protein (2 +) but glucose (). because the patient showed high levels of total protein in the serum, we considered a differential diagnosis of multiple myeloma. further tests revealed serum igg levels of 4389 (8701700) mg / dl, with igg - kappa - type m protein., the patient was diagnosed with multiple myeloma (durie salmon stage 2a). clinically, the patient showed the complete range of multiple myeloma symptoms (hypercalcemia, renal dysfunction, anemia, and bone disease, such as osteolytic pelvic bones and ribs), which had been unrecognized until hospital admission. during the first week after admission, the patient was able to communicate but was somehow in a state of a mild disturbance of consciousness (japan coma scale ; jcs - i-2 ; disoriented) and had difficulty imbibing oral fluids ; thus, he was hydrated with 10001500 ml of peripheral parenteral nutrition solution (soldem 3 : sodium [na ] concentration, 35 mmol / l ; chloride [cl ] concentration, 35 mmol / l). after the diagnosis of multiple myeloma with hypercalcemia, specific treatment was instigated, according to the following protocol (figure 2) : intravenous normal saline (na, 154 mmol / l ; cl, 154 mmol / l ; 500 ml 2/day ; daily), dexamethasone (8 mg per day 2 consecutive days), nahco3 (80 mmol / l per day ; daily), bortezomib (1.3 mg / m per dose, weekly), elcatonin (40 units per day for 5 consecutive days), and zoledronic acid hydrate (4 mg per dose, monthly). all drugs were intravenously administered in normal saline (n / s) ; thus, the patient received 200 mmol / l of sodium per 24 hours over a period of 6 days. as shown in figure 2, the patient experienced a gradual increase in serum sodium and chloride levels, which peaked at 183 mmol / l and 153 mmol / l, respectively. at this point, the patient was then switched to an intravenous infusion (1500 ml) of soldem 1 (na, 90 mmol / l ; cl, 70 mmol / l), soldem 3, and potassium chloride (kcl) in 5% glucose, which helped gradually resolve the hypernatremia / hyperchloremia over the next 7 days, but the patient continued to have difficulty in swallowing and a low oral fluid intake. the patient s disturbed consciousness persisted for almost a month, then the situation improved significantly thereafter. during the entire period of hypernatremia / hyperchloremia, the former was associated with the excretion of high levels of potassium (38 mmol / l ; normal levels, 160 mmol / l)3 has been described in various case reports, as high as a serum na concentration of 196 mmol / l, 201 mmol / l, 202 mmol / l, or 211 mmol / l.3,5,18,19 in addition, in a reported case of fatal ingestion of sodium hypochlorite bleach, the patient showed hypernatremia (serum na, 169 mmol / l) and hyperchloremia (cl, 143 mmol / l) in association with metabolic acidosis,20 and a patient with acute salt poisoning, due to an overdose of sodium chloride, showed a na load of 400 mmol / l over 12 hours.21 here, we report a case of severe hypernatremia (na, 183 mmol / l) in a patient with multiple myeloma. to date, however, few cases of hypernatremia in association with multiple myeloma have been described.16 in the present case, hypernatremia occurred after the patient was hydrated intravenously mainly with n / s as well as all necessary drugs (bortezomib, dexamethasone, elcatonin, and nahco3) were given in n / s, as he was unable to take fluids orally. besides the high - saline burden, nahco316 and dexamethasone administration might have accelerated the hypernatremia. in calculation, he received a salt load of 200 mmol / l over 24 hours, which was nearly comparable to one - fourth of the dose in a fatal case of household bleach ingestion reported by ross.20 the peak serum concentrations of sodium and chloride in the current patient reached 183 mmol / l and 153 mmol / l, respectively. although the patient suffered some mild disturbances of consciousness, he made a full recovery within approximately 1 month, with no residual adverse effects. in our case, the cause of the hypernatremia / hyperchloremia was likely to be linked to the renal tubular damage due to multiple myeloma, as the laboratory data showed a normal blood anion gap and a high urine anion gap, both of which are indicative of hyperchloremic acidosis (base excess < 2 mmol / l ; anion gap < 17 mmol / l).22 in general, decreases in plasma hco3 levels are associated with hyperchloremic acidosis and lactic acidosis. both type 1 (distal) and type 2 (proximal) renal tubular damage have been reported in patients with multiple myeloma and other related diseases.615 in particular, distal renal tubular acidosis is characterized by hyperchloremic metabolic acidosis23 and hypokalemia.24 on the other hand, sakaue and uchida described igg - kappa - induced proximal renal tubular acidosis, as observed in fanconi syndrome, in patients with igg - kappa type myeloma.14,15 although the patient in the present study showed persistently low levels of hco3, hypokalemia, and hypophosphatasia, he did not show evidence of glucosuria or generalized aminoaciduria, indicating that he did not have fanconi syndrome. therefore, we concluded that the extreme hypernatremia / hyperchloremia was due to a combination of excess saline and nahco3, dexamethasone administration in association with poor oral fluid intake, on top of the distal renal tubular damage caused by multiple myeloma. considering the rarity of hypernatremia regardless of renal tubular damage in patients with multiple myeloma, it remains unclear how much myeloma - associated renal damage played a role in causing severe hypernatremia in this patient. minemura reported that renal tubular acidosis was not improved by treating the myeloma with chemotherapy agents ; indeed, in their case, the patient died from an exacerbation of the disease. on the other hand, uchida reported a case in which renal tubular acidosis was corrected after chemotherapy for multiple myeloma, and sakaue reported that serum igg levels and urine sugar levels decreased, and serum potassium levels returned to normal, in a patient receiving chemotherapy for multiple myeloma. in the current case, the patient recovered from the electrolyte disturbances and acidosis within a month, responding to the myeloma therapy. the improvement in the electrolyte imbalance was associated with a return to normal levels of consciousness. in summary, there appears to be an increased risk of hypernatremia in elderly patients with renal tubular damage associated with multiple myeloma ; therefore, clinicians must exercise caution and not prescribe excess salt, particularly in elderly patients who have difficulty taking oral fluids. written, informed consent to publish this case report was obtained from the caregiver of the patient involved. si, nk, and kk contributed equally to this work ; si, nk, and kk managed the patient ; kk performed bone marrow studies ; all authors prepared the manuscript ; all authors read and approved the final manuscript. | a 77-year - old male was admitted to hospital after suffering a pelvic bone fracture in a road traffic accident and was incidentally found to have igg - kappa - type multiple myeloma with hypercalcemia. the patient was also noted to be hypokalemic and had low hco3, with possible damage to the distal tubules in the kidneys. when the treatment was begun with bortezomib / dexamethasone / elcatonin and sodium bicarbonate (nahco3) in normal saline (equivalent to a daily sodium dose of 200 millimoles per liter [mmol / l ]), the patient was in a state of poor oral fluid intake. the patient developed hypernatremia and hyperchloremia, with a peak serum sodium and chloride levels of 183 mmol / l and 153 mmol / l, respectively, at the sixth day after the start of treatment. following the switch of the intravenous infusions from normal saline to soldem 1 and soldem 3 solutions, these high - electrolyte levels gradually returned to normal over the next 7 days. although the patient showed disturbed consciousness (japan coma scale = jcs - i-3) during the period of electrolyte abnormality, he eventually fully recovered without sequelae. in this patient, we successfully managed the severe hypernatremia / hyperchloremia, caused by the combined effects of intravenous saline burden in a state of poor oral fluid intake, during the treatment for igg - kappa type multiple myeloma. |
pilsicainide is a pure sodium channel blocker with slow recovery kinetics, and it is known to be effective in converting recent - onset atrial fibrillation to sinus rhythm.1)2) on the other hand, high plasma concentrations of class ic antiarrhythmic drugs, including pilsicainide, have been known to induce life - threatening tachyarrhythmias, such as ventricular tachycardia, torsades de pointes (tdp) and ventricular fibrillation.3) we report a case of sudden cardiac death ; the patient received only three low oral doses of pilsicainide (100 mg / day) to convert paroxysmal atrial fibrillation to sinus rhythm and developed tdp only two days later during holter electrocardiogram (ecg) monitoring. this case may present a concern about the proarrhythmic effects of an oral pilsicainide for treatment of atrial fibrillation in octogenarian patients. an 84-year - old male visited our hospital due to palpitations and shortness of breath that began two days previously. he underwent physical examination, 12-lead ecg, chest x - ray, and blood tests. the patient 's heart rate was 150 beats / min, and his blood pressure was 126/76 mm hg. an ecg examination showed atrial fibrillation, which was considered to be of recent onset (within 48 hours). although ecg showed a tendency for poor progression of r in v 1 - 3, the patient had no chest pain and no history of coronary artery disease. in addition, an echocardiography, which was performed in a private clinic two months before the patient visited our hospital, showed normal left ventricular wall motion ; during the past two months, there were no significant differences of st - t changes in ecg. routine laboratory tests revealed serum sodium of 142 meq / l, potassium of 4.8 meq / l (no hypokalemia and hyperkalemia) and creatinine of 1.0 mg / dl, which were indicative of mild renal dysfunction. thus, oral low - dose pilsicainide at 50 mg twice daily was prescribed to convert atrial fibrillation to a sinus rhythm, and a holter ecg was ordered in the outpatient clinic. in addition, he started anticoagulant treatment with warfarin but did not receive any other drugs, including any other antiarrhythmic drugs. one day after receiving an oral administration of pilsicainide, he visited our hospital again, and a holter monitor was fitted to evaluate the efficacy of pilsicainide and initiated at 13:30 in the afternoon. at this time, he felt no palpitations and showed a sinus rhythm with normal qt and qtc intervals (320 msec and 400 msec, respectively) in the cm5 lead of the holter ecg (fig. 1). however, the patient died during his sleep 18 hours after the holter ecg was started. the analysis of the holter ecg recording revealed that the cause of sudden death was an episode of tdp (fig. the holter ecg showed that the patient developed atrial fibrillation followed by repetitive non - sustained and sustained tdp (figs. 2 and 3). 4, the qtc interval in the holter ecg showed gradual elongation from 13:30 at the start until 6:00 the next day. at 7:00 the next day, tdp was observed, and it continued for approximately one hour ; at 8:00, cardiac arrest was detected in the holter ecg. the segments with excessive noise, ectopic activity and atrial fibrillation were excluded from the analysis. the qtc interval was clearly prolonged compared with that of initial holter ecg. at the same time, the width of the qrs complexes was slightly prolonged compared with that of the initial holter ecg. pilsicainide, one of the class ic antiarrhythmic drugs, is very popular in japan and is also available in korea for terminating paroxysmal atrial fibrillation partially due to the favorable effects reported in the pilsicainide suppression trial on atrial fibrillation clinical study.2) an increase in the rate of sudden death in patients taking a class ic drug after myocardial infarction was reported in the cardiac arrhythmia suppression trial study;4) however, it is unknown whether pilsicainide also increases the rate of sudden death for patients with atrial fibrillation. pilsicainide is rapidly absorbed from the gastrointestinal tract, and most of it is excreted from the kidney ; therefore, the half - life of elimination is prolonged in patients with severe renal failure. in fact, previous studies reported arrhythmic events in patients after taking pilsicainide, and almost all these patients had renal function failure. in our case, the level of serum creatinine was in the normal range, but the patient was 84 years old, and his body weight was 65 kg. thus, the estimated creatinine clearance was 46 ml / min, and the estimated glomerular filtration rate was 54.4 ml / min/1.73 m, which were considered to represent mild renal dysfunction even though the serum creatinine was 1.0 mg / dl. although the terminal electrical events leading to sudden cardiac death are rarely recorded on holter ecg monitoring, in this case the patient happened to be wearing the holter ecg by chance during the cardiac event. the holter ecg monitor revealed a gradually prolonged qtc interval just before tdp, which was considered to indicate an elevated plasma concentration of pilsicainide, although the actual level was not measured. while orally administered pilsicainide is an effective drug for terminating atrial fibrillation, the dose should be adjusted cautiously in octogenarian patients even if their serum creatinine levels are within normal ranges. in addition, when pilsicainide is prescribed for the octogenarian, the patient should be admitted to the hospital for monitoring. | an 84-year - old male received oral pilsicainide, a pure sodium channel blocker with slow recovery kinetics, to convert his paroxysmal atrial fibrillation to a sinus rhythm ; the patient developed sudden cardiac death two days later. the holter electrocardiogram, which was worn by chance, revealed torsade de pointes with gradually prolonged qt intervals. this drug is rapidly absorbed from the gastrointestinal tract, and most of it is excreted from the kidney. although the patient 's renal function was not highly impaired and the dose of pilsicainide was low, the plasma concentration of pilsicainide may have been high, which can produce torsades de pointes in the octogenarian. although the oral administration of class ic drugs, including pilsicainide, is effective to terminate atrial fibrillation, careful consideration must be taken before giving these drugs to octogenarians. |
the approval of the institutional review board was obtained for the study and the research adhered to the tenets of the declaration of helsinki. the information on donor cornea tissues and the recipient details were collected from the eye bank and the medical records department of our tertiary eye care center for the year 2013. at our eye bank, the in situ excision is performed by trained technicians and the retrieved corneas are preserved in the mccarey - kaufman (mk) medium, which is a short - term preservation medium. although the standard practice is to utilize corneas within the dtpt of 6 h as the upper limit, some corneas with dtpt > 6 h were utilized as these were graded suitable for transplantation and hence could be analyzed in this study. details of the time of donor 's death were documented from the records of the deceased, and the time from death to excision and preservation into the mk medium were noted from the eye bank records. the donor corneas were evaluated on slit lamp (sl 115, carl zeiss meditec ag, jena, germany) and specular microscope (eye bank keratoanalyzer, konan inc., hyogo, japan) to grade the tissues and determine the suitability of transplantation. the slit lamp grading (optical vs. therapeutic grade) of the corneas whose dtpt was > 6 h, primary indication of transplants, and adverse events following transplants with these corneas were reviewed retrospectively. outcome of the optical transplants (optical penetrating keratoplasty [pk ] and endothelial keratoplasty [ek ]) was analyzed for primary graft failure and long - term graft survival. few parameters such as donor age, utilization rate, proportions of optical keratoplasty versus therapeutic keratoplasty, and endothelial cell density (ecd) of corneas with dtpt > 6 h were compared with those of corneas with dtpt 6 h. the data analysis was performed using the software origin version 7.0 (originlab corporation, northampton, ma, usa) and stata v11.0 (statacorp lp, college station, texas, usa). descriptive measures included mean and standard deviation for data with normal distribution, whereas those that were not normally distributed were described using median and interquartile range (iqr). categorical data were described in proportions and compared using chi - square test. mann whitney u - test (2 groups) and kruskal wallis test (> 2 groups) were used for comparisons between nonparametric data. the total number of corneas retrieved in the year 2013 was 4648, of which 2190 (47.1%) were utilized for transplantation. a total of 1204 transplants were performed at our tertiary eye care center, of which 65 (5.4%) transplants were performed using donor corneas that had dtpt > 6 h (upper limit 9.8 h). based on the standard guidelines of donor cornea evaluation which involves clinical slit lamp examination and specular microscopy, 44 (67.7%) of 65 corneas were evaluated as optical grade and 21 (32.3%) were evaluated as therapeutic grade. there was no relationship between dtpt and the quality grading of corneas into optical and therapeutic (p = 0.10) (optical grade corneas : median dtpt 7.25 h [iqr, 6.58 h ] vs. therapeutic grade corneas : median dtpt 6.75 h [iqr, 6.257.25 h ]). of the 44 optical grade corneas, 35 (79.6%) were used for optical transplants and 9 (20.4%) were used for therapeutic indications. the median age of the recipient at the time of surgery was 53.2 years (iqr, 29.860.7 years). (ek and optical pk), 23 (65.7%) had a follow - up duration of at least 3 months, the mean duration being 15.1 6.7 months (3.226.5 months). among these 23 transplants, all were secondary graft failures but for one case of primary graft failure, which was a complicated descemet 's stripping ek performed for a complex pseudophakic corneal edema [table 1 ]. there was a case of interface infection (1.5%), following a descemet 's stripping ek. the mate pair of this donor was used for an optical pk with no adverse event in the postoperative period. survival analysis this figure shows the survival probability of the corneal grafts (death - to - preservation time > 6 h : minimum follow - up of 3 months) done for purely optical indications summary of graft failures table 2 summarizes the comparison of donor - related characteristics between corneas with dtpt > 6 h and 6 h. the age of the donors with a dtpt > 6 h was slightly younger compared to those with a dtpt 6 h. however, there was no difference in the primary indication of corneal transplants. similar to corneas with dtpt > 6 h, there was no relationship between dtpt and ecd (p = 0.57) in corneas with dtpt 6 h. the utilization rate (number of corneas utilized / number of corneas retrieved) was lower (45.6%) in corneas with dtpt > 6 h. comparison of donor corneas : death - to - preservation time > 6 h versus death - to - preservation time 6 h the total number of corneas retrieved in the year 2013 was 4648, of which 2190 (47.1%) were utilized for transplantation. a total of 1204 transplants were performed at our tertiary eye care center, of which 65 (5.4%) transplants were performed using donor corneas that had dtpt > 6 h (upper limit 9.8 h). based on the standard guidelines of donor cornea evaluation which involves clinical slit lamp examination and specular microscopy, 44 (67.7%) of 65 corneas were evaluated as optical grade and 21 (32.3%) were evaluated as therapeutic grade. there was no relationship between dtpt and the quality grading of corneas into optical and therapeutic (p = 0.10) (optical grade corneas : median dtpt 7.25 h [iqr, 6.58 h ] vs. therapeutic grade corneas : median dtpt 6.75 h [iqr, 6.257.25 h ]). of the 44 optical grade corneas, 35 (79.6%) were used for optical transplants and 9 (20.4%) were used for therapeutic indications. the median age of the recipient at the time of surgery was 53.2 years (iqr, 29.860.7 years). (ek and optical pk), 23 (65.7%) had a follow - up duration of at least 3 months, the mean duration being 15.1 6.7 months (3.226.5 months). among these 23 transplants, all were secondary graft failures but for one case of primary graft failure, which was a complicated descemet 's stripping ek performed for a complex pseudophakic corneal edema [table 1 ]. there was a case of interface infection (1.5%), following a descemet 's stripping ek. the mate pair of this donor was used for an optical pk with no adverse event in the postoperative period. survival analysis this figure shows the survival probability of the corneal grafts (death - to - preservation time > 6 h : minimum follow - up of 3 months) done for purely optical indications summary of graft failures table 2 summarizes the comparison of donor - related characteristics between corneas with dtpt > 6 h and 6 h. the age of the donors with a dtpt > 6 h was slightly younger compared to those with a dtpt 6 h. however, there was no difference in the primary indication of corneal transplants. similar to corneas with dtpt > 6 h, there was no relationship between dtpt and ecd (p = 0.57) in corneas with dtpt 6 h. the utilization rate (number of corneas utilized / number of corneas retrieved) was lower (45.6%) in corneas with dtpt > 6 h. comparison of donor corneas : death - to - preservation time > 6 h versus death - to - preservation time 6 h in this study, we examined the outcomes of corneal transplantations where the donor tissue, after in situ excision, had a dtpt of more than 6 h and up to 10 h. understanding the influence of key donor factors such as dtpt on the outcomes of transplants helps in providing a rationale for the standard functioning of eye banks. studies have been done to evaluate the relationship between dtpt and the quality of donor tissue where the analysis included all the donor tissues (enucleation and excision). attributed the good utilization rate of in situ excised donor corneal tissues to shorter dtpt (12 h was shown to be unrelated to the degree of endothelial cell loss, following pk for endothelial disorders. following eye bank specular microscopy and assessment of endothelial cell morphology, some tissues were found to remain good at longer time (> 6 h) after death. studies that have examined the quality of donor tissues and the outcome of transplants with donor corneas with dtpt > 6 h are few. the statistics at our eye bank for the year 2013 revealed that the donor corneas with dtpt > 6 h had a higher ecd than those 6 h (range, 6.19.8 h), optical- and therapeutic - graded tissues had comparable dtpts, showing that dtpt does not impact the quality grading significantly. the cell density of the corneal endothelium of the in situ excised tissues was not found to be related to dtpt in our study. the long - term outcome of optical transplants with these corneas had a survival rate of ~64% at the end of 2 years, following the surgery. the success of optical keratoplasty with these corneas at 2 years follow - up was found to be somewhat better compared to our historical control data (success rate of 59% at 1 year from an earlier recent study on the outcomes of all optical transplants in the year 2012 : unpublished data). except one case (1.5%) of interface infiltrate where it could not be ascertained that it was donor - related infection, no other adverse event is noted. it is intuitive to understand that, following death, the metabolic processes cease to exist and provide a healthy environment to the donor endothelium. the tissue should ideally be retrieved quickly and transferred into a healthy medium. however, the delay in recovery is a practical reality in some instances and because workforce and serological testing costs exist for all tissues harvested (regardless of whether or not the tissue is utilized), the economic ramifications of discarding a tissue are not small. discarding all such corneas with dtpt more than 6 h may lead to a shortage of tissues suitable for surgery, particularly in developing countries. although every possible measure should be taken to facilitate the recovery and preservation process, the decision for utilization for clinical use of such corneas (with dtpt up to 10 h) can be made if they meet the criteria of tissue acceptance for optical use on the basis of standard guidelines of donor cornea evaluation. in summary, the donor corneas with dtpt 6 h to 10 h can be utilized for optical indications provided that they meet the criteria of tissue acceptance for optical use. | purpose : in tropical countries, physicians are skeptic in using corneas with death - to - preservation time (dtpt) > 6 h, concerns being endothelial cell viability and microbial contamination on prolonged dtpt. the objective of the study was to investigate these concerns by analyzing the outcomes of corneal transplants performed using donor corneas with dtpt > 6 h.materials and methods : the study was a retrospective case series of 65 transplants performed in 2013 with donor corneas that had dtpt > 6 h (range, 6.19.8 h). the information on donor cornea tissues and the recipient details were collected from the eye bank and the medical records department of our tertiary eye care center. the main outcome measures were slit lamp assessment of the donor corneas, primary graft failure, graft survival, and postoperative adverse reactions, especially infections, if any.results:median dtpt was 7 h. forty - four (67.7%) corneas were evaluated as optical grade and 21 (32.3%) were deemed as therapeutic grade ; 36 (55.4%) were used for optical indications. there was no relationship between dtpt and the tissue grading of corneas or endothelial cell density. of the 23 keratoplasties for purely optical indications with a minimum follow - up of 3 months, 15 (65.2%) remained clear whereas 7 (30.4%) failed (mean follow - up 15.1 6.7 months). the causes of failure were primary graft failure (n = 1) and secondary graft failure (n = 6).conclusion : the donor corneas with dtpt 6 h to 10 h can be utilized for optical indications provided that they meet the criteria of tissue acceptance for optical use. |
as a biomaterial is exposed to in vitro condition or in vivo physiologic environment, proteins that are present in the cell culture media or fluids of the body adsorb to its surface in less than 1 second. then, the functional groups (ligands) of the adsorbed protein bond with cell surface receptors (integrins).1 particularly, adsorption of vit - ronectin and fibronectin on the surface, results in formation of an intermediate layer that enhances cell adhesion.2,3 a hydrophilic surface shows greater protein adsorption than a hydrophobic surface4 which leads to an increase of desirable cellular behavior compared with a hydrophobic surface.58 the presence of nanoscale features on the surface of implants can enhance the growth and attachment (measured as cell density) of osteoblast bone - forming cells.9,10 such enhancement is due to an increase of surface area that provides more area for cell substrate interaction, more surface energy, more protein adsorption, integrin clustering, and as a result, higher cell adhesion.11,12 since nanometer features can mimic the natural environment to which cells are adapted,13 various surface modifications such as sandblasting, coatings, and plasma spraying have been introduced to enhance the biomimicry environment. however, commonly used ceramic coatings have undesirable mechanical properties such as cracking and delamination from the metal substrate. for instance, in titanium (ti) and hydroxyapatite (ha) plasma spraying, the resulting coating is not the same material as the bulk, due to vaporization, condensation, and substrate temperature variations during high temperature deposition. as a result, there will be residual stress in the coating / substrate interface due to expansion coefficient mismatch which leads to cracking and breakdown of the coating.14 hence, development of novel surface modifications with more robust and flexible structures containing nano features will be highly promising for better osseointegration, cell implant interaction, and implant life cycle. tio2 nanotubes that cover the surface of ti implants, and which are fabricated from the bulk material instead of as a coating, offer this flexibility and can relevantly mimic the natural environment of the bone - forming cells. considering that bone cells in the human body interact with fluid that flows around them in interstitial spaces,15,16 the presence of space between tubes is beneficial for transport of waste and nutrients, and therefore cell metabolism.17 since these electrochemically etched nanotubes are from the bulk, there is no interface, leading to much better design and analysis. the electrochemical etching process is performed in ambient temperature, leaving no residual stress due to heating.18,19 the effect of nanotubes wettability, diameter, crystallinity, and alloying elements has been investigated in several studies.2024 wettability is sharply increased after anodization of flat (without nanotube) ti, which enhances protein adsorption from body fluids in contact with implant surface and consequently enhances cell adhesion.2527 popat results indicated that in comparison to flat ti surfaces, nanotubular surfaces provided higher cell adhesion, alkaline phosphatase activity, and extracellular matrix (ecm) production as well as enhanced calcium (ca) and phosphorus (p). in addition, they investigated the biocompatibility of tio2 nanotubes by implanting disks with nanotubular titania surfaces in rats. their results showed no inflammatory response or fibrous tissue formation in the tissues surrounding the ti implant due to the presence of tio2 nanotubes. bjursten showed that tio2 nanotube surfaces have nine - fold higher bone - implant interlock compared with sandblasted surfaces. they compared three different surface structures including nanotubes, thin films, and foams of tio2 and concluded that tio2 nanotubes showed the best properties for higher cell density. yao compared three different surface structures (smooth, nanoparticulate, and nanotubular) and observed that the ca deposition by osteoblasts was highest on surfaces with tio2 nanotubular features. they explained that this finding is based on the fact that the nanotubes provide more surface area and reactive sites for fibronectin protein adsorption which mediates osteoblast adhesion. diameter of the nanotubes drastically affects cellular response31,32 since it defines the position of transmembrane integrins of attached cells. integrins transmit the force to actin filaments and cause cytoskeletal tension and consequently cell morphology and signaling is affected.33 reports on the effect of nanotube diameter on cell growth and adhesion are often contrasting. park reported the optimized nanotube diameter to be 15 nm for adhesion of mesenchymal stem cells (msc) while oh and brammer reported that 100 nm diameter nanotubes provide the highest mc3t3-e1 mouse osteoblast cell density. oh also reported that the 100 nm tube diameter resulted in extremely elongated human mscs, guiding them to differentiate into specific osteoblast cells. bauer investigated the effect of change in dimension and constructive material on mscs response attachment and proliferation. they concluded that change in diameter is more effective on cell response compared to change in surface chemistry and length. the present investigation focuses on the effects of substrate chemistry and atomic ordering on the cell growth and attachment on commercially pure (cp)-ti and alloy - ti surfaces with and without nanotubes. mc3t3 osteoblast cells were cultured on the substrates and their growth was characterized by optical microscopy, scanning electron microscopy (sem), and focused ion beam (fib) milling and imaging. this is the first study that utilizes the fib technique to investigate the biophysical behavior of cells on nano textured surfaces such as tio2 nanotubes. in particular, the architecture of the cell / substrate interface was visualized by sem and fib to obtain better insight into the morphological and functional features of interfacial osteoblast cell attachment. the substrates for cell growth were 23 cm flat sheets with thickness of 0.25 mm. prior to nanotube formation through anodization etching, the polished substrates were sequentially sonicated in acetone, isopropyl alcohol, and methanol, then rinsed with deionized water, and dried in an n2 stream. the electrolyte was 0.2 weight (wt) % solution of nh4f in 49 ml ethylene glycol and 1 ml deionized water. after anodization, the samples were rinsed with deionized water and dried in an n2 stream. the experimental setup consisted of a two - electrode arrangement, with copper (cu) mesh as the counter electrode during different sets of anodization. a heating / cooling rate of 308c / min was used, and the substrates were annealed in air at 450c for 3 hours. to investigate cell density of mc3t3-e1 mouse osteoblasts (crl-2593, subclone 4, atcc, manassas, va, usa),37 each 1 ml vial of purchased cell was mixed with 10 ml of alpha minimum essential medium (-mem ; invitrogen, usa), 10 vol % fetal bovine serum (fbs ; invitrogen), and 1 vol % penicillin streptomycin (ps ; invitrogen). the cell suspension was incubated at 37c and 5 vol % co2 environment until saturation point of approximately 3,000 cells / mm was reached after 72 hours of incubation. confluent cells were seeded onto the substrates of interest, placed on a 30-well polystyrene plate and stored in a 37c co2 incubator for 24, 48, and 72 hours to observe the cell morphology and count the number of cells attached as a function of incubation time. field emission scanning electron microscopy (fe - sem) hitachi s-4700 was used for surface characterization of the substrates. fe - sem was used to image the adhered cells. using energy dispersive spectroscopy (eds) detector of the fe - sem, chemical composition data were collected before and after cell culture to investigate the composition of individual nanotubes, confirm alloy nanotube composition characteristics of ti, al, and v, and to detect ca and p deposition after cell adhesion. the samples were washed several times to eliminate any entrapped media. after evaluating the cell density and adhesion on tio2 nanotubes, samples were rinsed with phosphate buffered saline (pbs), fixed, rinsed again and immediately imaged. the high - resolution imaging gave the capability of distinguishing between non - flat and well - spread flat cells and the rinsing followed by rapid fixation removed the non - adherent cells, resulting in a more accurate cell count assay. five random fields were counted per substrate and all experiments were run in triplicate, repeated at least three times. the mean numbers of total attached cells were calculated from the total cell number counted from five random square areas (1.01.0 mm) of each of the 18 different substrates. the mean numbers of attached cells on the surfaces of the control and nanotube covered samples were compared for 24, 48, and 72 hours of incubation. for the fib investigations, substrates with fixed cells were inserted into the specimen chamber of a hitachi sa-2000 fib. the rough milling conditions to open a trench in the cell, used ion currents of 5 to 7 na at 30 kv. lower beam currents of 100 to 670 pa were used to polish the cross section. samples of cp - ti and ti6al4v were used to investigate the effect of nanotubes on adhesion and cell density of osteoblasts due to their biocompatibility and preferred mechanical and biological properties.3840 in order to investigate the biphasic nature of nanotubes, the samples were etched for 30 seconds in 0.5% hf (figure 1). at the initial stage of anodization of cp - ti samples, a uniform porous structure was formed (figure 1a b), which eventually led to the formation of nanotubes (figure 1c). l shows the nanotubes after being carefully peeled off from the substrate using a diamond knife to observe their bottom and top morphologies. these images indicate a non - collapsed nanotube structure after anodization duration of 4 hours. no significant morphological difference can be seen between amorphous nanotubes (figures 1h and k) and crystalline nanotubes (figures 1i and l). in order to confirm the presence of atomic ordering in annealed nanotubes, transmission electron microscopy (tem) and x - ray diffraction (xrd) was utilized (figure 2). figure 2a shows a bright field tem image of amorphous nanotubes. the fuzzy diffraction pattern with no obvious spots of rings (the inset in figure 2a) indicates lack of crystallinity. high resolution tem imaging (figure 2b) confirms the absence of atomic ordering at nanoscale. similar tem imaging for annealed nanotubes (figure 2c and d) shows the presence of diffraction spots and atomic ordering at nanoscale which indicates formation of crystalline structures. the xrd data collected from amorphous and crystalline nanotubes (figure 2e) indicate that the crystalline nanotubes have an anatase phase. since the tem investigation of nanotubes grown on alloy - ti and cp - ti did not show any significant difference, the tem images shown in figure 2 are applicable for both. figure 3a f shows sem images of osteoblast cells after 72 hours of incubation on prepared substrates. figure 3 g shows a quantitative comparison on the effects of nanotube crystallinity and chemistry on cell density as a function of incubation times (24, 48, and 72 hours). following our observation that the crystallinity of the nanotubes is effective on the cell spreading, the efforts in this task were focused on better understanding the interaction of cells with amorphous and crystalline nanotubes. the filopodia extension and anchorage to the nanotubes is clear from figure 4, where it shows the interaction of mc3t3 osteoblast cells with tio2 nanotubes. a single cell sitting on the bed of cp - amorphous tio2 nanotubes is shown in figure 4a. an area close to cell filopodia in figure 4a was selected and enlarged to obtain figure 4b. as can be seen, figure 4c clearly shows the opening of the nanotubes and figure 4d shows that filopodia is grabbing the nanotubes as anchors for high attachment. these observations are represented as a schematic in figure 4e where cell layers are shown to grow on the surface, however, little filling inside the nanotubes can be seen. the interaction of osteoblast cells with crystalline nanotubes is documented in figure 4f j. figure 4f shows a single cell on the surface of cp - crystalline tio2 nanotubes. the enlarged image of the dotted box in figure 4f is shown in figure 4 g. as can be seen in this image, the cell has a much more spread out morphology compared to figures 4a and b. the well spreading is even more obvious from figure 4h which shows that filopodia of the cells enters inside the nanotubes and uses them as anchoring site to enhance spreading and attachment. figure 4i shows that filopodia has penetrated and grown inside the opening of the nanotubes and thus the opening of the nanotubes no longer remains open. these observations are summarized in the schematic figure 4j where it is shown that the cell layer grows inside the opening of the nanotubes resulting in their spreading and better attachment to the surface. in order to investigate the growth of filopodia within the hollow section of the amorphous and crystalline nanotubes,. low energy beam of ga ions was used to carefully cut through sections of cells and reveal the extent of interaction with the underlying nanotube surface. figure 5a shows an fib milled osteoblast cell that is attached to cp - amorphous tio2 nanotubes. closer examination demonstrates that the nanotubes have kept their morphology intact under the cells (figure 5b), while filopodia has not grown into the hollow section of the nanotubes (figure 5c). however, the free end of cp - crystalline nanotubes is clogged (figures 5d and e) and the cell filopodia has grown into the nanotubes (figure 5f). figure 6 shows sem images of the fib milled cells revealing that even after removal of the cell top section by fib the nanotubes remain clogged (figures 6a and b). the eds chemical analysis showed that the bond between the nanotubes and the adjacent osteoblast cell layer is composed of ca and p elements (figure 6c), which appears to mimic the bond in the bone tissue itself. figures 5 and 6 indicate that on the substrate surface, cell filopodia and the ecm remained attached following separation from the milled top section of the cell. these observations reveal a very close interfacial contact or direct contact between the osteoblasts and the nanotubes followed by ca and p elements deposition on the nanotubes (figure 6c). these elements are the primary components of bone matrix and their deposition onto nanotubes, which is an indication that nanotubes have maintained osteoblast functionality. the proposed morphological structure of deposited ca and p elements under sem imaging is in agreement with the conclusions made by das.41 the low intensity of the peaks is due to the fact that the eds data were taken after only 3 days of culture. in addition, it should be noted that the ca and p elemental peaks do not originate from the media. the concentration of ca and p elements in the media is much lower than other inorganic components (table 1). however, the eds analysis does not indicate any peaks regarding other elements such as potassium (k) and sodium (na). moreover, the samples were washed several times to eliminate any entrapped media in nanotubes before eds analysis. therefore, the only reason for the presence of ca and p elemental peaks is stimulation of cells by nanotube. following our observation that the crystallinity of the nanotubes is effective on the cell spreading, the efforts in this task were focused on better understanding the interaction of cells with amorphous and crystalline nanotubes. the filopodia extension and anchorage to the nanotubes is clear from figure 4, where it shows the interaction of mc3t3 osteoblast cells with tio2 nanotubes. a single cell sitting on the bed of cp - amorphous tio2 nanotubes is shown in figure 4a. an area close to cell filopodia in figure 4a was selected and enlarged to obtain figure 4b. as can be seen, figure 4c clearly shows the opening of the nanotubes and figure 4d shows that filopodia is grabbing the nanotubes as anchors for high attachment. these observations are represented as a schematic in figure 4e where cell layers are shown to grow on the surface, however, little filling inside the nanotubes can be seen. the interaction of osteoblast cells with crystalline nanotubes is documented in figure 4f j. figure 4f shows a single cell on the surface of cp - crystalline tio2 nanotubes. the enlarged image of the dotted box in figure 4f is shown in figure 4 g. as can be seen in this image, the cell has a much more spread out morphology compared to figures 4a and b. the well spreading is even more obvious from figure 4h which shows that filopodia of the cells enters inside the nanotubes and uses them as anchoring site to enhance spreading and attachment. figure 4i shows that filopodia has penetrated and grown inside the opening of the nanotubes and thus the opening of the nanotubes no longer remains open. these observations are summarized in the schematic figure 4j where it is shown that the cell layer grows inside the opening of the nanotubes resulting in their spreading and better attachment to the surface. in order to investigate the growth of filopodia within the hollow section of the amorphous and crystalline nanotubes, low energy beam of ga ions was used to carefully cut through sections of cells and reveal the extent of interaction with the underlying nanotube surface. figure 5a shows an fib milled osteoblast cell that is attached to cp - amorphous tio2 nanotubes. closer examination demonstrates that the nanotubes have kept their morphology intact under the cells (figure 5b), while filopodia has not grown into the hollow section of the nanotubes (figure 5c). however, the free end of cp - crystalline nanotubes is clogged (figures 5d and e) and the cell filopodia has grown into the nanotubes (figure 5f). figure 6 shows sem images of the fib milled cells revealing that even after removal of the cell top section by fib the nanotubes remain clogged (figures 6a and b). the eds chemical analysis showed that the bond between the nanotubes and the adjacent osteoblast cell layer is composed of ca and p elements (figure 6c), which appears to mimic the bond in the bone tissue itself. figures 5 and 6 indicate that on the substrate surface, cell filopodia and the ecm remained attached following separation from the milled top section of the cell. these observations reveal a very close interfacial contact or direct contact between the osteoblasts and the nanotubes followed by ca and p elements deposition on the nanotubes (figure 6c). these elements are the primary components of bone matrix and their deposition onto nanotubes, which is an indication that nanotubes have maintained osteoblast functionality. the proposed morphological structure of deposited ca and p elements under sem imaging is in agreement with the conclusions made by das.41 the low intensity of the peaks is due to the fact that the eds data were taken after only 3 days of culture. in addition, it should be noted that the ca and p elemental peaks do not originate from the media. the concentration of ca and p elements in the media is much lower than other inorganic components (table 1). however, the eds analysis does not indicate any peaks regarding other elements such as potassium (k) and sodium (na). moreover, the samples were washed several times to eliminate any entrapped media in nanotubes before eds analysis. therefore, the only reason for the presence of ca and p elemental peaks is stimulation of cells by nanotube. fe - sem images (figure 1) confirm formation of nanotubes on both cp - ti and alloy - ti. the nanotubes have an average diameter of 100 nm and an average length of 1 m. in agreement with the reports from macak only the al rich phase of ti alloy showed stable porous structures. the v rich phases etch faster and dissolve, leaving behind dark areas indicated by arrows in figure 1f. irrespective of the compositional elements in the substrate, the grown nanotubes are uniform and homogeneous. tem and xrd results (figure 2) show that right after anodization, as - anodized nanotubes have an amorphous structure which transforms into an anatase phase during annealing. fe - sem images of osteoblasts incubated over surfaces demonstrate that the presence of nanotubes on cp - ti substrate has been effective in increasing the number of attached cells (compare figure 3a with b). comparison between figures 3b and c shows a considerable increase in cell density and spreading for crystalline nanotubes. a similar observation can be made for the cells grown on an alloy - ti surface (figure 3d f) however the density of cells is much lower on alloy - ti. also, the cell density is always higher on cp - ti surfaces. the highest cell density (~2,500/mm) was obtained after 72 hours incubation of crystalline nanotubes grown on a cp - ti surface which is ~60% higher than amorphous nanotubes grown on cp - ti. the cell density after 72 hours of incubation on crystalline nanotubes grown on cp - ti is about 20% higher than similar nanotubes that were grown on alloy - ti. although the difference is not statistically significant, this observation is also consistent for the cells incubated for 24 and 48 hours suggesting that the chemistry of nanotubes can have an effect on cell density. the reduction in cell density on alloy - ti surfaces may be due to slightly lower biocompatibility of the alloying elements (al and v) and their oxides.42,43 in addition, the difference in cell density between the amorphous and crystalline nanotubes can be explained by hydrophilicity of the nanotubes as discussed in the next section. droplet contact angle is highly affected by surface treatments.44 after the anodization process, the droplet contact angles decreased in the range of 55%~70% compared to the bare substrate. this indicates that the creation of nanotube structures makes the surface more hydrophilic. remarkably, the crystalline nanotubes had the most hydrophilic properties among all the other substrates. on the crystalline nanotube substrates, water droplet contact angles decreased 40%~56% compared to the amorphous nanotube substrates. in general, annealing metal generates a hydrophobic surface.45 conversely, more hydrophilic surfaces were produced by annealing in this study which results in higher cell density. in view of nanotube structure, the crystalline nanotubes are more effective in enhancing cell growth compared with amorphous nanotubes probably due to increased hydrophilicity. the rationale behind such a conclusion is that nanotube surfaces enhance surface energy as well as hydrophilicity of the surface due to the presence of functional hydroxyl groups on the surface.44 considering that proteins are better absorbed on the hydrophilic tio2 nanotube surfaces, cell adhesion is enhanced. this hypothesis is in agreement with yao s study46 which reported that the initial adsorption of proteins on anodized nanotube ti was increased compared to anodized nanoparticles and un - anodized ti. the importance of the interaction between osteoblast filopodia and tio2 nanotubes has been the subject of several investigations.28,35,41 popat investigated the effect of ti nanotubes with different crystallographic structures on mc3t3-e1 osteoblast cells. they postulated that the cell filopodia grew inside the nanotubes, making a strong interlock between the cell and substrate. however, their discussion on interlocked interactions between the nanotubes and cell filopodia is not conclusive enough due to lack of any structural evidence. here, we have provided evidence of such an interlocked mechanism by means of sem and fib analysis. it is reasonable to conclude that the existence of atomic ordering in nanotubes and the resulting hydrophilicity would be essential to increase cell filopodia growth inside the nanotubes (figure 4). the investigation of cell filopodia interaction with nanotubes by means of sem has also been implemented by others. for instance, popat also reported the filopodia extension on the nanotubes based on high magnification sem image after 7 days of culture on a nanotubular surface. they discussed that their sem results show that cell extensions are protruding into the nanotubular architecture. nanotube interaction by means of sem to clarify the evidence of filopodia anchorage to nanotubes. based on this sem evidence, they reported that filopodia from human osteoblast cells use the nanoporous tio2 surface as anchorage sites for attachment. in addition, as can be seen from figure 5, the fib results revealed that the cells sustained very high vacuum without visible damage and they physically survived ion beam milling and imaging. moreover, ion milling revealed many internal features of cells at the submicron scale as well as the high interaction and growth of the cells among the nanotubes. this showed that fib and sem could be applied on dried biological material that is not prepared following standard cryopreservation methods, glutaraldehyde or a combination of glutaraldehyde and formaldehyde procedures.47 eds analysis results (figure 6) are in agreement with previous studies. popat also reported deposition of ca and p as the indication of bone matrix formation. yao also observed more ca deposition by osteoblasts on anodized nanotube - like ti compared with un - anodized ti. the results of the current investigation indicate the formation of ecm on the nanotubes initiated by deposition of ca and p.28 this production of ecm is the result of osteoblast migration and healthy cellular activity in both crystalline and amorphous nanotubular surfaces. in fact, it is possible to consider the nanotubes as anchors for the cell filopodia to grab onto and have a facilitated migration along the surface. this anchorage benefit of nanotubes together with the strong hydrophilic properties in crystalline nanotubes appear to have increased osteoblast density in annealed nanotubes and higher interactions with them. therefore, the observations made in the present investigation can be promising for the development of implants with high - cohesion osseointegration. the osteoblast cell culture experiments indicated that the presence of nanotube morphology increased the total cell density and spreading. this increase was correlated with the anchoring effect of nanotubes for the cell filopodia to grab onto, facilitating migration along the surface. the early deposition of ca and p onto nanotubes indicates that nanotubes have maintained osteoblast functionality. it was observed that the chemical composition of the substrate strongly affected the cell density. in particular, the total cell density of nanotubes grown on a cp - ti surface was higher in comparison to the cell density of nanotubes grown on an alloy - ti surface. this behavior can be explained by slightly lower biocompatibility of the alloying elements (al and v in this case) and their oxides. interestingly, the high surface wettability due to crystallinity is highly influential on further cell spreading and extension of the cell filopodia into the hollow space of nanotubes. in particular, the cell s filopodia extended into the crystalline nanotubes while much less penetration was observed for the cells grown on amorphous nanotubes. this was explained by the super hydrophilicity of crystalline nanotubes surfaces in comparison to amorphous nanotubes. the anchorage benefit of nanotubes together with their strong hydrophilic properties appear to cause increased osteoblast density in annealed nanotubular surfaces in comparison with anodized ones. this research can enable the determination of an optimal implant surface modification where implant bone interlock and bone - cell density is highest. | after the implantation of a biomaterial in the body, the first interaction occurs between the cells in contact with the biomaterial surface. therefore, evaluating the cell substrate interface is crucial for designing a successful implant. in this study, the interaction of mc3t3 osteoblasts was studied on commercially pure and alloy (ti6al4v) ti surfaces treated with amorphous and crystalline titanium dioxide nanotubes. the results indicated that the presence of nanotubes increased the density of osteoblast cells in comparison to bare surfaces (no nanotubes). more importantly, our finding shows that the chemistry of the substrate affects the cell density rather than the morphology of the cells. a novel approach based on the focused ion beam technique was used to investigate the biophysical cell substrate interaction. the analysis revealed that portions of the cells migrated inside the crystalline nanotubes. this observation was correlated with the super hydrophilic properties of the crystalline nanotubes. |
anatomical cerebral arterial variations are a very frequent finding in the general population due to the complex embryology of intracranial circulation. the most common normal variants of cerebral circulation include : fenestrations and duplications, persistent primitive fetal arteries, hypoplasia, and aplasia of arterial segments. moreover, the occlusion of an azygos or bihemispheric anterior cerebral artery (aca) may result in ischemia of both hemispheres. furthermore, patients with fetal origin of posterior cerebral artery (pca) and concomitant atherosclerotic disease of the carotid artery are prone to ischemic events in the pca territory. although the majority of normal variations have no major clinical impact, their appreciation may aid in surgical planning and can be useful in preventing complications during endovascular treatment. although digital subtraction angiography remains the standard reference procedure for detection of intracranial vascular variations, it is an invasive technique with potential complications. computed tomography angiography (cta) has emerged as an important noninvasive diagnostic tool in the evaluation of intracranial circulation. previous studies investigating radiation dose in cta examination reported the effective dose ranges between 0.6 and 1.2 msv depending on the scanning protocol. however, local doses in cta examination do not reach thresholds for the development of cataract formation, induction of thyroid malignancy, or hair loss. moreover, further developments, including reduced tube voltage and reducing scanning time, may provide a decrease in effective dose. cta allows reliable evaluation of intracranial arterial pathology, including aneurysms, stenosis, and occlusions. moreover, cta provides useful information about anatomical variations of cerebral circulation, with reported high sensitivity and specificity (8190% and 93%, respectively), approaching the diagnostic accuracy of digital subtraction angiography. at present, there are several reports regarding the use of cta for the assessment of intracranial arterial variations. however, these reports were mostly focused on fenestrations, duplications, and their association with aneurysm formation. nevertheless, the prevalence of some of the normal variants, such as azygos aca, hypoplastic vertebral artery terminating as posterior inferior cerebellar artery, and bihemispheric aca, has not been previously reported using cta. thus, the purpose of this study was to provide analysis of the prevalence and characteristic features of intracranial arterial variations and insight into associated vascular lesions. this prospective study was conducted between january 2012 and may 2013 and included 517 patients who underwent cta. most patients had or were suspected to have subarachnoid hemorrhage, ischemic cerebrovascular disease, or prolonged unexplained headache. sixty - two patients were excluded from the study due to suboptimal image quality, leaving 455 cta studies for detailed analysis. the study was approved by an institutional review board and written informed consent was obtained from all participants. cta examinations were performed at ge medical system (waukesha, wi, usa). images were obtained from c3 to the vertex using the following scanning parameters : detector rows, 16 ; collimation, 0.625 mm ; pitch, 1.375 ; gantry rotation time, 1.0 s ; slice thickness, 0.625 mm ; tube load, 380 ma ; and tube voltage, 120 kv. a total volume of 80100 ml of ultravist 370 was injected at a rate of 4.04.5 ml / s through an antecubital vein. the scan delay time was determined by a bolus tracking technique with a region of interest at 1 internal carotid artery. as soon as a threshold of 100 hu was exceeded, the spiral scan was automatically started. coronal and sagittal multiplanar reformatted, maximum intensity projections and 3d volume - rendered images were created at a ge advantage workstation. the demographic data, including sex, age at the time of presentation, and indication for the cta, were recorded. a prospective evaluation of ct examinations patients was performed independently by 2 radiologists (a.s. 15 years of experience) using axial and multiplanar reformatted images with attention to the presence of intracranial vascular variations : fenestrations, duplications, hypoplasia, aplasia, and persistent carotid - basilar anastomosis. independent - samples t - test was used for statistical evaluation. a value of p less than 0.05 was considered as significant. intracranial arterial fenestrations were found in 11 (2.4%) patients. the most common location was the vertebrobasilar system, with 5 (1.1%) fenestrations. the fenestrations of the basilar artery were observed in 4 patients (0.8%) and were located at the proximal segment with small slit - like configuration (figure 1a). the median length of the fenestrated segment was 11.6 mm, with 2 channels of 2.0 mm and 3.2 mm. the associated intracranial arterial variations were observed in 1 patient, who had aca trifurcation and bilateral fetal pca. vertebral artery fenestration was found in 1 patient (0.2%) (figure 1b), and was located at the v4 segment, with a length of 5.6 mm. the remaining fenestrations were found at the middle cerebral artery m1 segment in 1 patient (0.2%), anterior communicating artery (acoa) in 2 patients (0.4%), and aca a1 segment in 3 patients (0.6%) (figure 2a2c). the patient with fenestration of the middle cerebral artery had fetal pca as an associated variation. arterial duplication was observed in 2 patients (0.4%), who had duplicated acoa and duplicated pca p1 segment, respectively (figure 3a, 3b). the prevalence of an azygos aca was 1.5% (7 patients) (figure 4a). other associated vascular variations were fetal pca in 3 patients, and hypoplasia of an aca a1 segment in 2 patients. bihemispheric aca was noted in 4 patients (0.9%) (figure 4b). in 3 cases hypoplasia of the a1 segment was found in 17.6% of patients (figure 4a, 4b), while congenital absence of a1 segment was observed in 2 patients (0.4%) (figure 5a). among patients whit hypoplastic a1 segment, seven patients had associated vascular variants : 4 patients had hypoplastic a1 segment, and 3 patients had fetal pca. fetal origin of pca was found in 106 patients (23.3%), 51 cases on the left and 37 cases on the right, while bilateral location was present in 18 patients (figure 6). associated aneurysm was detected in 27 patients : 18 patients had an aneurysm of the middle cerebral artery, 4 patients of the internal cerebral artery c7 segment, and 5 patients of the acoa. hypoplastic vertebral artery terminating as posterior inferior cerebellar artery was observed in 9 cases (1.9%) (figure 7a). transversal anastomosis between vertebral arteries was seen in 1 patient (0.2%) (figure 7b). a lateral saltzman type 2 persistent trigeminal artery was found in 1 patient (0.2%) (figure 8). in our study population the aneurysms were located at acoa (n=39), middle cerebral artery (n=44), internal cerebral artery c7 segment (n=28), aca (n=2), and basilar artery (n=2). among patients with aneurysms, the rate of vascular variations was not significantly different in patients who had and did not have aneurysms (p>0.05). similarly, there was no significant difference in the rate of aneurysms among patients who had and did not have vascular variants (p>0.05). intracranial arterial fenestrations were found in 11 (2.4%) patients. the most common location was the vertebrobasilar system, with 5 (1.1%) fenestrations. the fenestrations of the basilar artery were observed in 4 patients (0.8%) and were located at the proximal segment with small slit - like configuration (figure 1a). the median length of the fenestrated segment was 11.6 mm, with 2 channels of 2.0 mm and 3.2 mm. the associated intracranial arterial variations were observed in 1 patient, who had aca trifurcation and bilateral fetal pca. vertebral artery fenestration was found in 1 patient (0.2%) (figure 1b), and was located at the v4 segment, with a length of 5.6 mm. the remaining fenestrations were found at the middle cerebral artery m1 segment in 1 patient (0.2%), anterior communicating artery (acoa) in 2 patients (0.4%), and aca a1 segment in 3 patients (0.6%) (figure 2a2c). the patient with fenestration of the middle cerebral artery had fetal pca as an associated variation. arterial duplication was observed in 2 patients (0.4%), who had duplicated acoa and duplicated pca p1 segment, respectively (figure 3a, 3b). the prevalence of an azygos aca was 1.5% (7 patients) (figure 4a). other associated vascular variations were fetal pca in 3 patients, and hypoplasia of an aca a1 segment in 2 patients. bihemispheric aca was noted in 4 patients (0.9%) (figure 4b). in 3 cases hypoplasia of the a1 segment was found in 17.6% of patients (figure 4a, 4b), while congenital absence of a1 segment was observed in 2 patients (0.4%) (figure 5a). among patients whit hypoplastic a1 segment, 25% had an associated aneurysm of acoa. seven patients had associated vascular variants : 4 patients had hypoplastic a1 segment, and 3 patients had fetal pca. fetal origin of pca was found in 106 patients (23.3%), 51 cases on the left and 37 cases on the right, while bilateral location was present in 18 patients (figure 6). associated aneurysm was detected in 27 patients : 18 patients had an aneurysm of the middle cerebral artery, 4 patients of the internal cerebral artery c7 segment, and 5 patients of the acoa. hypoplastic vertebral artery terminating as posterior inferior cerebellar artery was observed in 9 cases (1.9%) (figure 7a). transversal anastomosis between vertebral arteries was seen in 1 patient (0.2%) (figure 7b). a lateral saltzman type 2 persistent trigeminal artery was found in 1 patient (0.2%) (figure 8). the aneurysms were located at acoa (n=39), middle cerebral artery (n=44), internal cerebral artery c7 segment (n=28), aca (n=2), and basilar artery (n=2). among patients with aneurysms, the rate of vascular variations was not significantly different in patients who had and did not have aneurysms (p>0.05). similarly, there was no significant difference in the rate of aneurysms among patients who had and did not have vascular variants (p>0.05). although most of these variations have no significant clinical importance, some may predispose the patient to development of aneurysms or ischemic events. moreover, their recognition on cta examinations is important for surgical and endovascular treatment planning. fenestration consists of 2 arterial channels that fuse at the distal end and develop as a consequence of fusion failure during early gestation. the most common site of fenestration is the vertebrobasilar system, with high prevalence reported in previous studies [810 ]. reported a 2.8% overall rate of intracranial vertebrobasilar fenestrations and observed intracranial vertebral artery fenestrations in 0.40% of patients, consistent with the 0.38% found in the current study. however, the current study result of 0.8% ba fenestrations is significantly lower than the previously observed prevalence, and was closer to the results of the conventional angiographic studies (0.6%) reported by takahashi.. this discrepancy might be attributable to the larger study population and different clinical diagnoses of selected patients in prior ct and mr angiographic studies. due to the complex embryology and anatomy of the anterior communicating system, fenestrations and duplications in this region are frequent findings. ct angiographic prevalence of anterior communicating region fenestrations was reported to be 5.36.9%, but the frequency of anterior communicating region fenestration in our study was much lower (1.2%). the difference is likely due to the different inclusion criteria concerning clinical indications, and inclusion of more patients with normal cta findings in the present study. to our knowledge although the observed frequency of this variation in previous anatomic reports is very high (18%), it was detected in only 1 patient (0.2%) in our study population. pathologically, it could be explained by defects in the vessel wall at each end of the fenestration, which results in hemodynamic stress and structural degenerative changes in the vessel. in this regard, bayrak. found that 27.5% of patients with fenestrations had aneurysms, but only 2 patients had an aneurysm at the fenestration site. furthermore, no significant difference was found in the rate of aneurysms between patients with and without fenestrations. in the present study, aneurysms were detected in 29% of patients. among these patients, 52% had associated vascular variations, but none had fenestrations. on the basis of these results, it could be assumed that although fenestration is recognized as a risk for aneurysmal development, significant association could not be established. previous studies concerning the frequency and clinical significance of an azygos aca are numerous [1416 ]. this vascular variation represents a single midline a2 trunk that supplies blood to both hemispheres. ct angiographic findings have not been previously published, but closely correlate with these results according to the present study (1.2%). since there is a single a2 trunk, its occlusion may result in ischemia of both hemispheres. the same clinical significance has bihemispheric aca, where one a2 segment is hypoplastic, and the contralateral a2 segment supplies the medial portions of both hemispheres. to date, the prevalence of this vascular variant has not been reported on cta, and was found to be 1% in our study, which is in accordance with prior anatomic reports (18). among all aca variations, moreover, we found an associated acoa aneurysm in 25% of these patients. since an acoa aneurysm frequently occurs in patients with these patients, it is of great clinical importance to carefully examine contralateral the aca - acoa junction on cta examinations. our results are consistent with the findings of anatomic studies, reporting that the a1 segment is seldom absent. the least significant aca variation is its trifurcation or persistent median artery of the corpus callosum, and was observed in 2.2% of our patients. the results from the present study are in accordance with those of uchino., who found this anomaly in 3% of their study population. fetal origin of pca is a very common vascular variant, in which the caliber of the pca is the same or greater than that of the ipsilateral p1 segment. it occurs when embryonic pca does not regress, resulting in the persistence of internal carotid artery blood supply to the occipital lobe. consistent with previous reports, the prevalence of this arterial variant was 37% in our study, occurring more commonly on the right side and with a slight male predominance. such a fetal anastomosis may predispose patients to an ischemic event in pca, since it allows thrombo - embolic material from the carotid artery to pass into the pca. the intracranial part of vertebral arteries is frequently the site of various vascular variations, with fenestrations, duplications, and aneurysms being the most common. although the prevalence of vertebrobasilar system fenestrations and duplications has been previously extensively studied, the number of ct and mr angiographic reports describing other vascular anomalies is very limited. in the current study, a transversal intervertebral anastomosis beneath the vertebrobasilar junction was observed in 1 patient. to the best of our knowledge, this is the first report of such a case diagnosed by cta. this unusual variant could be the result of fusion of anterior spinal artery branches, as hypothesized in previous anatomic studies. although the prevalence of anterior spinal artery originating from transverse intervertebral anastomosis was reported to be 6.3%, it was not detected in our study. however, due to the small diameter of this artery, the visualization of anterior spinal artery is not an obligatory finding on cta. hypoplastic vertebral artery terminating as posterior inferior cerebellar artery is a rare variation, detected in 0.21.3% of the population. since there is no connection between vertebral arteries, the compression or occlusion of one of these arteries can lead to ischemia of the cerebellum or lateral medulla. although this vascular variant has many clinical implications, its significance and prevalence have not been previously reported in ct and mr angiography studies. in the present study, hypoplastic vertebral artery terminating as posterior inferior cerebellar artery the main limitation of this study was the absence of conventional angiography findings as a standard reference procedure. moreover, most of examined patients had or were thought to have ischemic or hemorrhagic cerebrovascular disease, which could have introduced some selection bias. in addition, the relatively small number of patients included in the study precludes generalized conclusions. the results of our study provide comprehensive evaluation of intracranial vascular variations using cta, reporting the prevalence of a few of them for the first time. furthermore, this study demonstrated that the rate of aneurysms was not significantly different in patients who had and did not have vascular variants. | backgroundintracranial arterial variations are a frequent finding in the general population. knowledge of these vascular variations has significant clinical impact because some of them predispose patients to development of an aneurysm or cerebrovascular ischemic disease. the purpose of this study was to evaluate the frequency of intracranial vascular variations and associated vascular lesions on computed tomography angiography (cta) examinations.material/methodscta examinations performed by 16-detector computed tomography were prospectively reviewed in 455 patients for the presence of fenestrations, duplications, hypoplasia, aplasia, aneurysms, and other vascular lesions.resultsarterial fenestrations were found in 2.4% of patients, with the vertebrobasilar system as the most common location. the remaining fenestrations were located on the middle cerebral artery m1 segment (0.2%), anterior communicating artery (0.4%), and anterior cerebral artery a1 segment (0.6%). no associated aneurysms were noted in these patients. the prevalence of an azygos anterior cerebral artery was 1.5%. bihemispheric anterior cerebral artery was found in 0.9%, hypoplastic a1 segment in 17.6%, and congenital absence of a1 segment in 0.4% of patients. fetal origin of the posterior cerebral artery was found in 37% of cases. hypoplastic vertebral artery terminating as posterior inferior cerebellar artery was observed in 9 patients, while transversal anastomosis between vertebral arteries was seen in only 1 patient.conclusionscta precisely demonstrates the diversity of intracranial arterial variations, whose overall frequency in this study is similar to previous radiological reports. furthermore, our results do not show significant association between the frequency of aneurysms and cerebral arterial anomalies. |
clefts of the lip, alveolus and palate are among the most common congenital malformation of the head and neck. the prevalence per 1000 total birth of cleft lip and/or with cleft palate ranges from around two in mongoloid populations to about 0.5 in negroid groups, and in caucasian populations the prevalence is about 1.2 per 1000 total birth. the problems / challenges associated with cleft lip and palate deformities vary depending on the degree and location of the defect. these include feeding difficulties, speech and language delays, ear infections / hearing loss, aesthetic problem, dental anomalies, psychosocial problems and reduced quality of life.[35 ] to correct the problems, these defects need to be repaired as soon as the patient is fit for surgery. in our institution, patients undergoing clp surgery and other maxillofacial surgical procedures have traditionally been required to look for a replacement donor to donate at least 1 unit of homologous blood before surgery and this is preoperatively cross - matched, in case significant blood loss is expected. this practice is expensive, time consuming, and may in several cases, be unnecessary. the over - ordering of cross - matched blood to cover operation can result in blood shortages. a number of surgical procedures including hysterectomy, colostomy, thyroidectomy and a few others have been shown to seldom require transfusion, and blood is not usually cross - matched and tied down. however, for these procedures, the policy of " type and screen " is usually adopted such that blood can be quickly made available if for any reason the surgical intervention eventually demands a blood transfusion. the procedure of type and screen requires that : i) the abo and rhesus type of the patient is determined ; ii) the patient serum is also screened for presence of unexpected allo - antibodies. if the patient does not have any allo - antibody it will be safe to select abo and rhesus donor blood compatible with the patient if there is a need for transfusion during or following surgery without a necessity for cross - matching. such a non - cross - matched abo and rhesus identical blood can be released with 99.9% assurance of safety as long as the patient has no unexpected antibody. the aim of this study was to determine the frequency of homologous blood transfusion in patients undergoing cleft lip and/or palate surgery at the lagos university teaching hospital, nigeria, with a view to setting a guideline for blood transfusion protocol in clp surgical procedures. one hundred consecutive patients who had clp surgery done at the lagos university teaching hospital between march 2007 and november 2008 were recruited into the study. data collected included age, sex and weight of patients, type of cleft defects, type of surgery done, preoperative haematocrit, duration of surgery, amount of blood loss during surgery, and amount of blood transfused. induction was either inhalational with incremental halothane or intravenous using thiopentone or propofol. when the depth of anaesthesia was judged to be adequate, the trachea was intubated with the appropriate sized south - polar (for cleft lip repair only) or re - inforced (for palatal surgery) endotracheal tube under deep inhalational anaesthesia or muscle relaxants using external laryngeal pressure if needed. maintenance of anaesthesia was with isoflurane and all patients were ventilated after administration of a muscle relaxant. unilateral cleft lip was repaired using either millard 's rotation advancement technique or tennison - randall triangular technique. bilateral cleft lip was repaired using fork technique, and cleft palate repair was done with von langenbeck technique. each patient was made to look for a replacement donor to donate 1 unit of blood prior to surgery. for all the patients, 1 unit of homologous blood was cross - matched for the surgery. blood loss was calculated by weighing gauze, measuring suctioned blood, and adjusting for the volume of irrigation solution used during the operation. afterward, cross - match - to - transfusion ratio (the index of efficiency of ordering and usage), transfusion index (average number of units transfused for a given procedure) and probability of transfusion were calculated. blood volume was calculated for each patient and percentage blood volume loss was calculated data was analysed using the spss for windows (version 12.0 ; spss inc, chicago, il) statistical software package ; and presented in descriptive and tabular forms. induction was either inhalational with incremental halothane or intravenous using thiopentone or propofol. when the depth of anaesthesia was judged to be adequate, the trachea was intubated with the appropriate sized south - polar (for cleft lip repair only) or re - inforced (for palatal surgery) endotracheal tube under deep inhalational anaesthesia or muscle relaxants using external laryngeal pressure if needed. maintenance of anaesthesia was with isoflurane and all patients were ventilated after administration of a muscle relaxant. unilateral cleft lip was repaired using either millard 's rotation advancement technique or tennison - randall triangular technique. bilateral cleft lip was repaired using fork technique, and cleft palate repair was done with von langenbeck technique. each patient was made to look for a replacement donor to donate 1 unit of blood prior to surgery. for all the patients, 1 unit of homologous blood was cross - matched for the surgery. blood loss was calculated by weighing gauze, measuring suctioned blood, and adjusting for the volume of irrigation solution used during the operation. afterward, cross - match - to - transfusion ratio (the index of efficiency of ordering and usage), transfusion index (average number of units transfused for a given procedure) and probability of transfusion were calculated. data was analysed using the spss for windows (version 12.0 ; spss inc, chicago, il) statistical software package ; and presented in descriptive and tabular forms. there were 52 females and 48 males with a mean age of 64.4 101.1 months (range, 3 - 420 months). the most common cleft defect was isolated cp (45%) followed by unilateral cleft lip (28%) [table 1 ]. cp repair was the most common procedure (45%) followed by unilateral cleft lip repair ; ucl (41%) [table 2 ]. a hundred units (1 unit per patient) of homologous blood were donated and cross matched for surgery. types of cleft defects transfusion rate in different types of cleft surgery preoperative haematocrit values ranged between 22 and 43% (mean=30.8%, sd= 4). the mean estimated blood loss was 95.8 144.9 ml (range, 2 - 800ml). the mean estimated blood loss in unilateral cleft lip surgery was not significantly different from that of bilateral cleft lip surgery (p=0.22) but significantly lower than those of cleft palate surgery and combined cleft lip / palate surgery (p=0.000). types of cleft surgery and blood loss ten (10%) patients (cl=2 ; cp=5, bcl=1 ; clp=2) had blood transfusion. the mean blood transfused was 131.5 135.4ml (range, 35 - 500ml). in nine of the 10 patients, volume of blood transfused ranged between 35 and 140ml. only one female adult patient (cp) who lost 800ml of blood received 1 unit (500ml) of blood perioperatively [table 4 ]. only 4.9% of patients who had unilateral cleft lip surgery were transfused as compared with 50% for clp surgery, 11% for cp surgery, and 10% for bilateral cleft lip surgery. there was no significant difference in transfusion rate in patients with preoperative haematocrit of less than 30% and those with more than 30% [table 5 ]. characteristics of patients who were transfused transfusion rate and preoperative hematocrit value estimated duration of surgery ranged between 20 minutes and 240 minutes (mean=91.6 sd=37.2 minutes). the duration of surgery in cleft lip procedures was significantly lower than those of cleft palate and combined cleft lip and palate procedures (p=0.001). simple regression analysis showed significant positive correlation between duration of surgery and blood loss (n=100, r=0.472, adjusted r = 0.215, p=0.000). table 6 shows the estimated blood volume, amount of blood loss and appropriateness or otherwise of blood transfusion. the cross - match - to - transfusion ratio was 10 overall, 20.5 for unilateral cleft lip surgery and 2.0 for cleft lip and palate surgery [table 7 ]. the transfusion index for unilateral cleft lip surgery was 0.05 and 0.5 for cleft lip and palate surgery [table 7 ]. the overall probability of transfusion and blood - ordering quotient in cleft lip and/or palate surgery was 2% and 10 respectively. appropriateness of blood transfusion based on patients ' blood volume and blood loss ebv = estimated blood volume values were calculated based on units of blood opened for transfusion the ultimate goal of evidence based clinical research is to formulate a clinical practice guideline. to date, there are no reports on transfusion rate in cleft lip and/or palate in the literature. however, many authors have reported rate of blood transfusion in other more extensive maxillofacial surgical procedures to be low. therefore, there is a need to ascertain the need for blood transfusion in cleft lip and/or palate surgery. the 2000 - 2001 annual report of the serious hazards of transfusion (shot) steering group (united kingdom) highlighted the serious hazards of blood transfusion. most morbidity or mortality results from giving the wrong blood or blood products rather than transmission of infection. the national health service, united kingdom executive paper on " better blood transfusion " recommends that blood should only be given when necessary to save life or prevent deterioration, not simply to hasten recovery. ordering of too much cross - matched blood to cover operations can result in shortages of blood, wasted laboratory time, and it is also costly. risk free transfusion whether it be homologous or autogenous blood does not exist. in the present study, the overall mean estimated blood loss was 95.8 144.9 ml ; and most patients (77%) lost between 2 - 100ml of blood during surgery. hence, clp surgery may be classified a " small / low volume " blood loss surgery. estimated blood loss in unilateral cleft lip surgery was not significantly different from that in bilateral cleft lip surgery ; but was significantly lower than that in cleft palate surgery and combined cleft lip / palate surgery. estimated duration of unilateral / bilateral cleft lip surgery was significantly lower than those of cleft palate surgery and combined cl / p surgery ; and a significant positive correlation was found between duration of surgery and blood loss. it is noteworthy that the local anaesthetic agent (lignocaine) containing 1:200,000 adrenaline was routinely injected before surgical incisions were made in all cases performed in the present study. the use of vasoconstrictor agent to reduce bleeding is a recommended approach to the conservation of blood. other recommended approaches to reduction of blood loss during surgery include hypotensive anaesthesia, 20 - 30 head up tilt, perioperative use of tranexamic acid, and ligation and diathermy of vessels. a head up tilt of the patient improves diaphragmatic function and respiratory status while reducing venous return, leading to a fall in cardiac output and mean arterial pressure. in the present study, frequency of transfusion in cleft lip and/or palate surgery was 10%. the transfusion rate was highest in combined cleft lip and palate surgery (50%) and cleft palate surgery (11%) and lowest in unilateral (4.9%) and bilateral cleft lip (10%) surgeries. blood - ordering tariffs can be calculated using the principle that those operations that rarely require blood can be safely done when policy of type and screen is adopted. rationalised ordering of blood is safe and also saves money. in view of low blood transfusion rate in cleft lip surgery, preoperative homologous blood donation and cross - match for cleft lip surgery especially in children with hb of 10g / dl this study also demonstrated that there is no indication to cross - match blood for all patients undergoing cleft lip and palate surgery. it should be sufficient to type and screen blood preoperatively for cleft palate surgery ; this can be made available quickly if required. in patients with low haematocrit undergoing cleft lip surgery, a transfusion need may be predicted and have a cross - match performed preoperatively. pre - operative grouping and cross - matching of blood may be necessary in case of combined cleft lip and palate surgery. in the present study, there was no significant difference in transfusion rates in patients with preoperative haematocrit of less than 30% and those with more than 30%. this implies that homologous blood transfusion in the present series was not dependent on preoperative haematocrit value. a patient who had underwent cleft lip repair and who had the lowest preoperative haematocrit (22%) in the present study, received no blood transfusion. this can be ascribed to the fact that with a preoperative low haematocrit, the amount of red cells per unit volume of blood lost during surgery is minimal and this forms the rationale for presurgical haemodilution. length of surgery, based on the findings in this study, seems a greater risk factor for blood transfusion rather than low haematocrit in an otherwise healthy patient. measures like acute normovolaemic haemodilution, being currently explored, and which may be of use in adult cleft palate surgery show great promise. however, they incur additional cost and require additional time for the collection and storage. the cross - match : transfusion ratio (ctr), which is the number of units cross - matched for a procedure divided by the total number of units transfused, and is an index of the efficiency of ordering and use of blood, and it should be less than 2.5. in this study, ctr was 20.5, 10.0, 9.0 and 2.0 for unilateral cleft lip, bilateral cleft lip, cleft palate and combined cleft lip and palate surgeries respectively. the transfusion index (ti) is a measure of the amount of blood used for a given procedure. a value of less than 0.5 suggests that cross - matched blood is unlikely to be required. in the present study, ti was 0.05, 0.1, 0.1 and 0.5 for unilateral cleft lip, bilateral cleft lip, cleft palate and combined cleft lip and palate surgeries respectively. pot is a ratio of the number of appropriate transfusions for the total number of operations. the pot of 2% in the present study supports a group and save policy for clp (and/or) surgery. the blood - ordering quotient (boq) is the number of cross - matched units of blood per patient divided by the number of units transfused per patient. the value of the ctr as a gross determination of over - ordering has been challenged, and the use of boq recommended. a boq above 1.5 is considered unacceptable and it is advised that these procedures be grouped and saved. boq was 20.5, 10.0, 9.0 and 2.0 for unilateral cleft lip, bilateral cleft lip, cp and combined clp surgeries respectively. these values were not different from ctr values because all the patients had 1 unit of blood cross matched preoperatively and kept ready. of the 10 patients who had blood transfusion, transfusion was deemed inappropriate in eight and appropriate in two based on percentage blood volume loss. available evidence does not support the use of single criterion for transfusion such as haemoglobin concentration of < 10 g / dl. no single measure can replace good clinical judgment and accurate monitoring in the peri - operative period. the decision to transfuse should take into consideration the expected level and duration of anaemia, the intravascular volume, the duration of operation and the probability of massive blood loss. in the present series, it was suspected that in a few circumstances, blood was given by a junior (trainee) anaesthetist just because it was available. if transfusion was done for reason of volume depletion, and not for oxygenation, crystalloid should have sufficed in most cases. but, if blood was transfused due to reduced oxygenation (reduced haemoglobin concentration), and not for volume depletion, packed red cell would have sufficed. the risk associated with transfusion has been reported to be less in packed red cell than in whole blood. there is less risk of volume overload, plasma antigens load and also allergic reactions. patients were most commonly transfused for cleft palate surgery and least commonly transfused for cleft lip surgery. in view of low blood transfusion rate in cleft lip surgery, preoperative homologous blood donation for cleft lip surgery especially in children with hb of 10g / dl this study demonstrates that there is no indication to cross - match one unit of blood for all patients undergoing cleft lip and palate surgery. it is sufficient to group and save one unit of blood preoperatively for cleft palate surgery ; this can be made available quickly if required. in patients with low haematocrit, a transfusion need | aim : the study aims to determine the frequency of homologous blood transfusion in patientsundergoing cleft lip and palate surgery at the lagos university teaching hospital, nigeria.setting and design : a prospective study of transfusion rate in cleft surgery conducted at the lagos university teaching hospital, nigeria.material and methods : one hundred consecutive patients who required cleft lip and palate surgery were recruited into the study. data collected included age, sex and weight of patients, type of cleft defects, type of surgery done, preoperative haematocrit, duration of surgery, amount of blood loss during surgery, the number of units of blood cross - matched and those used. each patient was made to donate a unit of homologous blood prior to surgery.results:there were 52 females and 48 males with a mean age of 64.4 101.1 months (range, 3 - 420 months). the most common cleft defect was isolated cleft palate (45%) followed by unilateral cleft lip (28%). cleft palate repair was the most common procedure (45%) followed by unilateral cleft lip repair (41%). the mean estimated blood loss was 95.8 144.9 ml (range, 2 - 800ml). ten (10%) patients (cl=2 ; cp=5, bcl=1 ; clp=2) were transfused but only two of these were deemed appropriate based on percentage blood volume loss. the mean blood transfused was 131.5 135.4ml (range, 35 - 500ml). six (60%) of those transfused had a preoperative pcv of < 30%. only 4.9% of patients who had unilateral cleft lip surgery were transfused as compared with 50% for clp surgery, 11% for cp surgery, and 10% for bilateral cleft lip surgery.conclusions:the frequency of blood transfusion in cleft lip and palate surgery was 10% with a cross - match : transfusion ratio of 10 and transfusion index of 0.1. a " type and screen " policy is advocated for cleft lip and palate surgery. |
the influenza pandemic of 19181919, now known to have been caused by an h1n1 virus, was characterised by global spread, multiple waves and high mortality. the disease was particularly severe among younger adults, possibly because they were immunologically nave to the new virus, while older adults had some protective immunity. the great war (19141918) affected population mixing, contributing to widespread transmission of influenza in most parts of england and wales, where three welldefined waves were described. the first wave was unusual, in that it began in summer in late june 1918, rather than in the colder months typical of interpandemic (seasonal) influenza., although this outofseason onset indicates an unusual level of population susceptibility, the mortality rate was relatively low at around 003%. the autumn wave (wave 2) was most severe in terms of clinical complications and mortality, with an average mortality rate of 027% across regions of england and wales. the third (winter) wave was less severe, with an average mortality of 01%. areas experiencing higher rates of allcause mortality in earlier years typically experienced higher mortality from pandemic influenza,, presumably reflecting social disadvantage., pandemic mortality in other european cities was also exacerbated by overcrowding and poverty. variation in pandemic mortality between cities likely reflect the influence of social, geographical and climatic factors affecting viral spread, changes in viral virulence, as well as effects of population immunity., in the united states, public health interventions designed to reduce viral transmission apparently reduced pandemic mortality in some cities. however, there is no evidence that similar social distancing measures were formally introduced in england and wales in 19181919. we previously showed that attack rates for waves 1 and 2 of pandemic influenza in 19181919 in raf camps in the uk could be explained by a model incorporating prior immunity, a high rate of asymptomatic infection, and waning of immunity (or antigenic drift of the virus) between waves. to better understand how pandemic spread and mortality was affected by social and geographical factors, and by population immunity, we now reanalyse 19181919 influenza mortality rates from 333 cities and rural boroughs in england and wales for each of the three waves. we used weekly influenzaspecific mortality counts and annualised rates/1000 population for the weeks ending 29 june 1918 to 10 may 1919, collated by the registrar general s office in 1920. mortality data were available for 333 administrative units (populations) : 29 london boroughs, 84 metropolitan boroughs, 77 urban districts, 82 county boroughs, and 61 others, comprising county remainders or counties with no specified urban centre. population totals for each unit were calculated from the cumulative rates and numbers of deaths over the 46week period. acreages were obtained from the decennial 1931 census for england and wales, and population density was calculated as persons / acre, further categorised into quintiles and modelled as an ordinal variable. allcause mortality rates/1000, agestandardised by the direct method, were available for 329 matched administrative units, and averaged over 19111914 for men and women combined. also available from this source was the standardising factor for each population, calculated as the ratio of agestandardised to crude death rates. in the absence of population age structures within administrative units, we used this factor to represent variation in age structure between administrative unit populations, with higher values indicating younger populations. latitude and longitude coordinates assigned to geographical areas, such that coordinates approximated county centroids and urbanised areas were within designated historical county boundaries, were obtained from the lat long finder website (map data 2010 tele atlas, powered by google, url http://www.satsig.net/maps/latlongfinder.htm). the average of ward scores from the indices of deprivation 2000 : district level presentations for england was used as an indicator of current socioeconomic deprivation at district level [higher scores indicating greater deprivation ], and matched by name to 62/82 (76%) of 19181919 counties as identified in johnson. we used digitised historical 1921 county boundary data for england and wales, with geographical coordinate system osgb 1936 in esri shape file format, to assess coordinates of geographical areas and outline contour plots of week of onset and influenza mortality. the minimum total deaths between waves 1 and 2 occurred in week 12 (week ending 14 september 1918) and in week 31 (week ending 25 january 1919) between waves 2 and 3 (figure 1). for each administrative unit, cumulative deaths for wave 1 were summed from the start of data collection to the weekly minimum deaths within 1 week of the overall minimum deaths between waves 1 and 2. cumulative deaths for wave 2 were summed from these minima to the weekly minimum deaths within 1 week of the overall minimum between waves 2 and 3 ; and for wave 3, from these minima to the end of data collection. waves were thus defined to include all recorded influenza deaths between data collection start and end points, with betweenwave demarcation defined by minima for each administrative unit population. cumulative influenza mortality rates for each wave were calculated as the total deaths occurring/1000 population, and a measure of overall mortality was obtained by summing across waves. for each administrative unit, wave onset was defined as the week marking the 10th percentile of deaths from wave commencement ; and wave duration was defined as the number of weeks between the 10th and 90th percentile of deaths, inclusive, per wave. betweenwave intervals were calculated as the number of weeks between the 50th percentile deaths for each wave. time series plot of influenza mortality between the weeks ending 29 june 1918 and 10 may 1919 in england and wales, indicating schematically weeks of overall minimum deaths, wave duration and betweenwave intervals for each of 333 administrative units. geographical spread of influenza was assessed by correlating latitude and longitude coordinates for each population with measures of timing and mortality in each wave. betweenwave assessments used anova (friedman s test) and the wilcoxon signedrank test for matched data. multivariate linear regression models, by ordinary least squares, used population size as an analytical weight. quadratic trend surfaces (first and secondorder terms in latitude and longitude, and the interaction of firstorder terms) were fitted in regression models to represent spatial trends (see table 4). standardised beta coefficients were reported to allow comparison of contributions of individual explanatory variables. statistical analyses were performed using stata (version 10.1 intercooled, stata corporation, college station, tx, usa), and variables describing wave characteristics were derived using matlab r2009a (mathworks, natwich, ma). contour plots of cumulative influenza mortality and week of onset, clipped to digitised map boundaries of england and wales, were produced by applying a radial basis function to a raster generated by applying cubic inverse distance weighted interpolation to the nonuniformly spaced data points using arcinfo version 9.3 (environmental systems research institute, inc., california, usa). weighted regression model output for predictors of cumulative influenza mortality/1000 by wave (n = 329) likelihood ratio test comparing model with quadratic trend surface only with model with additional risk factors. natural logarithmic transform (ln). risk factors adjusted for geographical variation modelled as a quadratic trend surface using latitude and longitude coordinates to represent location. likelihood ratio test (lr) for model with interaction term compared to model without : lr 234, p < 0001. interaction terms wave 1 mortality younger age and wave 2 mortality younger age were not statistically significant ; lr 049, p = 0782. we used weekly influenzaspecific mortality counts and annualised rates/1000 population for the weeks ending 29 june 1918 to 10 may 1919, collated by the registrar general s office in 1920. mortality data were available for 333 administrative units (populations) : 29 london boroughs, 84 metropolitan boroughs, 77 urban districts, 82 county boroughs, and 61 others, comprising county remainders or counties with no specified urban centre. population totals for each unit were calculated from the cumulative rates and numbers of deaths over the 46week period. acreages were obtained from the decennial 1931 census for england and wales, and population density was calculated as persons / acre, further categorised into quintiles and modelled as an ordinal variable. allcause mortality rates/1000, agestandardised by the direct method, were available for 329 matched administrative units, and averaged over 19111914 for men and women combined. also available from this source was the standardising factor for each population, calculated as the ratio of agestandardised to crude death rates. in the absence of population age structures within administrative units, we used this factor to represent variation in age structure between administrative unit populations, with higher values indicating younger populations. latitude and longitude coordinates assigned to geographical areas, such that coordinates approximated county centroids and urbanised areas were within designated historical county boundaries, were obtained from the lat long finder website (map data 2010 tele atlas, powered by google, url http://www.satsig.net/maps/latlongfinder.htm). the average of ward scores from the indices of deprivation 2000 : district level presentations for england was used as an indicator of current socioeconomic deprivation at district level [higher scores indicating greater deprivation ], and matched by name to 62/82 (76%) of 19181919 counties as identified in johnson. we used digitised historical 1921 county boundary data for england and wales, with geographical coordinate system osgb 1936 in esri shape file format, to assess coordinates of geographical areas and outline contour plots of week of onset and influenza mortality. the minimum total deaths between waves 1 and 2 occurred in week 12 (week ending 14 september 1918) and in week 31 (week ending 25 january 1919) between waves 2 and 3 (figure 1). for each administrative unit, cumulative deaths for wave 1 were summed from the start of data collection to the weekly minimum deaths within 1 week of the overall minimum deaths between waves 1 and 2. cumulative deaths for wave 2 were summed from these minima to the weekly minimum deaths within 1 week of the overall minimum between waves 2 and 3 ; and for wave 3, from these minima to the end of data collection. waves were thus defined to include all recorded influenza deaths between data collection start and end points, with betweenwave demarcation defined by minima for each administrative unit population. cumulative influenza mortality rates for each wave were calculated as the total deaths occurring/1000 population, and a measure of overall mortality was obtained by summing across waves. for each administrative unit, wave onset was defined as the week marking the 10th percentile of deaths from wave commencement ; and wave duration was defined as the number of weeks between the 10th and 90th percentile of deaths, inclusive, per wave. betweenwave intervals were calculated as the number of weeks between the 50th percentile deaths for each wave. time series plot of influenza mortality between the weeks ending 29 june 1918 and 10 may 1919 in england and wales, indicating schematically weeks of overall minimum deaths, wave duration and betweenwave intervals for each of 333 administrative units. geographical spread of influenza was assessed by correlating latitude and longitude coordinates for each population with measures of timing and mortality in each wave. betweenwave assessments used anova (friedman s test) and the wilcoxon signedrank test for matched data. multivariate linear regression models, by ordinary least squares, used population size as an analytical weight. quadratic trend surfaces (first and secondorder terms in latitude and longitude, and the interaction of firstorder terms) were fitted in regression models to represent spatial trends (see table 4). standardised beta coefficients were reported to allow comparison of contributions of individual explanatory variables. statistical analyses were performed using stata (version 10.1 intercooled, stata corporation, college station, tx, usa), and variables describing wave characteristics were derived using matlab r2009a (mathworks, natwich, ma). contour plots of cumulative influenza mortality and week of onset, clipped to digitised map boundaries of england and wales, were produced by applying a radial basis function to a raster generated by applying cubic inverse distance weighted interpolation to the nonuniformly spaced data points using arcinfo version 9.3 (environmental systems research institute, inc., weighted regression model output for predictors of cumulative influenza mortality/1000 by wave (n = 329) likelihood ratio test comparing model with quadratic trend surface only with model with additional risk factors. natural logarithmic transform (ln). risk factors adjusted for geographical variation modelled as a quadratic trend surface using latitude and longitude coordinates to represent location. likelihood ratio test (lr) for model with interaction term compared to model without : lr 234, p < 0001. interaction terms wave 1 mortality younger age and wave 2 mortality younger age were not statistically significant ; lr 049, p = 0782. wave duration increased significantly over successive waves (friedman s and wilcoxon signedrank tests p < 0001). cumulative mortality was significantly higher in wave 2 than in waves 1 and 3 (p < 0001), and the interval between waves 1 and 2 was significantly longer than between waves 2 and 3 (p < 0001). prepandemic mortality was strongly associated with cumulative pandemic influenza mortality in waves 1 and 3 (table 2, figure 2), and overall (= 044, p < 0001). influenza mortality tended to be higher in younger populations in waves 1 and 3, but the age trend was reversed in wave 2. populations with higher influenza mortality rates in wave 1 also had higher rates in wave 3, although rates in these waves were not significantly correlated with rates in wave 2 (table 2). early wave onset was associated with higher wave mortality for wave 1, but not for waves 2 and 3. wave 2 had the highest average influenza mortality overall, but for all three waves, duration was shorter in populations with higher influenza mortality (table 2). higher wave 1 mortality delayed onset of wave 2, while wave 3 mortality increased in populations with shorter intervals between waves 2 and 3 (table 2). wave characteristics over the 46week period of data collection during the 19181919 influenza pandemic for individual 333 administrative units in england and wales data from : johnson. no deaths recorded in 1 administrative unit. correlation between cumulative influenza mortality/1000 population and other wave characteristics and potential risk factors investigated for 333 administrative units (spearman s correlation coefficient and pvalues) data from : johnson ; ons 2001. no deaths recorded in 1 administrative unit. 4 cases missing data. younger population age structure corresponds to an age standardisation factor of greater than 1, giving a general indication of age differences between populations. cumulative influenza mortality/1000 population in waves 1 (a), 2 (b) and 3 (c) plotted against prepandemic allcause mortality/1000. populations in the north and west tended to be younger in age with higher rates of prepandemic mortality (table 3). a similar geographical trend was evident for cumulative influenza mortality in waves 1 and 3, with higher mortality evident in largely corresponding geographical locations in the northwest of england and wales (table 3, figure 3). complex patterns of onset are evident in all three waves, with a clear directional spread (northward) only in wave 2, notably later in areas where wave 1 mortality was higher, and earlier where wave 1 mortality was lower (2, 3, 3, 4). in contrast, the interval between waves 1 and 2 increased northward while the interval between waves 2 and 3 increased southward (table 3, figure 5). correlations of risk factors and wave characteristics with latitude and longitude (spearman s correlation coefficient and pvalue) data from : johnson ; ons 2001. four cases missing data. no deaths recorded in 1 administrative unit. contour plots of equal ranges of natural logarithmic transforms of cumulative influenza mortality, backtransformed to rates/1000, in waves 1 (a), 2 (b) and 3 (c) in england and wales. contour plots of week of onset, numbered by week of data collection, in waves 1 (a), 2 (b) and 3 (c) in england and wales. scatterplots of betweenwave intervals 12 (a) and 23 (b) against latitude. population sizes of administrative units ranged from 10 477 to 1 033 038 (median 50 384), and areas ranged from 406 to 1 655 571 acres (median 5062). population density varied from 007 to 143 persons / acre (median 91 persons / acre). whereas population size was unrelated to geographical location, population density was significantly correlated with longitude (table 3). population density was also correlated with prepandemic allcause mortality [(pvalue) 037 (< 0001) ], but no association was detected with population size [003 (0625) ]. mortality rates in individual waves were significantly correlated with population density (table 2), as was overall mortality [019 (< 0001) ]. however, overall mortality was not correlated with population size [003 (0603) ]. for waves 1, 2 and 3, onset occurred earlier in more densely populated areas [025 (< 0001) ; 021 (< 0001) ; 013 (0020), respectively ]. the index of deprivation from the year 2000 for matched areas (n = 62) was correlated with cumulative influenza mortality in waves 1 and 3 in 19181919 [(pvalue) 041 (0001) and 044 (< 0001), respectively ], but not wave 2 [008 (0516) ]. it was also correlated with overall mortality [042 (< 0001) ], northern latitudes [048 (< 0001) ] and, notably, with prepandemic mortality [060 (< 0001) ]. in the weighted regression analyses for cumulative influenza mortality (table 4), prepandemic mortality was predictive in all three waves when we fitted a quadratic trend surface (5 d.f.) to extract geographical variance. the model for wave 1 explains 69% of the variance in influenza mortality, with younger populations at greater risk and the effect of population density modified by covariates. for wave 2 mortality, geographical factors explained much of the variance between populations, with a significant tendency for wave 2 mortality to be reduced in younger populations with higher mortality rates in wave 1 (see discussion). population density was not a significant predictor of mortality in waves 2 and 3 when adjusted for the effects of other covariates included in regression models, likely because of shared variance with prepandemic allcause mortality in particular. as this study compares data from populations rather than individuals, any conclusions about causal processes involving individuals are subject to ecological bias as were those from earlier related studies., however, our novel findings from regression analysis used improved measures of wave severity and onset for all three waves, including the milder wave 1, for which, despite the lower mortality rates, we identified previously undocumented social and spatial associations. a fundamental observation is that influenza mortality during the 19181919 pandemic was greater in populations with higher levels of prepandemic allcause mortality, (2, 3). this suggests that common factors, most probably linked to social disadvantage, contribute to both influenza mortality and allcause mortality.. found that global mortality from the 19181919 pandemic was significantly greater in poorer countries. we thus prepandemic mortality as a surrogate for social disadvantage and population vulnerability. prepandemic mortality tended to increase in a northerly and westerly direction, so it is not surprising that pandemic influenza mortality showed the same trend in waves 1 and 3 (table 3). geographical gradients in wave 2 were inconsistent, possibly because of depletion of the most vulnerable individuals through exposure in wave 1. on this view, the more consistent geographical trend in wave 3 could be because of a waning of protective immunity induced in susceptible individuals in wave 1. absolute influenza mortality was greatest in wave 2 (table 1), possibly because the virus had acquired greater virulence, or because waning humoral immunity, with an inflammatory cytokine response and/or complicating bacterial infections, led to worse clinical outcomes, particularly among young adults., wave 3 mortality was reduced compared with wave 2, possibly because of prior immunising exposure, or declining virulence. a decline in virulence over time has been previously invoked to explain why pandemic mortality in western samoa in november 1918 was much higher than in nearby american samoa, where the arrival of the influenza virus was delayed for many months by strict quarantine. similarly, pandemic mortality was higher in new zealand, affected from october 1918, than in australia, where pandemic influenza was excluded until january 1919., other factors, including those related to season, could contribute to mortality differences between waves. for example, the winter onset, arguably associated with increased viral exposures, lesser uv exposure, vitamin d deficiency and impaired innate immunity could help to explain why wave 3 mortality was greater than in wave 1.,, spread and severity of the three waves of the 19181919 influenza pandemic were also influenced by wartime privations and changes in population mixing. spontaneous or imposed social distancing in response to influenza deaths can also limit influenza transmission and bring an outbreak to an earlier end., spontaneous, or reactive, social distancing in response to high localised mortality helped to explain the variability in mortality patterns observed across us cities. in our analysis, the onset of wave 2 was somewhat delayed in populations with greater wave 1 mortality (table 2). although spontaneous social distancing can explain these findings, they could also be explained by a greater immune protection, as immunising exposures would be expected to increase with population mortality, provided that case fatality did not differ markedly between populations. although mortality in wave 2 was not predictive of the interval between waves 2 and 3, wave 3 mortality was higher in populations with a shorter interval between waves 2 and 3 (table 2). this observation is hard to explain in terms of social distancing, whereas it would be expected that populations with lower levels of immunity at the end of wave 2 would have both an earlier onset and higher mortality in wave 3. within each wave, wave duration was also negatively correlated with wave mortality (table 2), as might be expected with social distancing. however, the negative correlation is also consistent with greater population immunity leading to slower spread and lesser mortality. the interval between waves 1 and 2 increased with latitude, whereas the wave 23 interval decreased with latitude (figure 5), in part at least because of the south to north spread of wave 2. at a global level, latitude was not significantly associated with pandemic mortality when adjusted for income. however, at the finer resolution of england and wales, spatial and temporal dependence of the three waves has been characterised in earlier reports, and in our own analyses (table 3 ; 3, 4, 5). reported that transmissibility was higher and more geographically heterogeneous in wave 2 than in wave 3. the contour plots of onset in successive waves (figure 4) are consistent with patterns identified by smallmanrayner. using 83 london and county boroughs. our regression analysis of influenza mortality over 333 populations (table 4) simplifies highly complex processes depending on social factors, nutrition, environment and comorbidities.,,, the best indicator we had for social deprivation and population vulnerability in the 19181919 influenza pandemic was allcause mortality for the period 19111914. we found that the geographical index of deprivation, derived as late as 2000, was still strongly correlated with prepandemic mortality, validating our use of it and showing that geographical predictors of social disadvantage and allcause mortality have been stable over many decades. pandemic signature of heightened mortality among younger individuals was observed and younger populations were at greater risk, suggesting that older persons were protected by prior immunity. a more complex scenario during wave 2 is evidenced by the significant negative interaction term between age and wave 1 mortality (table 4). in areas with lower wave 1 mortality, younger populations were again more severely impacted in wave 2, while the risk of influenza death in the ensuing wave 2 was reduced in areas that experienced higher wave 1 mortality, suggesting that greater exposure in wave 1 served to boost immune protection. among older populations, however, wave 2 mortality tended to increase with wave 1 mortality, possibly because confounding by population vulnerability overrode any protective effect of prior exposure in this ecological dataset. wave 1 mortality also predicted increasing wave 3 mortality, again presumably because of confounding owing to increased population vulnerability associated with socioeconomic status, whereas increasing wave 2 mortality predicted lower mortality in wave 3 (table 4), consistent with the idea of wavetowave immunological protection. after taking mortality in previous waves into account, the decreased risk of mortality in wave 3 associated with age structure also supports acquired immunity among younger populations. further, the contour plots (figure 3) show that the areas with highest absolute mortality in wave 2 tend to differ from wave 3, as would be expected with immune protection. although the relatively low mortality rates in wave 1 may not necessarily reflect the severity of this outbreak, our regression model explained a large proportion of the variance across administrative units (table 4). further, and despite wave 2 having the highest absolute mortality (table 1), our regression model explained a lesser proportion of variance in mortality for wave 2 compared with waves 1 and 3 (table 4). one explanation for this would be that immunity induced by wave 1 tended to neutralise the geographical and social influences on population vulnerability, leaving less real variation between populations to be explained in wave 2. by the time of wave 3, some of the immune protection from wave 1 would have been lost, with populations reverting to their former vulnerable status. an ancillary explanation would be that mortality in wave 2 was dominated more by factors in the virus itself, rather than by factors related to the geography and populations concerned. these findings build on earlier work, suggesting that population immunity can help to explain the wavelike behaviour of pandemic influenza in 19181919. although socialdistancing models can also explain some examples of wavelike behaviour, the very high attack rates seen when influenza reaches previously isolated populations seem to require an immunological explanation. this is not to deny the very important role of social distancing in limiting influenza transmission, particularly in populations that are partially immune, as seen in the 2009 pandemic. indeed, we would go further, and suggest that in today s highly connected world, most populations are partially protected against any future pandemic by the heterosubtypic immunity induced by prior exposure to seasonal strains of influenza.,, in any future pandemics in partially immune populations, high levels of public compliance with social distancing measures, combined with antiviral use, could be sufficient to delay spread sufficiently to allow development and distribution of a targeted vaccine. the conclusions from this study will inform more definitive modelling of mortality in the three waves of the 19181919 pandemic to explore the causal processes generating the data, including the putative effects of immunity from wave to wave, effects of geographical proximity in determining spread, and possible changes in virulence over time. such model results will greatly enhance our understanding of influenza biology and transmission, and inform the development and implementation of improved interventions. | please cite this paper as : pearce. (2011) understanding mortality in the 19181919 influenza pandemic in england and wales. influenza and other respiratory viruses 5(2), 8998. background the causes of recurrent waves in the 19181919 influenza pandemic are not fully understood. objectives to identify the risk factors for influenza onset, spread and mortality in waves 1, 2 and 3 (summer, autumn and winter) in england and wales in 19181919. methods influenza mortality rates for 333 population units and putative risk factors were analysed by correlation and by regressions weighted by population size and adjusted for spatial trends. results for waves 1 and 3, influenza mortality was higher in younger, northerly and socially disadvantaged populations experiencing higher allcause mortality in 19111914. influenza mortality was greatest in wave 2, but less dependent on underlying population characteristics. wave duration was shorter in areas with higher influenza mortality, typically associated with increasing population density. regression analyses confirmed the importance of geographical factors and prepandemic mortality for all three waves. age effects were complex, with the suggestion that younger populations with greater mortality in wave 1 had lesser mortality in wave 2. conclusions our findings suggest that socially disadvantaged populations were more vulnerable, that older populations were partially protected by prior immunity in wave 1 and that exposure of (younger) populations in one wave could protect against mortality in the subsequent wave. an increase in viral virulence could explain the greater mortality in wave 2. further modelling of causal processes will help to explain, in considerable detail, how social and geographical factors, season, preexisting and acquired immunity and virulence affected viral transmission and pandemic mortality in 19181919. |
patients with ankylosing spondylitis have been shown to have low bone mineral density at spine and propensity for vertebral fractures. the loss of bone mineral density has been shown to be more marked in late than early disease. the bone loss may be related to inflammation, as in other chronic inflammatory diseases [2, 3 ]. indeed, ankylosing spondylitis is characterized by chronic inflammation, as evidenced by elevated proinflammatory cytokines like tumour necrosis factor- (tnf) and interleukin-6. these may lead to bone loss by increased expression of receptor activator of nf-b ligand (rankl) on osteoblasts and stromal cells and its soluble form (srankl). rankl and cytokines lead to osteoclast and other inflammatory cell activation and release of bone and cartilage degrading enzymes like cathepsin k and matrix metalloproteinases (mmps) [5, 6 ]. their natural antagonists, that is, osteoprotegerin (for rankl) and tissue inhibitor of metalloproteinase or timp (for mmp) oppose their actions. among the mmps, there is inconsistent data on circulating levels of these molecules in ankylosing spondylitis, especially in asian indians. thus this study was planned to look at levels of these molecules in this population. this cross - sectional study was carried out in a north indian university hospital between april 2010 and february 2011. consecutive patients with ankylosing spondylitis, attending the outpatient rheumatology clinic, and who gave consent were recruited. patients with comorbid conditions / intake of drugs known to affect bone mineral density like renal failure, use of bisphosphonates, hypo / hyperthyroidism, or inflammatory bowel disease were excluded. patients with bamboo spine (radiographs of the lumbar and thoracic vertebra showing syndesmophytes at all intervertebral levels from t6 to s1) were also excluded. in addition 20-age and disease activity was assessed using the bath ankylosing spondylitis disease activity index (basdai). this consists of 6 questions on 5 major symptoms of spinal pain, joint pain, entheseal pain, fatigue, and early morning stiffness. function was assessed by the bath ankylosing spondylitis functional index (basfi), which comprises 10 questions on function and ability to cope with everyday life. metrology was assessed using the bath ankylosing spondylitis metrological index, which comprises measurement of lumbar flexion (modified schober 's test), intermalleolar distance, cervical rotation, lumbar side flexion, and tragus - to - wall distance. most were tested for presence of the gene hla - b27 using polymerase chain reaction. soluble receptor activator of nf - kappa b (srankl) was determined by sandwich elisa (komabiotech, republic of korea). osteoprotegerin was determined using sandwich elisa (r&d, duo set) with range 62.54000 pg / ml. mmp-3 and timp-1 were estimated by elisa using a commercial kit (r&d systems, minneapolis, mn, usa). the mmp-3 kit detected both active and pro - mmp-3 (total mmp-3) with range of 31.252000 pg / ml. student 's t - test was used to compare means and correlation was done by pearsons correlation. the study included 85 patients (m : f = 82 : 3) of ankylosing spondylitis with mean age (sd) 33.0 10.0 years. mean duration of disease was 11.3 7.3 years and time since diagnosis was 3.8 5.1 years. the means (sd) of basdai, basfi, basmi, and esr were 4.0 2.2, 3.9 2.8, 3.0 2.8, and 59.2 31.2 mm, respectively. syndesmophytes were present in 36 (42%) patients and hla - b27 positive in 95% (59 of 63 tested). however, srankl were similar in both patients and controls, being detectable in only a quarter of both (table 1). even on categorizing patients by basdai into three categories of high, moderate, and low (basdai 4, basdai 46, basdai 6, resp.), srankl was detected in an equal proportion in all three groups. levels of both serum mmp-3 and timp-1 levels were higher in patients compared to controls. but, there was no difference in the mmp-3 to timp ratio (table 1). there was no correlation between basdai and levels of opg (r = 0.05), mmp-3 (r = 0.04), or timp-1 (0.01, p = ns). there was also no correlation between esr and opg (r = 0.12) or mmp-3 (r = 0.09, p = ns). however, timp-1 correlated with esr (r = 0.30, p = 0.009). this study found higher levels of osteoprotegerin (opg) in patients of ankylosing spondylitis than controls ; however, srankl was detected in only a minority in both. matrix metalloproteinase 3 (mmp-3) and tissue inhibitor of metalloproteinase 1 (timp-1) were both elevated in patients ; however, there was no difference in the mmp-3/timp-1 ratio. this is similar to what most of the previous studies have found [1214 ]. however, some studies have found no difference or lower levels compared to controls [15, 16 ]. the reason for these differences may be related to disease duration, which was around 10 years in our study as well as the other studies showing raised opg but was only 5 years in the latter study. indeed, even on immunohistochemistry, a high expression of opg has been shown in synovial macrophage - type synovial lining cells and endothelial cells in patients of spondyloarthropathy. thus, it seems the high serum levels do reflect the high levels in the joints. we did not find any correlation with disease activity, similar to most other studies [12, 18 ]. have looked at markers to predict radiographic damage progression using 2-year data scored using the modified stoke as spine score (msasss). it would have been interesting to look at the cross - sectional association of mmp-3 and opg with msasss ; however, we did not obtain this data. this is different from previous studies, most of which found higher levels in patients compared to controls [14, 15 ]. the reason may be related to the detection limit of srankl by the kit we used, which captured only free srankl which constitutes only 1/1000 of the total serum srankl (other being bound to proteins like opg). on the other hand, it is possible that srankl is not overexpressed in ankylosing spondylitis in serum and the local tissues. indeed, a study on spinal tissue obtained during surgery in as patients did not find srankl expression in any patient sample except one. we could not assess correlation of srankl with disease activity measures, because it was undetectable in a majority. previous longitudinal studies of anti - tnf agents over 36 months, with control of disease activity, have not found any change in levels of opg or srankl [22, 23 ]. high levels of mmp-3 have been shown in serum of ankylosing spondylitis patients compared to controls [7, 24 ]. immuno - histochemistry in peripheral synovitis of spondarthritis patients also shows high levels of mmp-3, with downregulation after biological treatment. it has been shown that apart from cathepsin k, mmps have a role in bone matrix degradation leading to bone loss. indeed, immunohistochemical studies on spinal tissue from as patients do show increased mononuclear cells expressing matrix metalloproteinase 1 and 3. we did not find any correlation with measures of disease activity. among previous studies, some studies found a correlation of mmp-3 with both basdai and acute - phase reactants, some with only acute - phase reactants not with disease activity, some with basdai but not acute - phase reactants and some with none. to conclude, this study did not find elevated circulating levels of srankl or elevation in the mmp-3/timp ratio in ankylosing spondylitis patients. however, a study of bone density and radiological damage with these markers may provide a better understanding of the role of these markers in bone loss in ankylosing spondylitis. | background. bone loss in ankylosing spondylitis may be related to inflammation. data from previous studies on circulating levels of srankl, opg, mmp3, and timp is inconsistent ; thus this study is planned to look at this aspect in asian indian patients. methods. cross - sectional study included patients with ankylosing spondylitis and age- and gender - similar controls. serum levels of srankl, opg, mmp-3, and timp-1 were measured by elisa. results. included 85 patients (m : f = 82 : 3) having mean age (sd) 33.0 10.0 years and disease duration 11.3 7.3 years. basdai, basfi, basmi, and esr were 4.0 2.2, 3.9 2.8, 3.0 2.8, and 59.2 31.2, respectively. patients had higher mean (sd) opg level (649.7 286.8, 389.3 244.8 pg / ml, p < 0.001). however, there was no difference in srankl (349.2 872.0, 554.7 1850.1, p = ns). serum mmp-3 (91.4 84.7, 55.9 37.1 ng / ml, p < 0.01) and timp-1 (520.6 450.7, 296.5 114.2 ng / ml, p < 0.001) levels were higher in patients ; however, there was no difference in mmp-3/timp-1 ratio. conclusion. circulating levels of opg were higher ; however, there was no difference in srankl in asian indian ankylosing spondylitis patients. although both mmp-3 and timp-1 were raised, their ratio was not different from that of controls. |
between september 2000 and august 2001, 135 consecutive patients with hepatic masses underwent pulse - inversion harmonic us. a confirmed diagnosis of hepatic mass in one of three categories - hepatocellular carcinoma (hcc), metastasis, or hemangioma - was the only criterion for inclusion. patients in whom hcc had been treated by transarterial chemoembolization or radiofrequency ablation prior to us examination (n=21), or had a dysplastic nodule (n=8), inflammatory eosinophilic granuloma (n=4) or focal nodular hyperplasia (n=2), were excluded, as were those (n=10) for whom chemotherapy was ongoing or had been performed within the previous 30 days. thus, 90 patients [60 men and 30 women ; mean age, 57 (range 23 - 79) years ] with focal hepatic lesions, including hcc (n=43), metastasis (n=30) and hemangioma (n=17), were included in this study. informed consent was obtained from all patients and the approval of our institutional review board was obtained. five patients with hccs and 22 with metastases had multiple (two to seven) lesions. in patients with multiple hccs or metastatic lesions, only one dominant lesion per patient was analyzed (determined on the basis of size). a diagnosis of hcc was confirmed by us - guided percutaneous needle biopsy in 24 patients and at surgery in three. in the remaining 16 patients, hcc was diagnosed on the basis of the typical imaging findings of three - phase helical ct, angiography and/or ct after intra - arterial injection of iodized oil, as well as elevated levels of serum alpha - fetoprotein (> 200 ng / ml). for metastatic liver tumors, the sites of primary disease were the stomach (n=13), colon (n=10), pancreas (n=5) and lung (n=2). radiological or histological study showed that no metastatic lesion was hypervascular. for 27 of the 30 patients with hepatic metastases, the diagnosis was histologically confirmed at surgery (n=7) or by biopsy (n=20). in the remaining three cases, diagnosis was based on follow - up serial ct findings showing that the lesions had progressed rapidly. all patients with hemangioma underwent dynamic contrast - enhanced ct (n=17) or mr (n=9) examination. for 16 of the 17 hemangiomas, the hccs ranged in size from 1.0 to 7.0 (mean, 3.5) cm, metastases from 1.0 to 6.0 (mean, 2.9) cm, and hemangiomas from 1.0 to 4.0 (mean, 2.4) cm. pulse - inversion harmonic us was performed by one investigator (k.h.y.), using a commercially available us system (hdi-5000 ; advanced technology laboratory, bothell, washington, u.s.a.) together with a 5 - 2 mhz curved linear - array transducer. to assess tumor vascularity, continuous low - mi imaging (200 ng / ml). for metastatic liver tumors, the sites of primary disease were the stomach (n=13), colon (n=10), pancreas (n=5) and lung (n=2). radiological or histological study showed that no metastatic lesion was hypervascular. for 27 of the 30 patients with hepatic metastases, the diagnosis was histologically confirmed at surgery (n=7) or by biopsy (n=20). in the remaining three cases, diagnosis was based on follow - up serial ct findings showing that the lesions had progressed rapidly. all patients with hemangioma underwent dynamic contrast - enhanced ct (n=17) or mr (n=9) examination. for 16 of the 17 hemangiomas, the hccs ranged in size from 1.0 to 7.0 (mean, 3.5) cm, metastases from 1.0 to 6.0 (mean, 2.9) cm, and hemangiomas from 1.0 to 4.0 (mean, 2.4) cm. pulse - inversion harmonic us was performed by one investigator (k.h.y.), using a commercially available us system (hdi-5000 ; advanced technology laboratory, bothell, washington, u.s.a.) together with a 5 - 2 mhz curved linear - array transducer. to assess tumor vascularity, continuous low - mi imaging (< 0.3) was performed immediately following injection and during the delivery of a microbubble contrast agent (the first 60-second period after contrast injection). five minutes later, high - mi (1.0 - 1.3) delayed phase images were obtained, and the acoustic emission effect thus determined. during the interval between the vascular phase and delayed phase, the display was frozen and ' interval - delay scanning ' was not performed. the microbubble contrast agent used was sh u 508 a (levovist ; schering, berlin, germany), a 2.5 gm bolus of which was injected twice, for vascular and delayed imaging, at a concentration of 300 mg / ml followed by a 10-ml normal saline flush using a 20-gauge peripheral intravenous cannula. when the area of interest was identified at conventional us, pihi was activated, with the focal zone set at or just deeper than the level of interest. all imaging was performed with the transducer in a constant position over the region of interest in order to depict the lesion and to include some normal liver. while the contrast agent was being delivered, during a period of shallow breathing, scanning was performed and the enhanced blood signals in both normal hepatic blood vessels and lesional blood vessels were depicted. during low - mi imaging, a medium frame rate was set (8 per second). high - mi scanning, on the other hand, was performed during the shortest possible period (and with breath - hold), in order to reduce the amount of microbubble disruption in the region of interest outside, and the display was then frozen. in order to destroy microbubbles with insonation and to identify white flashes of contrast break - up, we used a medium pulse length, the maximum mechanical index, a high frame rate (15 per second) and low line density. after freezing the display, we reviewed the images using a cine loop and stored the strongest effects seen during the first and second frames. all studies were recorded on s - vhs video, and with selected digital images and cine loops were stored on a magneto - optical disk., j.j.w.), who were unaware of the pathologic diagnosis or other radiologic imaging findings. each had more than five years of experience of ultrasonography involving the use of contrast agents. the reviewers determined the diameters and echogenicity of the tumors, as seen on unenhanced conventional and pre - contrast pulse - inversion harmonic images. the pattern of contrast enhancement of each tumor depicted by dualphase pulse - inversion harmonic us was evaluated by examining the images obtained during the vascular phase of enhancement (typically 20 - 60 seconds after contrast injection), and during the delayed phase (5 minutes after a separate injection of contrast agent). tumoral vascularity of the dominant lesion, as seen on vascular phase images, was classified as one of four patterns, as follows (fig. the presence of linear or branched internal vessels was categorized as the ' internal vessels ' pattern ; that of linear or dot - like marginal vessels at the periphery of the tumor, regardless of the presence or absence of central vessels, as the ' marginal vessels ' pattern ; that of a discrete and well - defined ring of peripheral enhancing nodules, as ' peripheral nodular enhancement ' ; and the absence of visible vessels or enhancement in either internal or peripheral areas of the tumor, as the ' minimal or no enhancement ' pattern. the acoustic emission effect within the tumor, as seen on delayed phase images, was also classified as one of four patterns (fig. inhomogeneous enhancement ' was defined as irregular and heterogeneous enhancement within the tumor ; ' hypoechoic, decreased enhancement ' as the presence of contrast, but decreased enhancement with respect to adjacent liver parenchyma ; ' isoechoic, homogeneous enhancement ' as homogeneous enhancement, similar to that of adjacent liver parenchyma ; and the term ' hypoechoic and reversed echogenicity ' was used to describe a tumor represented as a hyperechoic lesion on unenhanced us images and as a reversed hypoechoic lesion on delayed phase images. agreement regarding the pattern analysis of all lesions was reached by consensus ; disagreements were resolved by majority opinion. sensitivity and specificity were determined for the different histologic diagnoses, and positive predictive values (ppv) for the various observed enhancement patterns. vascular phase low - mi enhancement patterns for tumoral vascularity in the 90 patients with focal hepatic lesions are summarized in table 1. forty - four lesions [40 hccs (91%) and four metastases (9%) ] demonstrated the ' internal vessels ' pattern, while for 27 lesions [25 metastases (93%) and two hccs (7%) ], the ' marginal vessels ' pattern was observed. twelve lesions [12 hemangiomas (100%) ] showed peripheral nodular enhancement, and in seven cases [five hemangiomas (71%), one hcc (14%) and one metastasis (14%) ], the observed pattern was ' minimal or no enhancement '. table 2 summarizes the vascular phase enhancement patterns for tumor vascularity associated with each diagnosis, and the corresponding sensitivity, specificity, and ppv values. for hccs, three patterns were observed : ' internal vessels ' [93% (40/43) (fig. 3) ] ; ' marginal vessels ' [5% (2/43) ] ; and ' minimal or no enhancement ' [2% (1/43) ]. for metastases, the same three enhancement patterns occurred : ' internal vessels ' [13% (4/30) ] ; ' marginal vessels ' [83% (25/30) (fig. 4) ] ; and ' minimal or no enhancement ' [3% (1/30) ], while for hemangiomas, there were two : ' peripheral nodular enhancement ' [71% (12/17) (fig. 5) ] and ' minimal or no enhancement ' [29% (5/17) ]. the observed patterns were associated with a specificity of 91% or greater, and ppvs of 71% or greater : ' internal vessels ' for hccs ; ' marginal vessels ' for metastases ; ' peripheral nodular enhancement ' for hemangiomas. delayed phase high - mi acoustic emission effect patterns within the tumor are summarized in table 3. thirty - nine lesions [37 hccs (95%) and two metastases (5%) ] demonstrated inhomogeneous enhancement, while 33[28 metastases (85%) and five hccs (15%) ] showed hypoechoic, decreased enhancement. in 11 tumors, all of which were hemangiomas, hypoechoic and reversed echogenecity was observed, and in seven [six hemangiomas (86%) and one hcc (14%) ], isoechoic, homogeneous enhancement was noted. table 4 summarizes the delayed phase the acoustic emission effect patterns associated with each diagnosis, and the corresponding sensitivity, specificity, and ppv values. for hccs, three enhancement patterns were observed : ' inhomogeneous enhancement ' [86% (37/43) (fig. 3) ], ' hypoechoic, decreased enhancement ' [12% (5/43) ], and ' isoechoic, homogeneous enhancement ' [2% (1/43) ]. for metastases, two patterns were apparent : ' hypoechoic, decreased enhancement ' [93% (28/30) (fig. 6) ], and ' inhomogeneous enhancement ' [7% (2/30) ]. hemangiomas showed two enhancement patterns : ' hypoechoic and reversed echogenicity ' [65% (11/17) (fig. 7) ], and ' isoechoic, homogeneous enhancement ' [35% (6/17) (fig. the above patterns were associated with a specificity of 92% or greater, and ppvs of 85% or greater : ' inhomogeneous enhancement ' for hccs ; ' hypoechoic, decreased enhancement ' for metastases ; and for hemangiomas, ' hypoechoic and reversed echogenicity ' or ' isoechoic, homogeneous enhancement '. vascular phase low - mi enhancement patterns for tumoral vascularity in the 90 patients with focal hepatic lesions are summarized in table 1. forty - four lesions [40 hccs (91%) and four metastases (9%) ] demonstrated the ' internal vessels ' pattern, while for 27 lesions [25 metastases (93%) and two hccs (7%) ], the ' marginal vessels ' pattern was observed. twelve lesions [12 hemangiomas (100%) ] showed peripheral nodular enhancement, and in seven cases [five hemangiomas (71%), one hcc (14%) and one metastasis (14%) ], the observed pattern was ' minimal or no enhancement '. table 2 summarizes the vascular phase enhancement patterns for tumor vascularity associated with each diagnosis, and the corresponding sensitivity, specificity, and ppv values. for hccs, three patterns were observed : ' internal vessels ' [93% (40/43) (fig. 3) ] ; ' marginal vessels ' [5% (2/43) ] ; and ' minimal or no enhancement ' [2% (1/43) ]. for metastases, the same three enhancement patterns occurred : ' internal vessels ' [13% (4/30) ] ; ' marginal vessels ' [83% (25/30) (fig. 4) ] ; and ' minimal or no enhancement ' [3% (1/30) ], while for hemangiomas, there were two : ' peripheral nodular enhancement ' [71% (12/17) (fig. 5) ] and ' minimal or no enhancement ' [29% (5/17) ]. the observed patterns were associated with a specificity of 91% or greater, and ppvs of 71% or greater : ' internal vessels ' for hccs ; ' marginal vessels ' for metastases ; ' peripheral nodular enhancement ' for hemangiomas. delayed phase high - mi acoustic emission effect patterns within the tumor are summarized in table 3. thirty - nine lesions [37 hccs (95%) and two metastases (5%) ] demonstrated inhomogeneous enhancement, while 33[28 metastases (85%) and five hccs (15%) ] showed hypoechoic, decreased enhancement. in 11 tumors, all of which were hemangiomas, hypoechoic and reversed echogenecity was observed, and in seven [six hemangiomas (86%) and one hcc (14%) ], isoechoic, homogeneous enhancement was noted. table 4 summarizes the delayed phase the acoustic emission effect patterns associated with each diagnosis, and the corresponding sensitivity, specificity, and ppv values. for hccs, three enhancement patterns were observed : ' inhomogeneous enhancement ' [86% (37/43) (fig. 3) ], ' hypoechoic, decreased enhancement ' [12% (5/43) ], and ' isoechoic, homogeneous enhancement ' [2% (1/43) ]. for metastases, two patterns were apparent : ' hypoechoic, decreased enhancement ' [93% (28/30) (fig. 6) ], and ' inhomogeneous enhancement ' [7% (2/30) ]. hemangiomas showed two enhancement patterns : ' hypoechoic and reversed echogenicity ' [65% (11/17) (fig. 7) ], and ' isoechoic, homogeneous enhancement ' [35% (6/17) (fig. the above patterns were associated with a specificity of 92% or greater, and ppvs of 85% or greater : ' inhomogeneous enhancement ' for hccs ; ' hypoechoic, decreased enhancement ' for metastases ; and for hemangiomas, ' hypoechoic and reversed echogenicity ' or ' isoechoic, homogeneous enhancement '. this study has demonstrated that vascular phase pihi with a low mi and delayed phase pihi with a high mi after the administration of a microbubble contrast agent may be able to differentiate hepatic tumors such as hcc, hemangioma, and metastasis. wilson. (11) reported their initial experience of harmonic hepatic us with microbubble contrast agent for the characterization of focal hepatic lesions. although their study and ours showed similar results, ours was different in two respects : firstly, we used sh u 508 a, which consists of galactose microaggregates with a small admixture of palmitic acid. on the other hand, used a perfluorocarbon microbubble agent, which is a blood pool contrast agent comprising microbubbles stabilized by a protein shell. although we know of no comparative studies of the various microbubble contrast agents, perfluorocarbon agents are more highly regarded than levovist for vascular imaging because of their stability (16) ; levovist, however, because of its unique liver - specific action, is considered better at providing the stimulated acoustic emission effect at delayed phase imaging (18). secondly, for delayed phase pulse - inversion imaging we used a high mi, not the interval delay procedure. kim. (14) recently reported that pihi with an interval delay technique was useful for the characterization of hepatic tumors. although they found that the technique demonstrated specific enhancement features of hepatic hemangiomas, it was not suitable for demonstrating malignant hepatic tumors such as hccs and metastases. ko. (19), on the other hand, showed that delayed phase imaging with gray - scale stimulated acoustic emission might be useful for differentiating between hcc and metastatic adenocarcinoma of the liver. in our study, vascular phase imaging with a low mi detected the morphological characteristics of tumoral vessels. during delivery of the contrast agent, enhanced signals were emitted by both normal hepatic and lesional blood vessels. in hccs, the ' internal vessels ' pattern was striking ; the characteristics of these vessels were distinct, unlike those of hemangiomas and metastatic tumors. 15) found that at the vascular imaging phase of the coded harmonic angio technique, a majority of hccs were depicted as irregular branching vessels or showed randomly stippled vascularity. we believe that even though the us technique employed was somewhat different, their vascular phase imaging findings and ours were similar. for hemangiomas, the observed pattern was ' peripheral nodular enhancement ' or ' minimal or no enhancement ' ; in no case were internal or marginal vessels observed. as in previous studies (14 - 16), our results showed that ' peripheral nodular enhancement ', observed at vascular phase imaging, was a highly specific finding for the diagnosis of hemangiomas. however, for five of the 17 tumors fo this kind, the observed pattern was ' minimal or no enhancement ', a result different from that of previous studies (14 - 15). we believe our results were due to the relatively low sensitivity of the pihi technique itself when used to evaluate the vascularity of hemangiomas, the dosage of the contrast agent injected, and the histologic characteristics of the tumor. at delayed phase imaging with a high mi using pihi, the intensity of the echo was proportional to the number of microbubbles present within the ultrasound beam, reflecting the amount of microbubbles in a tumor 's sinusoids and kupffer cells (11). although not certain, it is thought that microbubbles are simply retained in the sinusoidal spaces or are encapsulated by kupffer cells (12). because the majority of hepatic tumors do not contain sinusoids or kupffer cells, these tumors appear at stimulated acoustic emission imaging as defects or signal loss compared to normal hepatic parenchyma. hccs do not usually contain kupffer cells, though some types, such as well - differentiated hcc and early hcc, may contain a portion of normal - appearing hepatocytes or kupffer cells. profuse intratumoral vessels, as well as the sinusoids and reticuloendothelial cells within some types of hcc, might thus appear as bright emission signals, the so - called ' inhomogeneous enhancement ' seen on delayed phase images. in our study, 86% of hccs showed inhomogeneous enhancement, a result somewhat different from those of previous studies (15, 16), in which a hypoechoic washout pattern or no enhancement was observed at interval delay or postvascular imaging. the majority of metastases were seen as ' hypoechoic, decreased enhancement ', indicating that metastatic tumors are usually hypovascular and do not contain reticuloendothelial cells. in some hepatic metastases, however, acoustic emission signals were present at their periphery ; this was thought to represent capillarization of the sinusoid, and peripheral fibrosis of the hepatic parenchyma (21). although a small minority of the metastases in our study showed inhomogeneous enhancement, in 93% of cases the observed pattern was ' hypoechoic, decreased enhancement ', a finding similar to those previously reported (14 - 17). all hemangiomas showed one of two patterns, namely ' decreased and reversed echogenicity ' or ' isoechoic, homogeneous enhancement '. the former indicated that a lesion which was echogenic to surrounding liver at baseline imaging became echo - poor at delayed imaging, suggesting that hemangiomas have fewer sinusoids than normal liver. hemangiomas showing ' isoechoic, homogeneous enhancement ', on the other hand, the histopathology of hemangiomas varies, however, according to their tissue components and vascular pools, and the imaging findings thus present a spectrum, with various patterns observed at dynamic contrast - enhanced ct or mr. we therefore believe that delayed phase imaging of hemangiomas may demonstrate various patterns, and in order to evaluate tissue characteristics in terms of the relationship between histologic features and delayed - phase pihi findings, further study may be warranted. firstly, sh u 508 a, used as a contrast agent, is known to have a weak harmonic response when insonated with an ultrasound beam at a low mi (16). the microbubbles are destroyed even at continuous vascular phase imaging, for which levovist is a less suitable agent. we therefore suggest that in our study, vascular phase imaging might not have depicted tumoral vascularity sufficiently. continuous vascular phase us scanning is possible in only one scanning plane, and in patients with multiple lesions it is therefore not possible to characterize all lesions simultaneously. nor can delayed phase scanning simultaneously include all lesions, and since its duration is very short, it is not easy to obtain the best images depicting the acoustic emission effect in the same area as vascular phase images. in conclusion, vascular and delayed phase pihi appears to be a useful imaging technique for the characterization of focal hepatic lesions. the depiction of tumoral vascularity and the acoustic emission effect at dual - phase pihi after the injection of a microbubble contrast agent can help differentiate hcc, metastasis, and hemangioma. | objectiveto analyze the contrast - enhancement patterns obtained at pulse - inversion harmonic imaging (pihi) of focal hepatic lesions, and to thus determine tumor vascularity and the acoustic emission effect.materials and methodswe reviewed pulse - inversion images in 90 consecutive patients with focal hepatic lesions, namely hepatocellular carcinoma (hcc) (n=43), metastases (n=30), and hemangioma (n=17). vascular and delayed phase images were obtained immediately and five minutes following the injection of a microbubble contrast agent. tumoral vascularity at vascular phase imaging and the acoustic emission effect at delayed phase imaging were each classified as one of four patterns.resultsvascular phase images depicted internal vessels in 93% of hccs, marginal vessels in 83% of metastases, and peripheral nodular enhancement in 71% of hemangiomas. delayed phase images showed inhomogeneous enhancement in 86% of hccs ; hypoechoic, decreased enhancement in 93% of metastases ; and hypoechoic and reversed echogenicity in 65% of hemangiomas. vascular and delayed phase enhancement patterns were associated with a specificity of 91% or greater, and 92% or greater, respectively, and with positive predictive values of 71% or greater, and 85% or greater, respectively.conclusioncontrast-enhancement patterns depicting tumoral vascularity and the acoustic emission effect at pihi can help differentiate focal hepatic lesions. |
there have been a few case reports on selective serotonin reuptake inhibitor (ssri)-induced hyperprolactinemia,123) which may lead to secondary amenorrhea ; however, to our knowledge, this is the first case in which a false - positive urine pregnancy test was observed following initiation of escitalopram treatment. this case report suggests that women of child - bearing age taking antidepressants should be closely monitored. miss l is a 34-year - old caucasian female with a history of premenstrual dysphoric disorder and panic disorder. she then returned to the clinic in 2013 for worsening signs of depression and anxiety following the murder of her brother. she was started on alprazolam 0.5 mg four times a day when necessary (prn) and zolpidem 5 mg every hour of sleep (qhs) prn for anxiety and sleep disorders, respectively. maximum citalopram dosage was reached at 40 mg in the morning ; hence, mirtazapine was added to the treatment regimen for worsening affective symptoms. citalopram treatment was discontinued because mirtazapine was effective, and her wellbeing was maintained for over 12 months on mirtazapine monotherapy. in 2014, subsequently, she was started on escitalopram 10 mg daily (qd) as an augmenting agent for her worsening depressive symptoms ; after 3 months, the escitalopram dosage was increased to 20 mg qd. she presented for follow - up at 1 month after starting on 20 mg qd escitalopram and reported that she had missed her period after initiating escitalopram and had subsequently completed a home pregnancy test, which was positive. upon evaluation by her primary care physician, it was determined that her body mass index had not changed and there was no change in her mood. she was advised by her physician to discontinue escitalopram because it was the only new medication that she had recently started. there have been a few reported cases of ssri - induced hyperprolactinemia and development of galactorrhea while on escitalopram.123) serotonin may stimulate prolactin release either directly through postsynaptic 5-hydroxytrypamine (5-ht) receptors in the hypothalamus or indirectly via 5-ht mediated - inhibition of tuberoinfundibular dopaminergic neurons.4) one study comparing citalopram and escitalopram showed that after a single dose, escitalopram acted centrally (and not peripherally) and increased prolactin and cortisol levels while also exhibiting successful blockade of cortisol following administration of dexamethasone.5) miss l, who reports a history of regular menses, is thought to have developed amenorrhea as a result of escitalopram treatment. the mechanism behind the development of amenorrhea may be a result of serotonin 's effect of increasing prolactin levels. unfortunately, the patient 's prolactin levels were not ascertained at her follow - up appointment with her mental health provider (after having initiated escitalopram) because two months had already passed since discontinuation of escitalopram. elevated prolactin levels could cause amenorrhea by suppressing hypothalamic gonadotropin - releasing hormone, thereby decreasing production of gonadotropins such as luteinizing hormone (lh) and follicle - stimulating hormone. lh may play a role in false - positive urine pregnancy tests in patients receiving chlorpromazine.6) one study showed secretion of the posterior pituitary (neurohypophysial) hormones vasopressin and oxytocin through stimulation of serotonergic receptors in rats ; in particular, the 5-ht1a receptor was involved in serotonergic stimulation of oxytocin secretion.7) most studies investigating the role of oxytocin in lh regulation have been conducted in rats and show that oxytocin elicits concentration - dependent secretion of lh.8) in a small study, an oxytocin antagonist affected the ovulatory cycle of non - pregnant women, pointing to a role for oxytocin in the physiological processes of lh regulation in women.9) in the case of miss l, the serotonergic effects of escitalopram (which may be unique in its central - acting nature) may have been involved in an increase in oxytocin and subsequently an increase in lh. lh apparently cross - reacts with the antichorionic gonadotropin antibody used in immunologic urine pregnancy tests and can cause a false - positive result.6) potential causes of false - positive urine pregnancy tests include medical conditions such as malignancy and proteinuria. this case report suggests that women of child - bearing age should be carefully monitored while they are on an antidepressants. | escitalopram is a selective serotonin reuptake inhibitor antidepressant approved by the food and drug administration for the treatment of major depressive disorder and generalized anxiety disorder. a 34-year - old female patient with major depressive disorder developed amenorrhea and had a false - positive urine pregnancy test after initiation of escitalopram treatment. to our knowledge, no published case report of amenorrhea and false - positive urine pregnancy tests in women taking escitalopram exists. this case report suggests that women of child - bearing age should be carefully monitored for amenorrhea while they are on an antidepressant treatment regimen. |
autologous chondrocyte transplantation (act) was first described in 1994 and has become an accepted therapy for symptomatic full - thickness cartilage defects of the knee joint and osteochondritis dissecans. recently, structural and clinical superiority compared with arthroscopic microfracture has been reported, which supports the hypothesis that using well - characterized cell - based therapies can predict a better structural cartilage repair that can also result in a beneficial clinical outcome. several studies have also reported midterm and even some long - term results with success rates between 80% and 90%. since its introduction, many adaptations have been made to the original surgical technique using a cell suspension. originally, an autologous piece of periosteum was harvested and sutured over the debrided cartilage lesion. the cell suspension this procedure was tedious and required additional surgical time, and the periosteal flap was often fragile, difficult to manipulate, and susceptible to holes, tears, and leaks. it was subsequently observed that the periosteal flap was also associated with graft hypertrophy, frequently requiring additional surgical intervention. more recently, resorbable biomaterials have been developed, for example, a porcine collagen type i / iii membrane (chondro - gide, fa. not only does this biomaterial preclude the tedious harvesting of the periosteum, it is also more robust and less predisposed to tears. this development is today considered as the second generation of act. following the second generation of act, many attempts have been undertaken to combine cultured autologous chondrocyte with 3-dimensional matrix systems prior to implantation (third generation). demonstrated that the results from patients randomized to receive cell suspension based autologous chondrocyte implantation versus matrix - induced autologous chondrocyte implantation were equivalent at the 1-year follow - up. because the cells are retained and seeded within the matrix, a covering layer (periosteal or biomembrane) is no longer required. furthermore, some matrices are bioadhesive and allow for direct implantation without suturing. at the present time, the majority of the clinical studies using this technology have been large case series observations demonstrating a significant clinical improvement over pre - surgery assessments with observations up to 5 years. in a recent review of 18 studies (total of 731 patients) with an average follow - up of 27.3 months kon. reported that only 2 were found to be randomized controlled studies, and the majority (11) were prospective cohort studies or case series. the authors concluded that the limitations in the overall study designs and quality make definitive conclusions regarding the efficacy of this technique difficult. there are also some indications that basal and lateral integration with the surrounding normal cartilage may be lower compared with first - generation cell suspension techniques. in contrast to cell suspension, these cells are cultured into the matrix for several weeks prior to implantation, but there is a risk of the accumulation of extracellular matrix and cell maturation hampering integration. recently, a small randomized clinical trial compared the clinical outcomes of patients with symptomatic cartilage defects treated with matrix - induced autologous chondrocyte implantation versus those treated with microfracture. matrix - induced autologous chondrocyte implantation was significantly more effective at 24 months versus baseline than the microfracture treatment was, according to 4 different validated outcome measures. although there is still some debate over the comparative efficacy of the first- and second-/third - generation products, the advantages of each should be considered in optimizing a therapeutic modality for the patient and surgeon. accordingly, we have adopted a new procedure which combines the potential benefits of the former generations. we modified a second - generation product : the cell suspension is directly seeded onto the collagen membrane prior to implantation into the defect (autologous chondrocyte transplantation cell seeded [act - cs ]). this cell - seeded membrane is then sutured into the cartilage defect (cell - side down) and sealed with fibrin glue. this technique has been described by matthias steinwachs in 2009 and the first clinical results have been recently reported. this procedure allows the patient and surgeon to select their preferred cell expansion provider, retains the robustness and surgical ease of handling of a biomaterial, as opposed to a periosteal flap, enhances the homogeneity of cell distribution on to the membrane surface, requires a less tedious suturing procedure and avoids the increased incidence of hypertrophy associated with the periosteal flap covering for act. because the technique may be open to various interpretations of methodology of implementation and the possible variations that may be introduced by individual surgeons, a panel of experts convened during an act - cs consensus meeting. the goal was to incorporate the best clinical practices from the attendees and come to a consensus on standard operational procedures. therefore, the primary purpose of this article is to provide a detailed description of the surgical procedure to be adopted by the orthopedic community. the authors of the present article are aware that many of the recommendations provided are expert opinions. some of them are supported by scientific publications ; others are opinions with regard to personal experience (expert opinions, ebm level iv). the authors acknowledge that this should be an evolving document and encourage any scientific work that may be conducted in the future to further test and elaborate on the recommendations given in the present publication. the authors consider it to be important to share their opinion and experience in order to further unify surgical techniques of act and initiate a scientific and clinical open forum to further improve this technique. cell - seeded collagen matrix supported autologous - chondrocyte transplantation (act - cs) is a technique that combines a suspension of in vitro expanded articular chondrocytes seeded onto a porcine collagen type i / iii membrane (chondro - gide, fa. the idea behind this technique is that in using act - cs it is possible to combine the benefits of cells delivered as a cell suspension with the benefits of matrix - associated act technique. furthermore, by having isolated adherent cells on the membrane rather than embedded within the matrix, it is thought that the cells would not be encumbered by a cell matrix barrier to allow basal and lateral integration with the surrounding native tissue. the membrane in the act - cs technique is only used as a cell carrier to deliver chondrocytes to the chondral defect. figure 1 demonstrates the characteristic distribution of chondrocytes using act - cs 510 minutes after cell seeding. notably, the chondrocytes are densely distributed along the surface of the membrane and not embedded within a matrix, in contrast to matrix - associated autologous chondrocyte implantation (maci). in addition, the occlusive side of the chondro - gide membrane also acts as a barrier to an influx of surrounding cells (fibroblasts, synoviocytes, etc.) from being incorporated into the regenerative cartilage. because the cells are already adherent, the possibility of cell loss due to leaks and seepage may be minimized and accordingly requires less stitching of the membrane to the surrounding tissue. this may be a particular advantage when repairing lesions that are anatomically less accessible, such as posterior parts of the tibia or femoral condyles. a further theory about the act - cs technique is that cell seeding could result in a more homogenous distribution of the cells within the defect compared with cell suspension techniques, but this theory still lacks scientific evidence and has not been proven so far. cell distribution of chondrocytes 10 minutes following seeding on porcine collagen i / iii (magnification 10x) cell - seeded collagen matrix supported autologous - chondrocyte transplantation (act - cs) is a technique that combines a suspension of in vitro expanded articular chondrocytes seeded onto a porcine collagen type i / iii membrane (chondro - gide, fa. the idea behind this technique is that in using act - cs it is possible to combine the benefits of cells delivered as a cell suspension with the benefits of matrix - associated act technique. furthermore, by having isolated adherent cells on the membrane rather than embedded within the matrix, it is thought that the cells would not be encumbered by a cell matrix barrier to allow basal and lateral integration with the surrounding native tissue. the membrane in the act - cs technique is only used as a cell carrier to deliver chondrocytes to the chondral defect. figure 1 demonstrates the characteristic distribution of chondrocytes using act - cs 510 minutes after cell seeding. notably, the chondrocytes are densely distributed along the surface of the membrane and not embedded within a matrix, in contrast to matrix - associated autologous chondrocyte implantation (maci). in addition, the occlusive side of the chondro - gide membrane also acts as a barrier to an influx of surrounding cells (fibroblasts, synoviocytes, etc.) from being incorporated into the regenerative cartilage. because the cells are already adherent, the possibility of cell loss due to leaks and seepage may be minimized and accordingly requires less stitching of the membrane to the surrounding tissue. this may be a particular advantage when repairing lesions that are anatomically less accessible, such as posterior parts of the tibia or femoral condyles. a further theory about the act - cs technique is that cell seeding could result in a more homogenous distribution of the cells within the defect compared with cell suspension techniques, but this theory still lacks scientific evidence and has not been proven so far. according to accepted recommendations for all act procedures, any damaged cartilage should be removed completely and debrided until the defect is surrounded by a well - defined rim of healthy cartilage. 15) for the first cut to sharply define the edge of the defect. afterwards, sharp instruments such as a sharp spoon or a ring curette should be used to carefully remove all damaged cartilage. bleeding from the subchondral bone should be avoided to minimize the risk of infiltration of bone marrow derived cells into the implant, which may result in fibrocartilage tissue. a negative effect of blood on biochemical and morphological properties of regenerated cartilage has been demonstrated. in addition, direct contact of the seeded cellular layer of the membrane, unencumbered by a bone marrow derived cellular infiltrate, may allow for better basal integration. in case subchondral bone bleeding occurs during debridement, one may address it in one of several ways. for example, one approach is to apply a drop of fibrin glue to the bleeding surface to induce clotting. the glue can be pressed into the opened subchondral bone using a small swab, removing any excess glue after the bleeding is controlled. in case of intralesional osteophytes, often observed following prior bone marrow stimulating techniques such as microfracture, osteophytes should be removed and resected to the level of the surrounding subchondral bone plate. no sclerotic subchondral bone should remain, since it is considered to inhibit basal integration of the implantation with normal tissue. following debridement of the lesion, a template of the defect is traced along its edges. once seeded with the cell suspension, an enlargement of the collagen membrane by approximately 10% occurs. however, the enlargement is not uniform and is influenced by the orientation of the collagen fibers. a space of approximately 1 mm should be left between the template and the surrounding cartilage rim, and the size should be checked again before suturing. during shipment, the cell suspension commonly settles into a pellet at the bottom of the tube. the total volume of suspension fluid can be adapted to the size of the membrane. to do so, excess volume of suspension medium should be discarded prior to cell resuspension. often, a gentle rocking back and forth of the container will be sufficient to resuspend the cells. a plastic (not metal) 18-gauge (or larger) cannula may also be used. alternatively, a plastic (not metal) cannula system can be used for resuspension, and a minimum size of 18 gauge is recommended in order to not harm the cells. the optimal number of chondrocytes per square centimeter may be product - specific and the product insert should be referred to for each preparation. there are no well - controlled clinical trials comparing various doses of cells per square centimeter and clinical outcomes. most frequently, the dosing studies have been based on large - scale animal data. in general, commercially available sources of chondrocyte suspensions recommend a dose between 0.5 and 2.0 10 cells per cm. in the clinical experience of the authors using act - cs, membranes have been seeded at approximately 2 to 3 10 for a 4-cm collagen membrane. after resuspension of the delivered chondrocytes in a small amount of fluid (approximately 3001000 l), cells are applied on the rough side of the collagen membrane using a plastic cannula (18-gauge) by gently dripping the cell suspension until the membrane is saturated (also see fig. 2). it is preferable to start cell application on the edges of the membrane and move towards the middle of the membrane. cell seeding should be performed until the entire membrane is covered with cell suspension and it should be stopped before any fluid runs from the membrane. using the act - cs technique, chondrocytes are directly applied to the membrane immediately before implantation into the prepared cartilage defect specific time for cell adherence to the membrane has not been rigorously tested, but 5 to 10 minutes has been found as sufficient to allow cells to adhere to the membrane. in act - cs, total cell number per ml is determined by the suspension volume absorbed by the membrane. if a volume is used that exceeds the capacity of the membrane to absorb the suspension, then the solution with cells will run off the membrane. the authors recommend applying sufficient fluid to the membrane, such that a liquid film is visible on the membrane but not all of the fluid is absorbed completely. once the cell suspension has been applied and allowed to adhere, small amounts of additional fluid (such as nacl 0.9%) or a moist compress saturated with media may be added to keep the membrane moist. it is important that any materials or liquids coming into contact with the cells be isotonic and buffered. cell distribution on the membrane after 10 minutes is demonstrated in figure 1. after the 10-minute adhesion time the authors restrict the adhesion / seeding time to 1015 minutes as they believe this optimizes the ability to more effectively integrate with the subchondral bone plate layer. the seeded membrane must be handled with care as it is implanted into the defect. the authors strongly recommend using a small pair of tweezers or forceps. on implantation, there should be a direct contact between the cell - seeded surface of the membrane and the subchondral bone. sutures to fix the membrane to the adjacent cartilage is the classic technique that has previously been described in the original first - generation method. in the original technique, a distance of 46 mm between suture knots was recommended to ensure stable fixation allowing the injection of the cell suspension beneath the membrane. the number of sutures (hence time of surgery) can be reduced using the act - cs technique. the spacing and number of sutures should be sufficient to just allow mechanical stability of the cell - seeded membrane to the defect edges and onto the bottom of the defect. gaps between the seeded membrane and adjacent cartilage should be avoided, depending on the lesion size, and often 612 sutures per defect are sufficient to achieve adequate positioning and stability of the implant. the authors recommend a monofilament suture material (i.e., pds 6 - 0, fa. ethicon, nordersted, germany) as monofilament sutures are considered more compatible and less of an irritant to normal cartilage and membrane. although the degradation time for the sutures could play a critical role in clinical outcome, the authors are unaware of any detailed studies that have examined this in order to find an optimal material. demonstrated that suturing was associated with some local degeneration based on a histological evaluation, but it remains unclear if these observations are of clinical relevance. the authors recommend positioning the needle insertion close to the bottom of the defect, in close proximity to the subchondral bone. positioning the membrane on top of the cartilage should be avoided since this could result in delamination or disintegration to the borders and lifting of the membrane from the defect with any shear force. cell - seeded membrane is placed on the bottom of the prepared defect, cell - loaded side of the membrane directed toward and in direct contact with the subchondral bone plate pds 6 - 0 is recommended as a suture material to fix the cell / membrane construct into the adjacent cartilage. a strict position of the needle close to the subchondral bone needs to be regarded in order to provide a close contact of cells and subchondral bone there is general agreement that knots can potentially cause problems, especially in a mechanically active joint. placing the knots on the cartilage surface increases local shear forces and should be avoided. the authors agree that the best position for knots is beneath the surface of adjacent cartilage, directly on the transplanted membrane. this position also forces the membrane to the bottom of the defect directly opposed to the subchondral bone plate. knots are typically placed under the surface of the adjacent cartilage in order to avoid any irritation of the adjacent cartilage after fixation of the cell - seeded membrane by sutures, the authors recommend an additional sealing of the border using fibrin glue (see fig. the cell compatibility of fibrin glue and the ability of fibrin to support chondrogenic phenotype has been reported in various studies. this has also been demonstrated for the combination of fibrin glue and the chondro - gide membrane. any fibrin glue used in act - cs should have demonstrated compatibility with chondrocytes and the collagen membrane used. tissucol (baxter, unterschleiheim, germany) has been shown to have good chondrocyte compatibility, and this type of fibrin glue has also been used in the act - cs study. there is also consensus on the fact that the entire defect should not be filled or covered with fibrin glue. fibrin glue is carefully placed at the interface of membrane and adjacent cartilage in order to seal this intersection. the amount of fibrin glue should be limited in order to reach an appropriate sealing act - cs as described in the present paper uses a porcine collagen type i / iii membrane (chondro - gide, fa. geistlich) and represents an adoption of the initially described technique using a cell suspension injected beneath the identical membrane. for this technique and for the maci technique, which also represents an adoption using the identical biomaterial, various studies report safety, and midterm clinical outcome has been reported in several case series in the treatment of cartilage defects and osteochondritis dissecans. all these studies do not report any specific side effects and adverse events in context with the application of the collagen membrane. using the collagen membrane seems to further reduce the incidence of graft hypertrophy as demonstrated in a prospective randomized trial versus periosteum - covered act as well as in large retrospective studies including more than 400 patients with act. compared with conventional periosteum - covered act, as a possible disadvantage a higher rate of malfusion of the regenerative tissue into the adjacent cartilage has been reported, but this observation does not seem to be specific for the collagen membrane, and it has also been observed in other artificial biomaterials. act - cs represents an adoption of the initial technique using the collagen membrane for act. the first patients were treated using the act - cs technique in october 2005, and the principles of the technique were described in 2009. until preparation of the present manuscript, the authors (m.s. clinical 2-year results of the first 59 patients treated with act - cs have been reported recently, demonstrating a success rate (icrs a and b at 24 months) of 89% and a rate of 94 % improved knee function in the subgroup of patients with single defects. no technique - related complications were observed during the application of act - cs so far. nevertheless, long - term follow - ups are not yet available and results of act - cs used for the treatment for larger defects are still elusive. according to accepted recommendations for all act procedures, any damaged cartilage should be removed completely and debrided until the defect is surrounded by a well - defined rim of healthy cartilage. 15) for the first cut to sharply define the edge of the defect. afterwards, sharp instruments such as a sharp spoon or a ring curette should be used to carefully remove all damaged cartilage. bleeding from the subchondral bone should be avoided to minimize the risk of infiltration of bone marrow derived cells into the implant, which may result in fibrocartilage tissue. a negative effect of blood on biochemical and morphological properties of regenerated cartilage has been demonstrated. in addition, direct contact of the seeded cellular layer of the membrane, unencumbered by a bone marrow derived cellular infiltrate, may allow for better basal integration. in case subchondral bone bleeding occurs during debridement, one may address it in one of several ways. for example, one approach is to apply a drop of fibrin glue to the bleeding surface to induce clotting. the glue can be pressed into the opened subchondral bone using a small swab, removing any excess glue after the bleeding is controlled. in case of intralesional osteophytes, often observed following prior bone marrow stimulating techniques such as microfracture, osteophytes should be removed and resected to the level of the surrounding subchondral bone plate. no sclerotic subchondral bone should remain, since it is considered to inhibit basal integration of the implantation with normal tissue. following debridement of the lesion, a template of the defect is traced along its edges. once seeded with the cell suspension, however, the enlargement is not uniform and is influenced by the orientation of the collagen fibers. a space of approximately 1 mm should be left between the template and the surrounding cartilage rim, and the size should be checked again before suturing. during shipment, the cell suspension commonly settles into a pellet at the bottom of the tube. the total volume of suspension fluid can be adapted to the size of the membrane. to do so, excess volume of suspension medium should be discarded prior to cell resuspension. often, a gentle rocking back and forth of the container will be sufficient to resuspend the cells. a plastic (not metal) 18-gauge (or larger) cannula may also be used. alternatively, a plastic (not metal) cannula system can be used for resuspension, and a minimum size of 18 gauge is recommended in order to not harm the cells. the optimal number of chondrocytes per square centimeter may be product - specific and the product insert should be referred to for each preparation. there are no well - controlled clinical trials comparing various doses of cells per square centimeter and clinical outcomes. most frequently, the dosing studies have been based on large - scale animal data. in general, commercially available sources of chondrocyte suspensions recommend a dose between 0.5 and 2.0 10 cells per cm. in the clinical experience of the authors using act - cs, membranes have been seeded at approximately 2 to 3 10 for a 4-cm collagen membrane. after resuspension of the delivered chondrocytes in a small amount of fluid (approximately 3001000 l), cells are applied on the rough side of the collagen membrane using a plastic cannula (18-gauge) by gently dripping the cell suspension until the membrane is saturated (also see fig. it is preferable to start cell application on the edges of the membrane and move towards the middle of the membrane. cell seeding should be performed until the entire membrane is covered with cell suspension and it should be stopped before any fluid runs from the membrane. using the act - cs technique, specific time for cell adherence to the membrane has not been rigorously tested, but 5 to 10 minutes has been found as sufficient to allow cells to adhere to the membrane. in act - cs, total cell number per ml is determined by the suspension volume absorbed by the membrane. if a volume is used that exceeds the capacity of the membrane to absorb the suspension, then the solution with cells will run off the membrane. the authors recommend applying sufficient fluid to the membrane, such that a liquid film is visible on the membrane but not all of the fluid is absorbed completely. once the cell suspension has been applied and allowed to adhere, small amounts of additional fluid (such as nacl 0.9%) or a moist compress saturated with media may be added to keep the membrane moist. it is important that any materials or liquids coming into contact with the cells be isotonic and buffered. after the 10-minute adhesion time, the membrane may then be implanted into the debrided lesion and sutured. the authors restrict the adhesion / seeding time to 1015 minutes as they believe this optimizes the ability to more effectively integrate with the subchondral bone plate layer. the seeded membrane must be handled with care as it is implanted into the defect. the authors strongly recommend using a small pair of tweezers or forceps. on implantation, if it does, it is important to trim the membrane to size. there should be a direct contact between the cell - seeded surface of the membrane and the subchondral bone. sutures to fix the membrane to the adjacent cartilage is the classic technique that has previously been described in the original first - generation method. in the original technique, a distance of 46 mm between suture knots was recommended to ensure stable fixation allowing the injection of the cell suspension beneath the membrane. the number of sutures (hence time of surgery) can be reduced using the act - cs technique. the spacing and number of sutures should be sufficient to just allow mechanical stability of the cell - seeded membrane to the defect edges and onto the bottom of the defect. gaps between the seeded membrane and adjacent cartilage should be avoided, depending on the lesion size, and often 612 sutures per defect are sufficient to achieve adequate positioning and stability of the implant. the authors recommend a monofilament suture material (i.e., pds 6 - 0, fa. ethicon, nordersted, germany) as monofilament sutures are considered more compatible and less of an irritant to normal cartilage and membrane. although the degradation time for the sutures could play a critical role in clinical outcome, the authors are unaware of any detailed studies that have examined this in order to find an optimal material. demonstrated that suturing was associated with some local degeneration based on a histological evaluation, but it remains unclear if these observations are of clinical relevance. the authors recommend positioning the needle insertion close to the bottom of the defect, in close proximity to the subchondral bone. positioning the membrane on top of the cartilage should be avoided since this could result in delamination or disintegration to the borders and lifting of the membrane from the defect with any shear force. cell - seeded membrane is placed on the bottom of the prepared defect, cell - loaded side of the membrane directed toward and in direct contact with the subchondral bone plate pds 6 - 0 is recommended as a suture material to fix the cell / membrane construct into the adjacent cartilage. a strict position of the needle close to the subchondral bone needs to be regarded in order to provide a close contact of cells and subchondral bone there is general agreement that knots can potentially cause problems, especially in a mechanically active joint. placing the knots on the cartilage surface increases local shear forces and should be avoided. the authors agree that the best position for knots is beneath the surface of adjacent cartilage, directly on the transplanted membrane. this position also forces the membrane to the bottom of the defect directly opposed to the subchondral bone plate. knots are typically placed under the surface of the adjacent cartilage in order to avoid any irritation of the adjacent cartilage after fixation of the cell - seeded membrane by sutures, the authors recommend an additional sealing of the border using fibrin glue (see fig. the cell compatibility of fibrin glue and the ability of fibrin to support chondrogenic phenotype has been reported in various studies. this has also been demonstrated for the combination of fibrin glue and the chondro - gide membrane. any fibrin glue used in act - cs should have demonstrated compatibility with chondrocytes and the collagen membrane used. tissucol (baxter, unterschleiheim, germany) has been shown to have good chondrocyte compatibility, and this type of fibrin glue has also been used in the act - cs study. there is also consensus on the fact that the entire defect should not be filled or covered with fibrin glue. fibrin glue is carefully placed at the interface of membrane and adjacent cartilage in order to seal this intersection. act - cs as described in the present paper uses a porcine collagen type i / iii membrane (chondro - gide, fa. geistlich) and represents an adoption of the initially described technique using a cell suspension injected beneath the identical membrane. this technique has been introduced as the second - generation act. for this technique and for the maci technique, which also represents an adoption using the identical biomaterial, various studies report safety, and midterm clinical outcome has been reported in several case series in the treatment of cartilage defects and osteochondritis dissecans. all these studies do not report any specific side effects and adverse events in context with the application of the collagen membrane. using the collagen membrane seems to further reduce the incidence of graft hypertrophy as demonstrated in a prospective randomized trial versus periosteum - covered act as well as in large retrospective studies including more than 400 patients with act. compared with conventional periosteum - covered act, as a possible disadvantage a higher rate of malfusion of the regenerative tissue into the adjacent cartilage has been reported, but this observation does not seem to be specific for the collagen membrane, and it has also been observed in other artificial biomaterials. act - cs represents an adoption of the initial technique using the collagen membrane for act. the first patients were treated using the act - cs technique in october 2005, and the principles of the technique were described in 2009. until preparation of the present manuscript, the authors (m.s. clinical 2-year results of the first 59 patients treated with act - cs have been reported recently, demonstrating a success rate (icrs a and b at 24 months) of 89% and a rate of 94 % improved knee function in the subgroup of patients with single defects. no technique - related complications were observed during the application of act - cs so far. nevertheless, long - term follow - ups are not yet available and results of act - cs used for the treatment for larger defects are still elusive. in conclusion, although the clinical evidence is limited, the present paper provides concrete guidelines to surgeons on a standardized methodology for using the act - cs technique for the treatment of symptomatic full - thickness cartilage defects. all recommendations were based on a consensus meeting of the authors of the present article. the recommended procedures for act - cs are based on the authors clinical experience in treating more than 200 patients with act - cs over the past 5 years. a standardized methodology provides a framework for further comparative studies between various techniques to identify optimal treatment modalities, especially with evolving innovative regenerative medicine products. | objective : autologous chondrocyte transplantation has become an established therapy for full - thickness cartilage defects. cell - seeded collagen matrix supported autologous chondrocyte transplantation (act - cs) has been introduced as a modification of conventional act, which allows easier handling and is intended to combine the advantages of using a cell suspension (i.e., cell viability and mitotic activity) with the stability and self - containment provided by a matrix of biomaterials. unlike other techniques and products, this seeding step can be easily applied using a porcine collagen type i / iii membrane and autologous chondrocytes in an operating room setting. although some suturing is required, this technique provides the distinct advantage of not requiring a water - tight seal of the bilayer membrane, as is required using the classic cell suspension technique. comparable to other modifications of act, the act - cs procedure requires a specific surgical technique that focuses on the following important details : (1) accurate debridement of the cartilage defect ; (2) preparation of the cells, and seeding and containment of the cells within the transplantation site ; and (3) sealing and suturing around the defect.design:a consensus meeting of leading european orthopedic surgeons specializing in cartilage repair was convened to discuss and standardize the surgical aspects of this technique.results & conclusions : the present article describes and discusses the adoption of these best surgical practices for implementing the act - cs technique, including more detailed descriptions of each phase of the surgery in order to standardize and optimize patient outcomes. |
myelodysplastic syndromes (mds) are a group of disorders clinically characterized by peripheral cytopenia, followed by a progressive impairment in the ability of myelodysplastic stem cells to differentiate and an increasing risk of evolution into acute leukemia.1 mds represent one of the most common hematologic malignancies in western countries. they typically occur in elderly people with a median age at diagnosis of 70 to 75 years in most series, and their annual incidence exceeds 20 per 100,000 persons over the age of 70 years.1 the clinical course of the disease is very heterogeneous, ranging from indolent conditions spanning years to forms rapidly progressing to leukemia.2 this heterogeneity reflects the complexity of the underlying genetic defects.3 according to the prevailing dogma, clonal transformation in mds would occur at the level of a committed myeloid stem cell that can give rise to red cells, platelets, granulocytes and monocytes.4 the biologic hallmark of these stem cells is, rather, dysplasia, which indicates a defective capacity for self - renewal and differentiation and relies on various morphological abnormalities. karyotypic aberrancies (involving loss of genetic material and less frequently balanced translocations) are detected in about 50% of primary mds, and when present are a marker of clonal hematopoiesis.5 important steps have recently been made in characterizing the molecular basis of mds.3 mds del(5q) appears to derive from haplo - insufficiency of genes mapping to chromosome 5q32- q33, in particular from reduced expression of rps14 and mir-145/-146a, and from mutations of casein kinase 1a1 and tp53 genes.6 in addition, acquired somatic mutations have been detected in several genes, including tet2, asxl1, cbl, etv6, ezh2, idh1, idh2, kras, npm1, nras, runx1, and tp53.4 more recently, genes encoding for spliceosome components were identified in a high proportion of patients with mds. these genes include sf3b1, srsf2, u2af35 and zrsr2, and to a lesser extent, sf3a1, sf1, u2af65 and prpf40b.7 although most of the mutated genes in mds can be detected in different myeloid neoplasms and are not specific for mds, they may be of value to provide evidence for a clonal disorder in patients with suspected mds. in a recent comprehensive report,7 a total of 52% of patients with normal cytogenetics had at least one point mutation. these figures are even higher when accounting for mutations of the genes encoding for splicing factors. although the spread of massive genotyping methods will soon make possible for clinicians to detect a broad range of in peripheral blood at a reasonable cost, the screening of such molecular defects can not be recommended at this stage on a routine basis.7 to date, the morphological evaluation of marrow dysplasia represents the basis of the world health organization (who) classification of these disorders.8 this classification provides clinicians with a very useful tool for defining the different subtypes of mds and determining individual prognosis. the combination of overt marrow dysplasia and clonal cytogenetic abnormalities allows a conclusive diagnosis of mds. however, this combination is found only in some patients, who tend to be those with more advanced disease. in many instances, cytogenetics is not informative so that the diagnosis of mds is based entirely and exclusively on morphological evaluation.8 the who proposal has raised some concern regarding minimal diagnostic criteria for formulating the diagnosis of mds.9 morphology may be difficult to evaluate, because cellular abnormalities of bone marrow cells are not specific for mds and may be found in other pathological conditions.10,11 as a consequence, in clinical practice inter - observer reproducibility for recognition of dysplasia is usually poor, particularly in patients who do not have robust morphological markers such as ring sideroblasts or excess of blasts.11 moreover, poor technical quality of the specimen is a common obstacle in the accurate morphological diagnosis of mds and also has an influence on the diagnostic yield of conventional cytogenetics. finally, morphology may be difficult to evaluate in some patients either due to hypocellularity or fibrosis of the marrow.12 flow cytometry (fcm) immunophenotyping was introduced by who proposal for the classification of hematologic neoplasms as an indispensable tool for the diagnosis, classification, staging, and monitoring of several diseases, such as lymphoproliferative disorders and acute leukemias.13 in addition, immunophenotyping has been proposed in last years as a tool to improve the evaluation of marrow dysplasia. rationale for the application of fcm in the diagnostic work up of mds is that : i) immunophenotyping is an accurate method for quantitative and qualitative evaluation of hematopoietic cells (in this context it should be underlined that however, the morphologic definition of bone marrow cells is not equal to and can not be used in an exchangeable manner with flow cytometric nomenclature) and, ii) mds have been found to have abnormal expression of several cellular antigens.1315 flow cytometry immunophenotyping is able to identify specific aberrations in both the immature and mature compartments among different bone marrow hematopoietic cell lineages.1620 although no single immunophenotypic parameter has been proven to be diagnostic of mds, combinations of such parameters into scoring systems have been shown to discriminate mdss from other cytopenias with high sensitivity and acceptable specificity. flow cytometry was proven to be highly sensitive in identifying patients likely to be suffering from a clonal disease process (ie, an mds lacking specific diagnostic markers such as excess blasts, ring sideroblasts or karyotypic aberrations) rather than cytopenia of undetermined significance, which includes cases of sustained cytopenias in one or more lineages that do not meet the minimal criteria for mds and can not be explained by any other hematologic or nonhematologic disease.1620 in addition, flow cytometry is useful for distinguishing refractory anemia from refractory cytopenia with multilineage dysplasia by identifying immunophenotypic abnormalities in myeloid and monocytic compartments.1620 although further prospective validation of markers and immunophenotypic patterns against control patients with secondary dysplasia and further standardization in multicenter studies are required, at present, flow cytometry abnormalities involving one or more of the myeloid lineages can be considered as suggestive of mds. standard methods for cell sampling, handling, and processing, and minimal combinations of antibodies for flow cytometry analysis of dysplasia in mds have recently been established by the international flow cytometry working group within the european leukemianet.21 the integration of flow cytometry immunophenotyping following these standards is recommended in the workup of patients with suspected mds by the european leukemianet guidelines for diagnosis and treatment of primary mds,22 although the implementation of these procedures may not be immediately feasible in some hematologic centers. in this report, we reviewed the most relevant advancements in the evaluation of marrow dysplasia by fcm in mds. morphological granulocytic dysplasia as defined by who criteria is present in about 60% of mds patients at diagnosis.8,9 most significant morphological alterations on granulocytic lineage included hypogranularity on myeloid cells, the presence of pseudo - pelger neutrophils and increased prevalence in bone marrow of myeloid cells in the earliest stage of maturation.11 these abnormalities significantly affected the detection of physical parameters (i.e., side scatter, ssc and forward scatter, fsc) by fcm.23 defective capacity for self - renewal and differentiation by myelodysplastic stem cells also relies on various abnormalities of antigen expression on granulocytic cells, which may be easily detected by fcm due to a large availability of specific antibodies for myeloid lineage.1619 reported aberrancies of granulocytic lineage include the presence of antigens that are not normally present, such as lymphoid antigens, and altered expression of myeloid antigens, either in a single population of cells or within a generation of maturing cells. furthermore, monocytic compartment is also affected in mds.1619 davis studied for the first time the pattern of cd16 and cd11b expression by maturing granulocytes in the bone marrow of patients with mds and healthy controls.15 there was a highly consistent normal pattern of cd11b and cd16 expression in the granulocytic series in healthy subjects, while in mds patients an increased percentage of granulocytic cells with low cd16 or both low cd16 and low cd11b was noticed.15 in addition, an altered granulocytic maturation pattern can be demonstrated by plotting cd13 versus cd16.1619 during maturation ; myeloid cells normally acquire increasing levels of cd16 that are initially accompanied by a decrease in cd13 expression as cells mature from blasts through the myelocyte and metamyelocyte stages of maturation, followed by intermediate levels of cd13 in band forms and high levels in segmented neutrophils. several abnormalities on cd13/cd16 maturation pattern were described in mds patients, including an increase of cells in myelocyte and metamyelocyte stages of maturation and a decrease of cd13+cd16 + neutrophils.1619 although these investigations defined immunophenotypic abnormalities in mds, they did not address the potential contribution of fcm to the diagnosis of mds.. published in 2001 was the first to demonstrate that the identification of immunophenotypic abnormalities by fcm is useful in establishing a diagnosis of a mds, especially when the results of the morphologic evaluation and cytogenetic studies are indeterminate.16 in addition to maturation abnormalities, aberrancies in the expression of several antigens on granulocytes such as cd64, cd10, and cd56 were described in mds. lymphoid antigens, such as cd2, cd5, cd7, and cd19 may be abnormally expressed on myeloid progenitors and maturing myeloid cells. moreover, a common finding in these patients is the atypical expression of antigens on immature myeloid cells that are normally expressed on mature myeloid cells, such as cd11b and/or cd15.1719 as far as monocytic compartment is concerned, most frequent abnormalities observed in mds patients include altered expression of cd56, hla - dr, cd36, cd33, cd15, cd14, cd13, and cd11b.18,19,24 in general, the amount of abnormalities reported by fcm correlates with the degree of dysplasia assessed by morphology. although most of the studies have evaluated bone marrow cells, there is some evidence that fcm analysis of peripheral blood could also assist in the diagnosis of mds.25 scientific evidence suggests that aberrant antigen expression by myeloid cells is more frequent and carries more discriminant weight on detection of marrow dysplasia than altered expression of monocytic antigens.24 a single myeloid immunophenotypic abnormality was reported in about 3040% of patients affected with nonclonal cytopenia.1620 therefore, a single myeloid immunophenotypic abnormality is not a definitive finding for mds, and other abnormalities should be detected on granulocytic cells to conclude that myeloid dysplasia is present. multiparametric evaluation of myeloid and monocytic maturation and antigen expression pattern leads to the identification of two or more aberrancies in the great majority of mds cases (from 70% to more than 90% in different studies).1620,26 in general fcm is more sensitive in detection of myeloid dysplasia with respect to morphology, and immunophenotypic myeloid abnormalities are identified in a significant percentage of cases (from 20% to more than 90%) classified as refractory cytopenia with unilineage dysplasia or unclassifiable mds.1620,26 in addition, fcm was found to be useful for detection of marrow dysplasia in a proportion of patients with marrow hypocellularity, fibrosis or inadequate specimen collection, suggesting that variables related to sample quality are less significant in immunophenotypic analysis than in morphological evaluation.17 the great variability on the percentage of reported immunophenotypic abnormalities in mds patients reflect in part the biological heterogeneity within these disorders, but more likely, the lack of a standardized and reproducible procedure for the evaluation of these parameters.21 the most largely used approach to evaluate myeloid dysplasia by fcm is pattern recognition analysis.16 this is a qualitative method based on recognition of a deviation from normal antigen expression pattern. although similarly to morphological evaluation this approach is a good tool for expert operators (i.e., people with extensive knowledge of changes in antigen expression in normal and pathological hematopoietic cell differentiation) pattern recognition analysis presents several weak points. the numerical description of the results is difficult, thus quantitative analysis is not possible ; moreover, the precise definition of the normal pattern of reference may be complex.13 overall, fcm multiparametric approaches based on a quantitative evaluation of myeloid antigens allow to classify about 90% correctly of cases with suspected mds.1620,26 the eln working group for fcm in mds started a consensus process on how to standardize sample collection/ preparation and data acquisition, that is expected to significantly improve the fcm accuracy in detection of marrow dysplasia.21,2730 clonal transformation in mds occurs at the level of a myeloid committed stem cell which has a competitive advantage over normal stem cell compartment.1 these hematopoietic precursors (blasts) are morphologically defined as immature cells with uncondensed chromatin pattern, prominent nucleoli, low nuclear / cytoplasmic ratio, and no / few cytoplasmic granules.11 the evaluation of blast compartment has diagnostic relevance in the who system, and the percentage of marrow blasts has recognized to have prognostic effect by all the currently available prognostic scores.8 in the who guidelines, despite inaccuracies inherent in manual differential counting, morphological analysis is actually the gold standard for determining blast percentage.11 the first attempt of fcm immunophenotyping was to provide a quantitative estimation of bone marrow blasts with increased sensitivity and reproducibility with respect to morphological count. unfortunately, the quantitative evaluation of marrow blasts in mds by fcm presents both technical and intrinsic limitations.13 first, mds blasts are not predominant cells in the bone marrow making their reliable analysis difficult, and in addition they are identified in the cd45 versus ssc dotplot as cd45lowssclow cells ; however, hypogranular more mature myeloid cells may have decreased ssc and fall in this region, and it may be difficult to distinguish monoblasts from more mature monocytes.13 the percentage of cd34 + cells determined by fcm has been tested as a substitution for a visual blast count. however, although hematopoietic cells that express cd34 are blasts, not all blasts express cd34. it should be considered in addition that marrow samples for morphological evaluation can differ form that for fcm analysis in terms of cellularity. hence, the percent of cd34 + cells determined by fcm as substitution for a visual blast count in mds is discouraged by current who classification.8,31 more interesting results in the light of a diagnostic application of fcm in work - up of mds patients derive from the analysis of immunophenotypic abnormalities of blast cell compartment. the proportion of cd34 + cells is significantly higher in mds with respect to healthy subjects, and the great majority of cells are committed to the myeloid lineage (cd38+hla - dr+cd13+cd33+).14,32 in addition, a significant down - regulation of b - cell lineage - affiliated genes was observed in cd34 + hematopoietic precursors isolated from low - risk mds with respect to healthy controls and patients with nonclonal cytopenia, and a reduction in stage i hematogones is one of most consistent immunophenotypic findings in mds patients.33,34 in different studies considering patients performing bone marrow evaluation for peripheral blood cytopenia, a significant decrease of cd34 + b cell progenitors was observed in 4070% of subjects with a conclusive diagnosis of mds and in 2040% of patients with nonclonal cytopenia. the analysis of both percentages of cd34 + myeloblasts and cd34 + b cell precursors was found to have little interobserver variability.33,34 several other immunophenotypic abnormalities on mds blast cells were reported, including asynchronous co - expression of stem - cell and late - stage myeloid antigens (cd117, cd15, and cd11b) or abnormal expression of lymphoid markers (cd2, cd5, cd7, cd19, and cd56).18,19,32,33,35 however, most of these parameters do not have adequate reproducibility in the mds setting with the exception of lymphocytes- to - myeloblasts cd45 ratio that ensures acceptable interobserver variability by adjusting data on target cells with those on lymphocytes in the same sample. the analysis of percentage of cd34 + myeloblasts, cd34 + b - cell progenitors and myeloblast cd45 expression by fcm has little interoperator variability and appears to be applicable in many laboratories.36,37 when combined together with the evaluation of ssc on granulocytes, these parameters differentiate correctly the majority of mds and pathological controls, sensitivity ranging from 30 to 70% and specificity ranging from 80% to more than 90% in different studies.3638 (figure 1) all these findings strongly suggest that cd34-related parameters are good candidates for the identification of diagnostic markers that not only can be used for the diagnosis of mds patients but also are relatively stable and result in acceptable between - operator data variation. erythroid dysplasia is the milestone of the morphological diagnosis of mds. in fact, it is present in almost all patients with mds and is the only morphological abnormality in those with refractory or sideroblastic anemia.8,11 the evaluation of erythroid dysplasia represents a challenge in the immunophenotypic analysis of myelodysplastic marrows : the precise identification of marrow erythroid precursors is problematic, and there is a limited availability of specific markers.16 the first critical issue of erythroid compartment immunophenotyping is the gating strategy to identify marrow erythroid precursors.21,27,39 nucleated erythroid cells are characterized by reduced / absent cd45 and low ssc. to gate cd45dim to negative / ssclow cells is certainly simple and seems likely to be reproducible. however, this region also contains mature (anucleate) red cells, cellular debris, and nonhematopoietic cells, which are not discriminable on the basis of cd45 or scatter proprieties. alternatively, an immunological gate based on the antigens expressed by erythroid cells can be performed. during physiological development from the basophilic erythroblast to the erythrocyte, there is a progressive decrease in cd45 expression.21,27,39 an increase in glycophorin a (gly a) is observed early upon differentiation from the basophilic erythroblast to the orthochromic erythroblast. finally, cd71 is one of the earlier antigens expressed during erythroid maturation (which anticipates gly a expression), remains on the reticulocyte after enucleation and then is lost prior to the loss of the rna. from a theoretical point of view, gating erythroblast on the basis of cd71 expression would be preferable, gly a cells excluding a proportion of more immature erythroid precursors, which may be increased in mds.21,27,39 however, a dysregulation of cd71 expression is reported in mds, and gly a that has a very tight coefficient of variation of intensity from individual to individual should be preferentially adopted in gating erythroid precursors in the setting of mds. the lysis process is also critical, affecting nucleated as well as mature red blood cells to an unknown variable degree.27,30,39 although a no - lyse, no - wash system would provide the most accurate estimate of the nucleated red cell, a lyse no - wash approach is certainly simpler and more easily implementable in the diagnostic workup of mds patients.. demonstrated for the first time the feasibility of the evaluation of erythroid dysplasia by fcm.16 however, the only consistent erythroid abnormality in this study was a dys - synchronous expression of cd71 versus gly a on red cell precursors. in last years an increasing amount of studies addressed the issue of the immunophenotypic evaluation of erythroid compartment in mds.4043 flow cytometric aberrancies that have been reported to reflect mds- related dyserythropoiesis are : a) an increased number of nucleated erythroid cells within total nucleated cells ; b) an altered proportion of consecutive erythroid differentiation stages, such as an increased number of immature erythroid cells (cd117 + and/or cd105 +) or, by contrast, a decrease in erythroid progenitors ; c) an abnormal pattern of cd71 versus cd235a ; d) a reduced expression of cd71 and/or cd36 ; and e) an overexpression of cd105. most of these aberrancies are present in 7080% of mds cases.4043 the eln working group for fcm in mds recently reported the results of a multicenter study focused on defining those erythroid fcm parameters that enable distinction of dyserythropoiesis associated with mds from non - clonal cytopenias.43 analysis of the presence of aberrancies in the erythroid markers cd71 and cd36 (expressed as the coefficient of variation, cv), together with the mfi of cd71 and an abnormal percentage of cd117 + erythroid progenitor cells provided the best discrimination between mds and non - clonal cytopenia. a weighted score based on these four parameters yielded a specificity of 90% and a sensitivity of 33%. addition of erythroid aberrancies to flow cytometric models based on the evaluation of myeloid abnormalities may significantly increase the sensitivity to detect myelodysplastic changes in bone marrow.4043 the implementation of who classification of mds in clinical practice compels a refinement of the accuracy to detect marrow dysplasia.8 fcm immunophenotyping has been proposed as a tool to improve the evaluation of marrow dysplasia.13 to become clinically applicable, fcm analysis should be based on parameters with sufficient specificity and sensitivity, data should be reproducible between different operators, and the results should be easily understood by clinicians.13,15 with respect to this ideal situation, the results of the studies that pointed out the feasibility of immunophenotyping in diagnostic work - up of mds patients raise some concerns : no single marker has proved able to discriminate accurately between mds and other pathological conditions, no consensus exists on which diagnostic parameters are the most appropriate, and published protocols are mainly based on a qualitative analysis of cytometric variables thus limiting a wide clinical implementation.21,27,29 however, in recent years significant progresses were made. clonal transformation in mds occurs at the level of a cd34 + committed stem cell, and therefore cd34-related parameters are good candidates for identification of diagnostic markers for these disorders.4,31,32 consistent immunophenotypic aberrations reported in mds cd34 + cell compartment are an increase of cd34 + myeloblasts, a decrease of b cell progenitors, expression of lymphoid antigens and abnormal cd45 expression. increasing evidence suggests that these parameters have little interoperator variability and, when combined, are able in discriminating between mds and patients with nonclonal cytopenia.31,37 evaluation of erythroid dysplasia represents a challenge in the immunophenotypic analysis of myelodysplastic marrows due to a limited availability of specific markers.16 promising results are coming from recent studies, showing that the addition of erythroid aberrancies to flow cytometric models based on the evaluation of myeloid abnormalities may significantly increase the sensitivity to detect myelodysplastic changes in bone marrow.4043 a standardized application of fcm in the diagnosis of mds also requires a minimal variability in sample processing, antibody combinations, and data acquisition. the european leukemianet (eln) working group for fcm in mds started a consensus process on how to standardize sample collection / preparation and data acquisition. it is expected to significantly improve the diagnostic accuracy of fcm in mds.21,27,28,29 according to the available evidence and published diagnostic guidelines, in clinical practice immunophenotyping is strongly indicated in the screening evaluation of patients with peripheral blood cytopenia:13,22 in this clinical situations, it can provide a sensitive screen for the presence of hematologic malignancy and/or assist in demonstrating the absence of disease. in addition, when morphology and cytogenetics are indeterminate, an abnormal phenotype determined by fcm can help to establish a definitive diagnosis of mds.13,22 | the pathological hallmark of myelodysplastic syndromes (mds) is marrow dysplasia, which represents the basis of the who classification of these disorders. this classification provides clinicians with a useful tool for defining the different subtypes of mds and individual prognosis. the who proposal has raised some concern regarding minimal diagnostic criteria particularly in patients with normal karyotype without robust morphological markers of dysplasia (such as ring sideroblasts or excess of blasts). therefore, there is clearly need to refine the accuracy to detect marrow dysplasia. flow cytometry (fcm) immunophenotyping has been proposed as a tool to improve the evaluation of marrow dysplasia. the rationale for the application of fcm in the diagnostic work up of mds is that immunophenotyping is an accurate method for quantitative and qualitative evaluation of hematopoietic cells and that mds have been found to have abnormal expression of several cellular antigens. to become applicable in clinical practice, fcm analysis should be based on parameters with sufficient specificity and sensitivity, data should be reproducible between different operators, and the results should be easily understood by clinicians. in this review, we discuss the most relevant progresses in detection of marrow dysplasia by fcm in mds |
in vivo carcinogenicity testing is an expensive and time - consuming process, and as a result, only a relatively small fraction of new and existing chemicals has been tested in this manner. therefore, the development and validation of alternative approaches is desirable. we previously developed a mammalian in vitro assay for genotoxicity based on the ability of cells to increase their level of the tumor - suppressor protein p53 in response to dna damage. cultured cells are treated with various amounts of the test substances, and at defined times following treatment, they are harvested and lysed. the lysates are analyzed for p53 by western blot and/or enzyme - linked immunosorbent assay analysis. an increase in cellular p53 following treatment is interpreted as evidence for dna damage. to determine the ability of this p53-induction assay to predict carcinogenicity in rodents and to compare such results with those obtained using alternate approaches, we subjected 25 chemicals from the predictive toxicology evaluation 2 list to analysis with this method. five substances (citral, cobalt sulfate heptahydrate, d&c yellow no. 11, oxymetholone, and t - butylhydroquinone) tested positive in this assay, and three substances (emodin, phenolphthalein, and sodium xylenesulfonate) tested as possibly positive. comparisons between the results obtained with this assay and those obtained with the in vivo protocol, the salmonella assay, and the syrian hamster embryo (she) cell assay indicate that the p53-induction assay is an excellent predictor of the limited number of genotoxic carcinogens in this set, and that its accuracy is roughly equivalent to or better than the salmonella and she assays for the complete set of chemicals.imagesfigure 1figure 2figure 3figure 4 |
|
blindness secondary to ocular trauma occurs in approximately half a million people worldwide,1 and that affecting the cornea may result in significant ocular morbidity and visual difficulty.2 corneal injuries make up the vast majority of ophthalmic cases seen in emergency departments, both in the pediatric and adult age - groups.3 diagnosing and managing pediatric corneal injuries is much more challenging compared to adult patients, and it is also one of the commonest cases seen in pediatric emergency setting.2 corneal lacerations or perforations are usually caused by high - speed objects flying into the eye. missiles with stones, catapults, glass, knives, sport - related injuries, and fishing - related injuries are a few of the commonest causes of corneal lacerations that have been reported.46 corneal laceration caused by a river crab claw is an unusual case, and to the best of our knowledge it has not been reported in any literature thus far. close monitoring of the patient is important, as physical findings may be misleading and would result in poor visual outcome, especially in the pediatric age - group. a healthy 5-year - old boy presented with right eye pain associated with tearing and photophobia of 1 day s duration. he tried to fling the crab off, but the crab flew and hit his right eye. following the incident, he developed pain over the right eye associated with excessive tearing, photophobia, and poor vision. however, there were no floaters, flashes of light, or curtain field defects. according to his father, he had good premorbid vision in both eyes prior to the incident. there was a cornea ulcer centrally obscuring the visual axis region measuring 1.4 mm horizontally and 1.0 mm vertically (figure 1). there were fibrin clumps located beneath the corneal ulcer with hypopyon in the anterior chamber (figure 1). he was diagnosed with corneal ulcer of the right eye, most probably secondary to bacterial infection following trauma. he was treated with topical 0.9% gentamicin and 5% ceftazidime eyedrops hourly after performing corneal scrapping. on the day following the injury, the fibrin clumps beneath the corneal ulcer got dislodged and revealed a full - thickness corneal laceration wound centrally (figure 2). the full - thickness corneal laceration wound was 1.4 mm in length across the visual axis. the anterior chamber was noted to be very shallow, with fibrin clumps located in the superotemporal region and disappearance of the hypopyon. he was then diagnosed as having a right eye full - thickness corneal laceration wound with severe traumatic uveitis secondary to trauma. he underwent right eye examination under general anesthesia, and the corneal laceration was repaired with 10/0 nylon sutures. postoperatively, the patient was treated with topical 0.3% gentamicin, 5% ceftazidime, and 0.1% dexamethasone eye - drops, in addition to oral ciprofloxacin 250 mg 12-hourly for a week. culture from the corneal scrapping specimen grew citrobacter diversus and proteus vulgaris, which were sensitive to gentamicin and ceftazidime. the anterior - chamber inflammation resolved completely over several days. a week later, visual acuity in the right eye had improved to 6/36 and 6/12 with pinhole. the topical ceftazidime and dexamethasone eye - drops were tapered slowly within the next 4 weeks. corneal sutures were completely removed at 8 weeks postoperation. at 2 weeks post - suture removal, subjective refraction revealed right eye best - corrected vision of 6/9 and 6/6 with pinhole. trauma involving the cornea can be a devastating injury to the eye, and causes a significant visual difficulty and ocular morbidity.2,7 penetrating corneal injuries are one of the commonest form of ocular trauma seen at the emergency department, and they can vary from a small perforation to an extensive laceration.2 it has been reported that 6.8%14.7% of ocular traumatic injuries that present at emergency departments are corneal lacerations and perforations,810 and these are the commonest finding in the pediatric age - group.4,11 omobolanle found that males have a higher incidence of corneal lacerations compared to females, with a ratio of approximately 3:1 and mean age of 8.7 years.4 common symptoms of patients with corneal laceration include severe pain, tearing, photophobia, blurring or reduced vision, foreign - body sensation, and sometimes blood in the eye. detecting, diagnosing, triaging, and deciding on the treatment should be done as soon as possible to achieve the best result and outcome.12 culturing of the wound site is imperative if there are suspicions of an infectious etiology,13 which we did for our patient, as river crabs are present in soil and water that may be contaminated with organisms. in our patient, our patient probably sustained an injury to the cornea from the claws of the river crab when he was trying to swing the crab away from his clamped finger. following that, an acute inflammatory reaction set in, probably secondary to the impact of the trauma (perforation and concussion), and resulted in anterior - chamber exudation, and this intense inflammation sealed the lacerated corneal wound temporarily when he was seen shortly after the injury. somehow, the clumped fibrin dislodged the following day, revealing a full - thickness corneal laceration wound. the treatment of corneal laceration has evolved over the years, both medically and surgically. the standard surgical management for corneal laceration is still the interrupted 10 - 0 nylon, which is able to oppose the anterior and posterior edges well without any override or underride. however, placing a suture for a central corneal laceration interferes with vision, and the tension that sutures exert on tissue to pull it together may create irregular astigmatism. also, unlike a sealant or glue, which forms a continuous bond, sutures are spaced apart and create a bond that is weaker in some areas than others. aukerman mentioned that tissue adhesives in repairing superficial, linear, and low - tension lacerations are effective, and have proven results comparable to those with conventional suturing.14 velazquez suggested that either method of closure, adhesive or suture, is able to withstand the physiologic increases in intraocular pressure postoperatively, and that biodendrimer adhesives are able to seal large corneal lacerations.15 given that tissue adhesive can be very costly, several cases are scheduled together to save cost. in our patient, it was an emergency and his parents were unable to afford the cost of tissue adhesive, so we proceeded with suturing. however, the primary goal of initial repair is to restore the integrity of the globe, thus preventing hypotony and infection, and the secondary goal, which can be achieved during the primary repair or secondary procedures, is to restore vision by means of repairing damaged intraocular structures.10 corneal lacerations and perforations, especially in the pediatric age - group, are significantly challenging,2 right from the time the child presents at the eye clinic, through diagnosing, treatment, management, and compliance with therapy,16 to follow - up and finally visual rehabilitation and the outcome. corneal laceration is unlikely to cause mortality, but can result in significant morbidity.6 common associated complications are astigmatism due to corneal sutures and subsequent scarring, corneal opacity, and amblyopia. with the current alternative suture techniques and proper understanding of the cornea s refractive properties via topography, we were able to restore corneal curvature to its best.17 navon concluded that after suture removal, the cornea regains almost normal contour due to its unique topographic memory.18 in order to prevent amblyopia, early correction of refractive error, reestablishing a clear media, and ensuring the use of injured eye by patching the normal eye are very important.2 however, the management and prognosis of corneal lacerations varies, and strongly depends on the nature of the object involved and the wound characteristics, site and extent of the wound, time of presentation and treatment started, and other associated ocular injuries, such as lens opacity, vitreous hemorrhage, retinal detachment, limbal scleral laceration with uveal tissue prolapse, lid laceration, and optic nerve avulsion.3,4 if not treated promptly, ocular trauma in children can result in development of intractable amblyopia. many factors play an important role in managing corneal trauma in order to prevent poor vision. besides that, by educating parents and caregivers in the importance of supervising children while they play and immediate action when trauma takes place is important in optimizing visual outcomes.4 | a 5-year - old boy presented with right eye pain associated with tearing and photophobia of 1-day duration. he gave a history of playing with a river crab when suddenly the crab clamped his fingers. he attempted to fling the crab off, but the crab flew and hit his right eye. ocular examination revealed a right eye corneal ulcer with clumps of fibrin located beneath the corneal ulcer and 1.6 mm level of hypopyon. at presentation, the seidel test was negative, with a deep anterior chamber. culture from the corneal scrapping specimen grew citrobacter diversus and proteus vulgaris, and the boy was treated with topical gentamicin and ceftazidime eyedrops. fibrin clumps beneath the corneal ulcer subsequently dislodged, and revealed a full - thickness corneal laceration wound with a positive seidel test and shallow anterior chamber. the patient underwent emergency corneal toileting and suturing. postoperatively, he was treated with oral ciprofloxacin 250 mg 12-hourly for 1 week, topical gentamicin, ceftazidime, and dexamethasone eyedrops for 4 weeks. right eye vision improved to 6/9 and 6/6 with pinhole at the 2-week follow - up following corneal suture removal. |
otosclerosis was first described in 1741 by valsalva, who noted ankylosis of the stapes in an autopsy on a deaf patient and in 1894 politzer defined otosclerosis as a clinical condition. it is an illness that affects the optic capsule, which forms part of the group of osteodystrophies, caused by changes in bone metabolism. it is characterised by the presence of several spots of reabsorption and bone repositioning. despite significant research having been undertaken on the topic, the cause of otosclerosis remains unknown, although most authors consider it a multifactorial disease. there would appear to be a genetic predisposition with autosomal dominant inheritance, although it may occur sporadically. caucasians are most typically affected, although the prevalence rate of clinical otosclerosis is lower than 1% [13 ]. it is most common amongst the middle aged, with women most commonly affected [47 ]. it is one of the most common causes of conductive hearing loss and tends to be progressive in nature. treatment tends to be medical or surgical, with surgery being the preferred option, namely, a stapedotomy or stapedectomy. although uncommon, in certain circumstances, some health professionals treat the condition medically with bisphosphonates or fluorine compounds, normally during the initial stages of the illness, either separate from or in combination with calcium and vitamin d. the use of glucocorticoids has also been proposed when there is an associated sensorineural component [9, 10 ], and even the use of calcitonin has been suggested. dizziness is a complication of stapedial surgery mentioned in the literature, as it poses a significant risk of vestibular damage, perilymphatic fistula and prosthesis displacement, in addition to other complications [12, 13 ]. various studies have been carried out to assess the improvement in conductive hearing loss or sensorineural loss after stapedial surgery ; however, little research has been undertaken to assess vestibular functions after surgery. symptoms related to vestibular function disorders in patients with otosclerosis have also been documented prior to surgery [14, 15 ], with clinical evidence demonstrating the presence of dizziness in almost 17 to 23% of otosclerosis patients [15, 16 ]. the purpose of this study is to assess this presence and distinguish between dizziness before and dizziness after surgery in patients with otosclerosis. we have designed a prospective and observational study, which was approved by the ethics committee at our hospital. all patients on whom stapedial surgery was carried out at our hospital between october 2013 and december 2014 were invited to participate. as part of the service, partial posterior stapedotomy / stapedectomy are carried out under general anesthetic by employing a transcanal approach. by means of a horizontal incision in the skin of the outer ear canal 's posterior superior wall, 5 - 6 mm from the annulus, a tympanomeatal flap is created that makes it possible to access the tympanic cavity. to guarantee better control of the oval window, curettage or drilling is performed several times in the canal 's posterior superior wall, sparing the tympanic chord. after confirming the security of the stapes, the platinotomy, incudostapedial joint disarticulation, sectioning of the stapes muscle tendon, fracturing of the stapes superstructure, lengthening of the platinotomy / platinectomy, and installation of the causse - type polytetrafluoroethylene prosthesis (teflon) are carried out. for stapedial surgery, we accepted the recommendation of an air - bone gap equal to or greater than 30 db. the presence of dizziness was assessed before and 4 months after surgery. for instances of dizziness, the portuguese version of the dizziness handicap inventory questionnaire the questionnaire comprises 25 simple questions, grouped into three categories : physical, emotional, and functional, with a variable score of between 0 and 100. patients submitted to revision surgery and those that failed to attend follow - ups were excluded. for the purposes of statistical analysis, the spss program was used and a level of statistical significance of p < 0.05 was allocated. 140 patients were included in the study, 96 of whom (68.6%) were women. age varied between 23 and 66, with an average age of 42. in regard to the ear operated on, right ear was for 91 patients (65%) and left ear was for 49 patients (35%). 48 patients (34.3%) mentioned dizziness in one of the two study periods. before surgery, when questioned on the presence of any kind of vestibular disorder, 12 patients (8.6%) mentioned dizziness. 36 patients (25.7%) mentioned dizziness 4 months after surgery, of whom none had mentioned dizziness before surgery. 28.1% of the patients, without dizziness before surgery, developed vestibular complains after surgery. the total postoperation scores of the questionnaire on patients with dizziness varied between 2 and 18 points, with an average of 10 points. there was no significant statistical difference between gender and age and the presence or seriousness of dizziness, whether in the presurgical period or in the 4 months after surgery. women suffer otosclerosis more frequently than men, with a variable ratio of women to men of up to 2 : 1. amongst patients subject to stapedial surgery at our hospital, during the study period, 96 (68.4%) this data is similar to the figures obtained by other authors : 68.4% women in a study carried out in spain and 67% women in a french study. we obtained a ratio of women to men of 2 : 1, which is also consistent with the literature [3, 17, 18 ]. the most affected age range was between 40 and 49 years of age (56%). in a study carried out on 475 spanish patients, the most affected age range was between 15 and 45 years of age (62.2%) whereas, as part of a study carried out in england on 65 english patients, the most affected age range was between 40 and 49 years of age. therefore, our results with regard to the most affected age range align with the results of publications released by other authors in the literature. the condition was bilateral for 11 patients (7.9%) as part of our study, somewhat lower than in most of the studies published. as part of a study carried out in india, the condition was bilateral for 70% of study patients, whereas in an iranian study this figure stood at just 18.2%. the cause of a lower value having been recorded may be attributable to the fact that patients with previous stapedial surgery were excluded from the study. as part of this study, when asked regarding the presence of any kind of vestibular disorder before surgery, 12 patients (8.6%) mentioned dizziness. this amount is lower than what is provided in the literature, with most studies returning dizziness rates of ranges between 17% and 23% [15, 16 ], for patients with otosclerosis. amongst these patients, dizziness reduced after surgery, a phenomenon also identified by other authors. the likely mechanism suggested by other authors is the fact that the quick recession is attributable to the sudden ruptures in the membranous labyrinth that cause changes in intralabyrinthine pressure due to changes in the volume of inner ear fluids. after stapedial surgery, as a result of movement in the ossicles and due to the prosthesis, a compensatory mechanism is established that prevents changes in pressure, which could explain the disappearance of dizziness. as part of this study, in the 4th month following surgery, 36 patients (25.7%) reported dizziness. dizziness is a common case in the days following surgery on the stapes [23, 24 ] ; however, it rarely lasts longer than a week. generally speaking, patients affected develop a permanent vestibular hypofunction, and over time they get used to the condition and are asymptomatic. birch and elbrond reported a rate of dizziness lasting longer than one week of 4%, and plaza mayor recently reported a rate of persistent vertigo lasting 12 months of 2.6%. the results obtained from this study were higher than those reported by the above authors, but very few studies assessing the presence of dizziness in the months following stapedial surgery have been carried out. amongst these patients, as part of our study, all denied experiencing dizziness before surgery, which has made it possible to conclude that stapedial surgery could cause new vestibular disorders that are not related to the condition as such, which would remain in place after a 4-month period. as part of this study, the total questionnaire scores in the postoperating period for patients with dizziness varied between 2 and 18 points, with an average score of 10 points ; thus, dizziness can be considered light (whenever the result of the questionnaire is < 30), with no significant effect on day - to - day activities. our study demonstrates that vestibular disorders may persist after the immediate postoperating period and highlights the need for patient clarifications at the time of providing informed consent. | introduction and objectives. vertigo is a described complication of stapedial surgery. many studies have been conducted to assess the improvement of hearing loss, but there are few studies that assess vestibular function after stapedial surgery. the aim of this study was to evaluate the presence and characterize the vertigo after stapedial surgery. methods. we conducted a prospective observational study. patients undergoing stapedial surgery in our hospital between october 2013 and december 2014 were invited to participate. the vertigo was assessed before and 4 months after surgery, using the dizziness handicap inventory. results. we included 140 patients in the study. 12 patients (8.6%) reported vertigo before surgery, and all of them denied vertigo after surgery. 36 patients (25.7%) reported vertigo four months after surgery, and none of them had vertigo before surgery. postoperative total scores in patients with vertigo ranged between 2 and 18 points. conclusion. the study shows that vestibular disorders may remain after the immediate postoperative period and reinforces the need for clarification of the patient in the informed consent act. |
osteoarthritis (oa) is a highly prevalent joint disorder with a great pain and disability burden1. it is characterized by the loss of cartilage structure, subchondral bone sclerosis, synovial inflammation, and osteophyte formation with involvement of the whole joint (i.e., joint failure)2. different risk factors have been suggested for oa, such as age, female sex, and obesity. however, other potential risk factors have also been suggested, such as the presence of diabetes mellitus (dm), menopause, and high cholesterol levels3,4,5,6. atherosclerosis is also a highly prevalent chronic disorder that has a substantial impact on quality of life and leads to ever - increasing costs to society1, 7. several observational studies have reported an association between subclinical measures of atherosclerosis and oa of the hands and knees, predominantly among women8,9,10. similarly, a greater risk of cardiovascular death has been reported for patients with knee and/or hip oa11. however, it is unclear whether atherosclerosis and oa are associated as concurrent diseases due to a common etiology or whether they are causally related. the purpose of this study was to investigate the relationship between atherosclerosis and the progression of oa using ultrasonography (us) and plain radiography. a total of 70 female patients who visited a physical medicine and rehabilitation outpatient clinic were recruited for this study. all patients with a diagnosis of knee oa according to the american college of rheumatology criteria were enrolled12. the local ethics committee approved the study, and all participants provided written informed consent. patients with a history of myocardial infarction, percutaneous transluminal coronary angioplasty, surgery for ischemic heart disease, stroke, transient ischemic attack, carotid endarterectomy, inflammatory / infectious arthritis, knee surgery, or intra - articular injection within the previous month were excluded. none of the patients had redness, swelling, or joint instability upon physical examination. some had dm and/or arterial hypertension (ah) ; they did not use any drugs other than those specifically related to dm and ah. patients were clinically assessed for pain and functional status using a visual analog scale (vas) at rest and at motion and the western ontario and mcmaster universities arthritis index (womac), respectively. the womac is a three - dimensional, disease - specific, self - administered health status measure that evaluates pain, joint stiffness, and physical function in patients with knee oa. laboratory measures were determined using blood samples obtained after at least a 6-hour fast and included tests for glucose, complete blood count, erythrocyte sedimentation rate (esr), c - reactive protein (crp), and renal / liver function, as well as lipid profiles for total cholesterol (tc), high - density lipoprotein (hdl) cholesterol, low - density lipoprotein (ldl) cholesterol, and triglycerides (tg). knee radiographs were evaluated using the kellgren - lawrence (k - l) grading system (range 14), the most widely used method for diagnosing knee oa, focusing on osteophytes and/or joint space narrowing14, 15, and higher grades reflect greater severity of oa. patients with k - l grades 1 and 2 were included in group 1, while those with k - l grades 3 and 4 were included in group 2. all ultrasonographic measurements were performed by the same physiatrist using a 510-mhz linear probe (diagnostic ultrasound system, shimadzu, kyoto, japan). distal femoral cartilage assessment was performed as patients lay in a supine position with their knees at maximum flexion. cartilage thickness measurements were taken from the midpoints of the medial femoral condyle (mfc), intercondylar area (ica), and the lateral femoral condyle (lfc). cartilage thickness was measured as the distance between the thin hyperechoic line at the synovial space cartilage interface and the sharp hyperechoic line at the cartilage cartilage grading (range, 06) was performed by evaluating the sharpness, clarity, and thickness of the cartilage band16, and higher grades reflect greater severity of the disease. patients with cartilage 13 were included in group 1, while those with cartilage grades 46 were included in group 2. all measurements were made by the same examiner in a quiet room in which the patient had been resting for 15 minutes.. ultrasound analysis of both common carotid arteries was performed by the same radiologist, who was not informed of the participants histories or clinical / laboratory measurements. measurements were performed using a b - mode high - resolution hd 15 ultrasound (philips healthcare, bothell, wa, usa) with a 512 mhz linear probe. the distance between the lumen - intima interface and the leading edge of the media - adventitia interface of the far wall was taken to represent the intima - media thickness. after localization of the common carotid artery, cross - sectional measurements were performed 10 mm proximal to the carotid bulb. the mean for each side (right and left) was calculated and recorded. focal widening of the vessel walls by 50% relative to adjacent segments with protrusion into the lumen or an intima - media thickness of > 1.5 mm was defined as plaques. data were expressed as the mean standard deviation for nominal variables and as the median (minimum - maximum) for ordinal variables. the normal distribution and homogeneity of each parameter was tested with independent samples t - tests. the mann - whitney u - test was used for data with non - normal distributions. categorical variables (i.e., the presence of dm and/or ah) were evaluated with the test. the demographic characteristics of the patients are presented in table 1table 1.the clinical and demographic characteristics of the patients with oapatients with oa (n=70)age (years)61.6 8.2height (m)1.58 0.1weight (kg)78.1 14 dm, n (%) 19 (27.1)ah, n (%) 44 (62.9)duration of symptoms (years)8.87 4.5womacpain11.4 2.9stiffness4.64 1.5function42.9 6.8vasat rest4.47 0.9at motion7.5 0.9esr (mm / h)19.9 11.5crp (mg / l)0.53 0.5tc (mg / dl)230.6 40.7tg (mg / dl)183.4 82.2hdl (mg / dl)50.9 11ldl (mg / dl)134.2 34.5cimt (mm)0.81 0.2values are shown as the mean standard deviation or median (2575 interquartile range). oa : osteoarthritis ; dm : diabetes mellitus ; ah : arterial hypertension ; womac : western ontario and mcmaster universities arthritis index ; vas : visual analog scale ; esr : erythrocyte sedimentation rate ; crp : c - reactive protein ; tc : total cholesterol ; tg : trygliseride ; hdl : high - density lipoprotein ; ldl : low - density lipoprotein. values are shown as the mean standard deviation or median (2575 interquartile range). oa : osteoarthritis ; dm : diabetes mellitus ; ah : arterial hypertension ; womac : western ontario and mcmaster universities arthritis index ; vas : visual analog scale ; esr : erythrocyte sedimentation rate ; crp : c - reactive protein ; tc : total cholesterol ; tg : trygliseride ; hdl : high - density lipoprotein ; ldl : low - density lipoprotein patients were divided into two group based on k - l grade. the duration of symptoms (p=0.012), womac pain (p=0.049), womac stiffness (p=0.002), womac function (p<0.001), vas at rest (p=0.036), and vas at motion (p=0.005) values were found to be higher in group 2 than in group 1. cartilage thicknesses for the mfc, ica, and lfc (p<0.001) and height (p=0.004) were found to be higher in group 1 than group 2 (table 2table 2.radiographic evaluation of patients knees of according to the k - l grading systemgroup 1 (n=39)(grades 12)group 2 (n=31)(grades 34)age (years)60.5 7.863.0 8.6height (m)1.60 0.051.56 0.05weight (kg)78.9 9.877.0 18.2 dm, n (%) 9 (23.1)10 (32.3)ah, n (%) 21 (53.8)23 (74.2)duration of symptoms (years)7.7 4.410.4 4.3womacpain10.8 3.112.2 2.5stiffness4.18 1.55.22 1.3function40.5 6.646.0 5.8vasat rest4.28 0.94.71 0.8at motion7.21 0.97.87 0.8cartilage thickness (mm)mfc2.24 0.51.52 0.2ica2.48 0.41.89 0.3lfc2.41 0.41.7 0.3esr (mm / h)19.97 11.419.8 11.9crp (mg / l)0.54 0.40.53 0.5tc (mg / dl)229.2 38.3232.5 44.1tg (mg / dl)171.5 85.9198.2 76hdl (mg / dl)51.4 10.950.3 11.2ldl (mg / dl)131.9 32.8137.1 36.8p<0.05. values are shown as the mean standard deviation or median (2575 interquartile range). oa : osteoarthritis ; k - l : kellgren - lawrence ; dm : diabetes mellitus, ah : arterial hypertension ; womac : western ontario and mcmaster universities arthritis index ; vas : visual analog scale ; mfc : medial femoral condyle ; ica : intercondylar area ; lfc : lateral femoral condyle ; esr : erythrocyte sedimentation rate ; crp : c - reactive protein ; tc : total cholesterol ; tg : trygliseride ; hdl : high - density lipoprotein ; ldl : low - density lipoprotein). values are shown as the mean standard deviation or median (2575 interquartile range). oa : osteoarthritis ; k - l : kellgren - lawrence ; dm : diabetes mellitus, ah : arterial hypertension ; womac : western ontario and mcmaster universities arthritis index ; vas : visual analog scale ; mfc : medial femoral condyle ; ica : intercondylar area ; lfc : lateral femoral condyle ; esr : erythrocyte sedimentation rate ; crp : c - reactive protein ; tc : total cholesterol ; tg : trygliseride ; hdl : high - density lipoprotein ; ldl : low - density lipoprotein patients were also divided into two groups based on the ultrasonographic cartilage grading system. the duration of symptoms (p=0.002), womac pain (p=0.007), womac stiffness (p=0.001), womac function (p<0.001), vas at rest (p=0.002), and vas at motion (p=0.001) values were found to be higher in group 2 than in group 1. cartilage thicknesses for the mfc, ica, and lfc were found to be higher in group 1 than group 2 (p<0.001) (table 3table 3.ultrasonographic evaluation of patients knees of according to the cartilage grading systemgroup 1 (n=30)(grades 13)group 2 (n=40)(grades 46)age (years)60.3 8.362.6 8.1height (m)1.60 0.11.57 0.1weight (kg)79.0 10.277.4 16.4 dm, n (%) 6 (20)13 (32.5)ah, n (%) 19 (63.3)25 (62.5)duration of symptoms (years)7.0 4.310.3 4.2womacpain10.5 2.612.1 2.9stiffness4.0 1.55.12 1.3function38.9 6.346.0 5.6vasat rest4.1 0.94.75 0.8at motion7.03 0.97.85 0.8cartilage thickness (mm)mfc2.38 0.41.58 0.2ica2.58 0.41.95 0.3lfc2.55 0.31.75 0.3esr (mm / h)22.2 1118.2 11.7crp (mg / l)0.59 0.40.49 0.4tc (mg / dl)229.3 35.7231.7 44.5tg (mg / dl)164.7 79.4197.3 82.5hdl (mg / dl)52.65 10.949.6 11ldl (mg / dl)131.7 values are shown as the mean standard deviation or median (2575 interquartile range). oa : osteoarthritis ; k - l : kellgren - lawrence ; dm : diabetes mellitus ; ah : arterial hypertension ; womac : western ontario and mcmaster universities arthritis index ; vas : visual analog scale ; mfc : medial femoral condyle ; ica : intercondylar area ; lfc : lateral femoral condyle ; esr : erythrocyte sedimentation rate ; crp : c - reactive protein ; tc, total cholesterol ; tg : trygliseride ; hdl : high - density lipoprotein ; ldl : low - density lipoprotein). values are shown as the mean standard deviation or median (2575 interquartile range). oa : osteoarthritis ; k - l : kellgren - lawrence ; dm : diabetes mellitus ; ah : arterial hypertension ; womac : western ontario and mcmaster universities arthritis index ; vas : visual analog scale ; mfc : medial femoral condyle ; ica : intercondylar area ; lfc : lateral femoral condyle ; esr : erythrocyte sedimentation rate ; crp : c - reactive protein ; tc, total cholesterol ; tg : trygliseride ; hdl : high - density lipoprotein ; ldl : low - density lipoprotein cimt measurements were higher in group 2 than in group 1 according to both the k l and the cartilage grading systems (p=0.001 and p=0.002, respectively) (table 4table 4.carotid intima - media thickness according to k - l and cartilage grading system in patients with oak - l gradingcartilage gradinggroup 1(n=39)(grades 12)group 2(n=31)(grades 34)group 1(n=30)(grades 13)group 2(n=40)(grades 46)cimt (mm)0.76 0.20.88 0.2 0.75 values are shown as the mean standard deviation or median (2575 interquartile range).oa : osteoarthritis ; k - l : kellgren - lawrence ; cimt : carotid intima - media thickness). values are shown as the mean standard deviation or median (2575 interquartile range). - l : kellgren - lawrence ; cimt : carotid intima - media thickness there were positive correlations between cimt measurements and womac stiffness (r=0.265, p=0.026), womac function (r=0.265, p=0.027), vas at motion (r=0.309, p=0.009), ldl cholesterol (r=0.260, p=0.029), cartilage grade (r=0.369, p=0.02), and k l grade (r=0.387, p=0.01) (table 5table 5.correlation with carotid intima - media thickness in oa patientscorrelation coefficientage (years)0.266height (m)0.088weight (kg)0.059duration of symptoms (years)0.180womacpain0.141stiffness0.265function0.265vasat rest0.099at motion0.309esr (mm / h)0.103crp (mg / l)0.028tc (mg / dl)0.140tg (mg / dl)0.210hdl (mg / dl)0.299ldl (mg / dl)0.260cartilage thickness (mm)mfc0.339ica0.375lfc0.347cartilage grading (16)0.369k - l grading (14)0.387p<0.05. values are shown as the mean standard deviation or median (2575 interquartile range). oa : osteoarthritis ; womac : western ontario and mcmaster universities arthritis index ; vas : visual analog scale ; esr : erythrocyte sedimentation rate ; crp : c - reactive protein ; tc : total cholesterol ; tg : trygliseride ; hdl : high - density lipoprotein ; ldl : low - density lipoprotein ; mfc : medial femoral condyle ; ica : intercondylar area ; lfc : lateral femoral condyle ; k - l : kellgren - lawrence). p<0.05. values are shown as the mean standard deviation or median (2575 interquartile range). oa : osteoarthritis ; womac : western ontario and mcmaster universities arthritis index ; vas : visual analog scale ; esr : erythrocyte sedimentation rate ; crp : c - reactive protein ; tc : total cholesterol ; tg : trygliseride ; hdl : high - density lipoprotein ; ldl : low - density lipoprotein ; mfc : medial femoral condyle ; ica : intercondylar area ; lfc : lateral femoral condyle ; k - l : kellgren - lawrence there were negative correlations between cimt measurements and hdl cholesterol (r=0.299, p=0.012) and the following measures of cartilage thickness as assessed by ultrasonography : mfc (r=0.339, p=0.004), ica (r=0.375, p=0.001), and lfc (r=0.347, p=0.003) (table 5). this study evaluated the association between cimt and radiographic and ultrasonographic knee oa grades. the results showed that cimt measurements were positively correlated with both the k - l grade and cartilage grade. additionally, womac stiffness, womac function, and vas scores at motion were also found to be positively correlated with cimt measurements. arterial wall thickening has a strong prognostic value for cardiovascular diseases, and cimt assessment allows easy identification of patients at risk, as shown in a recent systematic review and meta - analysis18. today, there is widespread acceptance of cimt as a reliable and easily reproducible noninvasive marker of preclinical atherosclerosis and future cardiovascular disease risk19. early detection of atherosclerosis with a simple cimt measurement and the use of related precautions can be life - saving for most patients. different mechanisms have been postulated to explain the potential association between atherosclerosis and oa. one hypothesis is that they are concurrent diseases that share etiological features and risk factors8, 20, 21. systemic inflammation caused by visceral adipose tissue could explain a shared pathogenesis and may consequently highlight a target for the prevention and treatment of atherosclerosis and oa22, 23. another hypothesis is that atherosclerotic disease plays an initiating role by causing microcirculatory disturbances in the synovial membrane and subchondral bone that contribute to the cartilage destruction and pathophysiological processes of oa9, 24, 29. this hypothesis was put forth by johnsson., who demonstrated an association between hand oa and atherosclerosis in older women9, 24. however, several genes, including the klotho gene, have been found to be associated with both conditions, and a variety of other mechanisms may be involved, including inflammation and the accumulation of advanced glycation end - products25,26,27,28. johnsson. showed that when data were coupled to evidence of hand oa, there was clearly significant positive association in females, with those having total knee or hip replacements and hand oa exhibiting the highest level of atherosclerosis30. in another study, independent correlations were reported between measures of atherosclerosis and the prevalence of knee and/or hand oa in women after adjusting for cardiovascular risk factors10. in the present study, cimt measurements were found to correlate positively with the presence and progression of knee oa in women. cigarette smoking, a poor - quality diet, physical inactivity, excessive alcohol consumption, and obesity are all recognized as major preventable causes of coronary heart disease and premature mortality31,32,33,34. the risk of atherosclerosis is higher in men than in women, but its prevalence in women increases rapidly with certain risk factors, such as menopause, obesity, increasing age, and the presence of dm. in addition, knee pain, such as that caused by oa, can lead to a sedentary lifestyle, and cardiometabolic syndrome might also be present10. in our study, cimt, as a marker of preclinical atherosclerosis, increased in menopausal women with severe knee oa. notably, cimt measurements were significantly higher in patients who experienced pain during motion and/or functional disability in daily life. the imaging modalities most frequently used are conventional radiography and, within the last decade, ultrasonography35. radiographically diagnosed oa is believed to be most commonly manifested in the knee joint36. another major limitation of measuring the joint space width is that clinicians can only measure the sum of the two opposing cartilage layers and can not measure the individual cartilage layer thicknesses37, 38. it can be promptly performed, is easily accepted by patients, is a radiation - free diagnostic test, and does not have any contraindications39. a previous study reported good agreement between cartilage thickness measurements obtained by magnetic resonance imaging (mri) and those obtained by ultrasonography40,41,42. the present study showed an association between radiographic and ultrasonographic grades for clinical features of knee oa. at the same time, in patients with knee oa, there was a correlation between cimt and both radiographic and ultrasonographic grades. despite widespread interest in the community this is the first study to explore the relationship between subclinical atherosclerosis and ultrasonographic findings in patients with knee oa. this study showed that ultrasonography is a valid and reliable method for evaluating oa progression. further research into the pathogenesis of increased cardiovascular risk in patients with oa should be a high priority, as many risk factors are likely to be modifiable. | [purpose ] the aim of this study was to assess the relationship between atherosclerosis and knee osteoarthritis grade in women as assessed by both ultrasonography and radiography. [subjects and methods ] seventy women diagnosed with knee osteoarthritis were classified into two groups according to cartilage grading / radiographic grading. patients with kellgren - lawrence grades 1 and 2 were included in group 1, while those with kellgren - lawrence grades 3 and 4 were included in group 2. patients with cartilage grades 13 were included in group 1, while those with cartilage grades 46 were included in group 2. patients were clinically assessed using a visual analog scale and the western ontario and mcmaster universities arthritis index. radiographic osteoarthritis grade was scored using the kellgren and lawrence grading system. using ultrasonography, symptomatic knees were graded and evaluated for distal femoral cartilage thickness. carotid intima - media thickness and serum lipid levels were measured to assess atherosclerosis. [results ] carotid intima - media thickness measurements were higher in group 2 than in group 1 as determined by the kellgren - lawrence and cartilage grading systems. carotid intima - media thickness measurements were positively correlated with both the ultrasonographic cartilage grade and kellgren - lawrence. [conclusion ] the results of this study suggest that osteoarthritis as assessed by ultrasonography was successful and comparable to assessment with radiography. we showed a correlation between atherosclerosis and ultrasonographic knee osteoarthritis grade. |
this was a population - based, observational study in central illinois representing a population of about 1.4 million persons. this study utilized a well - administrated database of colonoscopy screening and surveillance from seven hospitals and medical centers, in which 28,782 colonoscopies were enrolled during the period from january 2010 to march 2014 and included their examination histories. the quality of preparation based on the boston bowel preparation scale was evaluated for each bowel preparation type. this study also examined the influence of preparation quality on exam completion and the time of withdrawal after insertion. shared reporting of colonoscopies (screening and surveillance) in central illinois the database was initially created by quality quest for health of illinois and included seven participating sites : the central illinois endoscopy center, decatur digestive disease center, decatur memorial hospital, methodist medical center of illinois, osf saint francis medical center, pekin hospital, and proctor hospital in the central illinois. each site was responsible for abstracting data through their own screening and surveillance colonoscopies and then entering data into the central illinois colonoscopy access database and electronically transferring data to quality quest for health of illinois. the database is currently managed by the department of medicine in the university of illinois college of medicine at peoria. the information in the database which was used in this study includes age in years (exam year), gender, previous and current procedure date, personal history of crc (yes / no), family history of crc including first and second degree relatives (yes / no), bowel preparation type, bowel preparation assessment (excellent, good, fair, and poor), examination completion (yes / no), american society of anesthesiology (asa) classification score with a range of 1 to 5, and time of withdrawal after insertion (minutes). this was a retrospective study where results would not change the course of patient care or current patient outcomes. no risk was involved in collecting patient data as information was the minimum necessary information for research purposes. also, this study was approved by the institutional review board (irb) at the university of illinois college of medicine at peoria. a total of 414 without bowel preparation assessments were excluded resulting in a final dataset that included 28,368 colonoscopies. in order to examine the influence of bowel preparation type on the quality of prep, ordinal logistic regressions were used to estimate odds ratio (or) and 95% confidence interval (95% ci) compared with its reference group. we employed a logistic regression model to analyze the association between exam completion and preparation quality. a log transformation was used for the time of withdrawal after insertion due to its skewed distribution. then we conducted a general linear model regression to see if high - quality bowel preparation could decrease the time of withdrawal after insertion. for all the above models, we also did multivariable analyses, which controlled age, gender, asa score, family history of colorectal cancer, personal history of colorectal cancer, and adenoma detection during the last colonoscopy. a secondary analysis was performed to examine the impact of preparation quality on detecting any adenoma or advanced adenoma. advanced adenoma was defined as a villous / tubulovillous adenoma, severely / high - grade dysplastic polyp, or colorectal cancer based on the polyp histopathology. we excluded those had inadequate preparation (poor) from the secondary analysis because of its low exam completion. after univariate analysis, multivariable logistic regression was used to calculate predicted detection rate of advanced adenoma, and adjusted or and 95% ci by the level of bowel preparation quality controlling for the above confounders and the time of withdrawal after insertion. variables were reported as mean, standard deviation, median, and range for continuous variables, and percentage for categorical variables. a two - tailed p - value was calculated for all tests and p0.05 was considered as being of statistical significance. this was a population - based, observational study in central illinois representing a population of about 1.4 million persons. this study utilized a well - administrated database of colonoscopy screening and surveillance from seven hospitals and medical centers, in which 28,782 colonoscopies were enrolled during the period from january 2010 to march 2014 and included their examination histories. the quality of preparation based on the boston bowel preparation scale was evaluated for each bowel preparation type. this study also examined the influence of preparation quality on exam completion and the time of withdrawal after insertion. shared reporting of colonoscopies (screening and surveillance) in central illinois was established by the development of a quality of health index database in 2010. the database was initially created by quality quest for health of illinois and included seven participating sites : the central illinois endoscopy center, decatur digestive disease center, decatur memorial hospital, methodist medical center of illinois, osf saint francis medical center, pekin hospital, and proctor hospital in the central illinois. each site was responsible for abstracting data through their own screening and surveillance colonoscopies and then entering data into the central illinois colonoscopy access database and electronically transferring data to quality quest for health of illinois. the database is currently managed by the department of medicine in the university of illinois college of medicine at peoria. the information in the database which was used in this study includes age in years (exam year), gender, previous and current procedure date, personal history of crc (yes / no), family history of crc including first and second degree relatives (yes / no), bowel preparation type, bowel preparation assessment (excellent, good, fair, and poor), examination completion (yes / no), american society of anesthesiology (asa) classification score with a range of 1 to 5, and time of withdrawal after insertion (minutes). this was a retrospective study where results would not change the course of patient care or current patient outcomes. no risk was involved in collecting patient data as information was the minimum necessary information for research purposes. also, this study was approved by the institutional review board (irb) at the university of illinois college of medicine at peoria. a total of 414 without bowel preparation assessments were excluded resulting in a final dataset that included 28,368 colonoscopies. in order to examine the influence of bowel preparation type on the quality of prep, ordinal logistic regressions were used to estimate odds ratio (or) and 95% confidence interval (95% ci) compared with its reference group. we employed a logistic regression model to analyze the association between exam completion and preparation quality. a log transformation was used for the time of withdrawal after insertion due to its skewed distribution. then we conducted a general linear model regression to see if high - quality bowel preparation could decrease the time of withdrawal after insertion. for all the above models, we also did multivariable analyses, which controlled age, gender, asa score, family history of colorectal cancer, personal history of colorectal cancer, and adenoma detection during the last colonoscopy. a secondary analysis was performed to examine the impact of preparation quality on detecting any adenoma or advanced adenoma. advanced adenoma was defined as a villous / tubulovillous adenoma, severely / high - grade dysplastic polyp, or colorectal cancer based on the polyp histopathology. we excluded those had inadequate preparation (poor) from the secondary analysis because of its low exam completion. after univariate analysis, multivariable logistic regression was used to calculate predicted detection rate of advanced adenoma, and adjusted or and 95% ci by the level of bowel preparation quality controlling for the above confounders and the time of withdrawal after insertion. all analyses were conducted with sas 9.4 (by sas institute inc., cary, nc, usa). variables were reported as mean, standard deviation, median, and range for continuous variables, and percentage for categorical variables. a two - tailed p - value was calculated for all tests and p0.05 was considered as being of statistical significance. a total of 28,368 colonoscopies ; half the patients were male, and the average age was 619 years. the majority (75%) lived with mild - to - moderate medical conditions (asa score=2). the most popular bowel preparation type was peg - based preparations (70.2%), followed by sodium sulfate based preparations (21.4%), sodium phosphate based preparations (2.5%), and magnesium - based preparations (0.4%). patients who selected sodium phosphate based preparations were a little younger than others. around 21.5% of patients who chose magnesium - based preparation were in poor conditions (asa>2), which was higher than others. subgroups a : peg - based preparations, n=19,912 ; b : magnesium - based preparations, n=107 ; c : sodium phosphate based preparations, n=6,081 ; e : other preparations, n=322 ; f : not recorded, n=1,257. american society of anesthesiology (asa) classification score was defined as five levels (1 = healthy, no comorbidities ; 2 = mild - to - moderate medical conditions controlled ; 3 = disease severely limits activities ; 4 = severe life - threatening disorders ; 5 = moribund). as it was shown in table 2, sodium sulfate based preparations and sodium phosphate based preparations had six and two times better quality of preparation than peg - based formulations, respectively (or=5.7, 95% ci 5.46.1 ; or=2.1, 95% ci 1.82.5). magnesium - based preparation was not as good as peg - based preps in the quality of preparations (table 3). influence of bowel preparations type on the quality of preparations ordinal logistic regression was used to calculate the odds ratio (or) and 95% confidence interval (95% ci) controlling for age, gender, american society of anesthesiology classification score, family history of colorectal cancer, personal history of colorectal cancer, and adenoma detection during the last colonoscopy. influence of bowel preparations subtype on the quality of preparations ordinal logistic regression was used to calculate the odds ratio (or) and 95% confidence interval (95% ci) controlling for age, gender, american society of anesthesiology classification score, family history of colorectal cancer, personal history of colorectal cancer, and adenoma detection during the last colonoscopy. in peg - based preparation, moviprep was better than other peg - based preparations (or=1.3, 95% ci 1.21.4). in magnesium - based preparation, no significant difference was found between magnesium citrate only and magnesium citrate with ducolax (or=0.6, 95% ci 0.31.6). in sodium sulfate based preparation, the effect of visicol tabs was very similar with osmoprep (or=0.9, 95% ci 0.71.2). a better bowel preparation significantly increased the rate of exam completion (table 4). only 88.4% completed exams when the bowel preparation was poor, whereas 99.5% completed exams when the bowel preparation was excellent. the rate of exam completion was also acceptable when the bowel preparation was good or fair. association between preparations quality and exam completion logistic regression was used to calculate the odds ratio (or) and 95% confidence interval (95% ci) controlling for age, gender, american society of anesthesiology classification score, family history of colorectal cancer, personal history of colorectal cancer, and adenoma detection during the last colonoscopy. the time of withdrawal after insertion was the shortest when bowel preparation assessment was excellent (10.45.5 min), followed by good bowel preparation (11.06.0 min), poor bowel preparation (12.28.5 min), and fair bowel preparation (13.57.7 min). general linear model was used to estimate the time of withdrawal after insertion at each level of bowel preparations quality controlling for age, gender, american society of anesthesiology classification score, family history of colorectal cancer, personal history of colorectal cancer, and adenoma detection during the last colonoscopy. our general polyp detection rate was 44.1% (12,525/28,386), while adenoma detection rate in fair, good, and excellent preps was 51.7, 58.3, and 54.7, respectively. although the differences of adenoma detection rate among them were not large, the good and excellent preps still increased the likelihood of adenoma detection than the fair prep (or=1.1, 95% ci 1.01.2 ; or=1.3, 95% ci 1.214, respectively). as shown in table 6, we found that the better quality of bowel preparation could significantly increase the detection rate of advanced adenoma (5.0, 3.6, and 2.9% for excellent, good, and fair, respectively). association between preparations quality and detection rate of advanced adenoma / adenoma detection rate logistic regression was used to calculate the odds ratio (or), 95% confidence interval (95% ci), and predicted detection rate controlling for age, gender, american society of anesthesiology classification score, family history of colorectal cancer, personal history of colorectal cancer, adenoma detection during the last colonoscopy, and time of withdrawal after insertion. a total of 28,368 colonoscopies ; half the patients were male, and the average age was 619 years. the majority (75%) lived with mild - to - moderate medical conditions (asa score=2). the most popular bowel preparation type was peg - based preparations (70.2%), followed by sodium sulfate based preparations (21.4%), sodium phosphate based preparations (2.5%), and magnesium - based preparations (0.4%). patients who selected sodium phosphate based preparations were a little younger than others. around 21.5% of patients who chose magnesium - based preparation were in poor conditions (asa>2), which was higher than others. subgroups a : peg - based preparations, n=19,912 ; b : magnesium - based preparations, n=107 ; c : sodium phosphate based preparations, n=6,081 ; e : other preparations, n=322 ; f : not recorded, n=1,257. american society of anesthesiology (asa) classification score was defined as five levels (1 = healthy, no comorbidities ; 2 = mild - to - moderate medical conditions controlled ; 3 = disease severely limits activities ; 4 = severe life - threatening disorders ; 5 = moribund). as it was shown in table 2, sodium sulfate based preparations and sodium phosphate based preparations had six and two times better quality of preparation than peg - based formulations, respectively (or=5.7, 95% ci 5.46.1 ; or=2.1, 95% ci 1.82.5). magnesium - based preparation was not as good as peg - based preps in the quality of preparations (table 3). influence of bowel preparations type on the quality of preparations ordinal logistic regression was used to calculate the odds ratio (or) and 95% confidence interval (95% ci) controlling for age, gender, american society of anesthesiology classification score, family history of colorectal cancer, personal history of colorectal cancer, and adenoma detection during the last colonoscopy. influence of bowel preparations subtype on the quality of preparations ordinal logistic regression was used to calculate the odds ratio (or) and 95% confidence interval (95% ci) controlling for age, gender, american society of anesthesiology classification score, family history of colorectal cancer, personal history of colorectal cancer, and adenoma detection during the last colonoscopy. in peg - based preparation, moviprep was better than other peg - based preparations (or=1.3, 95% ci 1.21.4). in magnesium - based preparation, no significant difference was found between magnesium citrate only and magnesium citrate with ducolax (or=0.6, 95% ci 0.31.6). in sodium sulfate based preparation, the effect of visicol tabs was very similar with osmoprep (or=0.9, 95% ci 0.71.2). a better bowel preparation significantly increased the rate of exam completion (table 4). only 88.4% completed exams when the bowel preparation was poor, whereas 99.5% completed exams when the bowel preparation was excellent. the rate of exam completion was also acceptable when the bowel preparation was good or fair. association between preparations quality and exam completion logistic regression was used to calculate the odds ratio (or) and 95% confidence interval (95% ci) controlling for age, gender, american society of anesthesiology classification score, family history of colorectal cancer, personal history of colorectal cancer, and adenoma detection during the last colonoscopy. the time of withdrawal after insertion was the shortest when bowel preparation assessment was excellent (10.45.5 min), followed by good bowel preparation (11.06.0 min), poor bowel preparation (12.28.5 min), and fair bowel preparation (13.57.7 min). general linear model was used to estimate the time of withdrawal after insertion at each level of bowel preparations quality controlling for age, gender, american society of anesthesiology classification score, family history of colorectal cancer, personal history of colorectal cancer, and adenoma detection during the last colonoscopy. our general polyp detection rate was 44.1% (12,525/28,386), while adenoma detection rate in fair, good, and excellent preps was 51.7, 58.3, and 54.7, respectively. although the differences of adenoma detection rate among them were not large, the good and excellent preps still increased the likelihood of adenoma detection than the fair prep (or=1.1, 95% ci 1.01.2 ; or=1.3, 95% ci 1.214, respectively). as shown in table 6, we found that the better quality of bowel preparation could significantly increase the detection rate of advanced adenoma (5.0, 3.6, and 2.9% for excellent, good, and fair, respectively). association between preparations quality and detection rate of advanced adenoma / adenoma detection rate logistic regression was used to calculate the odds ratio (or), 95% confidence interval (95% ci), and predicted detection rate controlling for age, gender, american society of anesthesiology classification score, family history of colorectal cancer, personal history of colorectal cancer, adenoma detection during the last colonoscopy, and time of withdrawal after insertion. this study compares the impact of bowel preparation type on the quality of colonoscopy in a large, population - based cohort of colonoscopies that were conducted in clinical practice. considering the fact that the national colorectal roundtable set a goal of increasing colorectal screening to 80% by 2018, the use of screening colonoscopies will continue to increase. this will save valuable resources and also play an essential part in improving clinical outcomes and reducing the disease burden of colorectal cancer (11, 16). a meta - analysis of 104 studies from 1985 to 2010 showed no difference in the efficacy between sodium phosphate based preparations and peg (or=0.82, 95% ci 0.561.21 ; p=0.36). in addition, peg - based preparations were found to provide a better cleaning of the proximal portion of the colon (12, 17). based preparations to be superior in terms of bowel preparation and rates of complete examination (12, 18). in addition to this, sodium- and magnesium - based preparations are slightly cheaper as compared with peg - containing preparations (19). based preparations had six and two times better quality of bowel preparation when compared with peg - based preparation, respectively (or=5.7, 95% ci 5.46.1 ; or=2.1, 95% ci 1.82.5). magnesium - based preparations were found to be inferior to peg - based formulations (or=0.6, 95% ci 0.40.9). peg- and sodium - based bowel preparations had acceptable levels of bowel cleanliness, mostly ranging from fair to good with a good response in 6070% of the patients, independent of the type of preparation being used. in our study, 19,912 patients used peg - based preparation and out of these only 14.5% had excellent preparations, where as 31.8 and 55.8% with sodium phosphate and sodium sulfate these results were consistent with several previous studies in which patients with sodium - based preparations had better results as well as superior completion rates (12, 20). excellent bowel preparation was found in 58% (ci 4967%) of the patients taking sodium phosphate tablets, 42.1% (ci 3351%) for sodium phosphate solution, and 33.7% (ci 2641%) for those who had used 4 l peg (20). in a meta - analysis of seven randomized trials comparing sodium phosphate and peg solution, the relative risk of having an excellent preparation was 1.28 (95% ci 1.111.48) in favor of sodium phosphate (nnt=10) (18). similarly, other studies have shown that sodium phosphate is also superior to sodium picosulfate in terms of bowel purging activity with a similar side - effect profile (21, 22). completion rates of colonoscopy did not vary significantly between fair, good, and excellent preparations (99.5, 99.4, and 99.1%, respectively) but had almost a 12% decline in patients with poor preparation (88.4%). these results are in contrast to a recent study in which completion rates were significantly lower in patients with fair and poor bowel preparations (75.4 and 72.1%, respectively) as compared with those with good and excellent bowel preparations (99.7 and 99.9%, respectively ; p<0.001) (23). this difference could possibly be because of smaller number of patients in their study (23). other studies have reported a completion rate of 90% in people with intermediate and high - quality preparations while completion rates of 70% in those with low - quality bowel preparations (24). our study clearly shows that withdrawal times were faster in patients with excellent and good preparation (10.45.5 and 11.06.0 min, respectively) while those with fair and poor preparation had longer withdrawal times after insertion (13.57.7 and 12.2 8.5, respectively). the longer withdrawal time in fair preparation could be due to higher completion rates and effort by physicians in patients with poor preparation of which only 88.4% underwent complete examination as compared with 99% of those with fair preparation who underwent complete exam. previous studies have shown that inadequate bowel preparation decreases the adenoma detection rate (2527). other studies have also suggested that the adenoma detection rate is comparable in patients with fair - quality bowel preparation and those with adequate bowel preparation (25, 28). the overall adenoma detection rate in our study was 44.1% which is comparable with other studies and well above the 30% mark set by the american college of gastroenterology task force (29). this supports the fact that the quality of colonoscopic examination in our study was consistent with national standards. we found better bowel preparations could increase the detection rate of adenoma, especially advanced adenoma. advanced adenoma detection rate was 2.9% in those with fair bowel prep as compared with 3.6 and 5% in those with good and excellent bowel preparations, respectively. our results clearly suggest that the odds of finding an advanced polyp in a patient with excellent bowel preparation was almost two times higher compared with those with fair prep (or=1.8, 95% ci 1.52.1) we did not record whether the patients received split - dose preparation or nightly preparations in the case of peg - based preparations. we did not measure true adenoma missed rates by performing follow - up colonoscopies in patients with poor or fair bowel preparation. other confounding factors that could have altered outcomes include concomitant laxative use, hydration variability, and differences in patient compliance across various groups and hospitals. in summary, while peg - based preparations continue to be used most commonly, the search for an ideal bowel purge, which is inexpensive, offers good outcomes with a high success rate, with relatively no side effects continues to be a goal for physicians. we recommend that sodium - based bowel preparations should be used whenever possible as sodium - based preparations appear superior to peg- and magnesium - based preparations according to our study results. further, the findings suggest that adequate bowel preparation not only improves withdrawal times but also enhances the adenoma detection rate in specifically advanced adenomas. in addition, our results also support the finding that patients with fair bowel preparation should also be screened earlier to enhance the chances of detecting any missed adenomas. with an increasing population of patients entering into colorectal cancer screening age, the volume of screening colonoscopies will increase. continued exploration for an optimal bowel preparation remains essential for continued reduction in colorectal cancer. all authors have disclosed that they have no significant relationships with or financial interests in any commercial companies related to this study or article. post presentation was at the digestive disease week (ddw) 2015 conference in washington, dc (may 1619, 2015). the manuscript was drafted and revised by sw, jr, dm, ca, sd, za, and sp. statistical analysis was done by jr, and data were acquired by ca and jr. | backgroundhigh - quality bowel preparation is crucial for achieving the goals of colonoscopy. however, choosing a bowel preparation in clinical practice can be challenging because of the many formulations. this study aims to assess the impact the type of bowel preparation on the quality of colonoscopy in a community hospital setting.methodsa retrospective, observational study was conducted utilizing a colonoscopy screening / surveillance database in central illinois during the period of january 1, 2010, to march 31, 2014. patients without bowel preparation assessment were excluded from this study. controlling for the confounders, generalized linear models were used to estimate the adjusted impact [odds ratio (or) ] of bowel preparation type on the quality of preparation (excellent, good, fair, and poor), and on the detection of advanced adenoma. the association between the time of withdrawal after insertion and the quality of preparation was also examined using a linear model.resultsa total of 28,368 colonoscopies ; half the patients were male, and the average age was 619 years. polyethylene glycol (peg) was used in the majority (70.2%) of bowel preparations, followed by sodium sulfate (21.4%), sodium phosphate (2.5%), magnesium sulfate (0.4%), and others. compared with peg, magnesium sulfate had a poorer quality of bowel preparations (or=0.6, 95% ci 0.40.9 ; p<0.05), whereas the quality of bowel preparation was significantly improved by using sodium sulfate (or=5.7, 95% ci 5.46.1 ; p<0.001) and sodium phosphate (or=2.1, 95% ci 1.82.5 ; p<0.001). for those who had adequate bowel preparation, the better quality of preparation significantly increased the detection rate of advanced adenoma (5.0, 3.6, and 2.9% for excellent, good, and fair, respectively).conclusionwhen possible, sodium sulfate based preparations should be recommended in the community setting for colonoscopy because of their high quality of bowel preparation. |
aldosterone is an endogenous mineralocorticoid receptor (mr) agonist synthesized in the adrenal glomerular layer as a final product of the renin - angiotensin - aldosterone system (raas) ; it is strongly involved in the development of hypertension due to excessive sodium retention. it has been reported that suppression of the renin - angiotensin system (ras) by angiotensin - converting enzyme inhibitors (ace - is) and angiotensin ii type 1 receptor blockers (arbs) provides an effective treatment against cardiovascular diseases such as hypertension and cardiac failure [1, 2 ]. several studies have also revealed that the blockade of mr by an mr antagonist (mra), such as spironolactone or eplerenone, offers an effective approach to treat cardiac disease, especially cardiac failure [36 ]. these facts indicate that raas may contribute to the underlying mechanisms of cardiac diseases for which its control may play a critical role in ameliorating the effectiveness of treatments. although the blockade of ras by ace - is or arbs (ras inhibitors) may be effective, the long - term treatment of hypertension by drugs classified as such often results in a diminished efficacy owing to the inadequate suppression of aldosterone synthesis.. an effective approach may therefore be to use an mra in addition to ras inhibitors to avoid such deterioration of the ace - i / arb efficacy due to aldosterone breakthrough. to this extent, however, mra use has been associated with an increased risk of fatal hyperkalemia, and the concomitant use of mras with ras inhibitors may have synergistic effects, potentiating the risk for hyperkalemia. in addition, aldosterone - related hypertension may also be caused by autonomous aldosterone secretion, such as primary hyperaldosteronism, which is often associated with severe hypertension and obesity. these findings indicate that it is necessary that the suppression of aldosterone production be considered as an alternative choice to control blood pressure. recently, several groups have reported that ca channel blockers (ccbs), which are another class of antihypertensive agent widely used to control blood pressure, may have inhibitory actions on aldosterone synthesis. here, we provide an overview of the effects of ccbs on the production of aldosterone and discuss clinical perspectives of their use to curb aldosterone production. a brief summary of steroid biosynthesis in human adrenal cells is provided here for the convenience of the reader. the biosynthetic pathways of adrenal steroids are summarized in figure 1 [12, 13 ]. briefly, steroid biosynthesis is initiated by steroidogenic acute regulatory protein (star), which transports cholesterol into the mitochondria. the side chain of cholesterol is then cleaved by cytochrome p450 side chain cleavage enzyme (cyp11a1) to produce pregnenolone. in the zona glomerulosa of the adrenal gland (solid line in figure 1), which does not express cytochrome p450 17-hydroxylase/17,20 lyase (cyp17), pregnenolone is converted to progesterone by 3-hydroxysteroid dehydrogenase type 2 (3-hsd2). cyp17, which is expressed by cells of the zona fasciculata (dotted line in figure 1) and the zona reticularis (broken line in figure 1), catalyzes the conversion of pregnenolone and progesterone to 17-hydroxypregnenolone and 17-hydroxyprogesterone, respectively. via a different pathway, progesterone is catalyzed to 11-deoxycorticosterone (doc), and 17-hydroxyprogesterone to 11-deoxycortisol, respectively, by hydroxylation with steroid 21-hydroxylase (cyp21a2). corticosterone is generated from doc by 11-hydroxylase (cyp11b1), which also generates cortisol from 11-deoxycortisol in zona fasciculata cells, and, in turn, aldosterone is generated from 18-hydroxycorticosterone by cyp11b2, which is also known as aldosterone synthetase. the regulation of cyp11b2 is mediated by ca - sensitive manner through mechanisms involving calmodulin and calmodulin - dependent kinases, and the 11-hydroxylase activity is also stimulated by ca [14, 15 ]. the previous experiments revealed that angiotensin ii - induced aldosterone synthesis is involved in activation of the low voltage - activated t - type ca channel [16, 17 ], and the expression of cyp11b2 mrna was suppressed by some dihydropyridine ccbs, which can inhibit the t - type ca channel [1820 ]. in the adrenal cells of the zona reticularis, 17-hydroxypregnenolone and 17-hydroxyprogesterone the dhea is further sulfated to dhea - sulfate (dhea - s) by sulfotransferase (sult2a1) and reversely sulfated from dhea - s by steroid sulfatase (sts). androstenedione on the other hand is converted to testosterone by 17-hydroxysteroid dehydrogenase type 3 (17-hsd3). both the expression of star at mrna and protein levels and its activity were shown to be increased by nifedipine and efonidipine in ma-10 mouse leydig cells and nci - h295r human adrenocortical carcinoma cells, but decreased by amlodipine, azelnidipine, or r(-)-efonidipine [21, 22 ]. likewise, cyp11b1 and cyp11b2 are decreased by azelnidipine, benidipine, and efonidipine (in figure 1) in nci - h295r human adrenocortical carcinoma cells [1820 ], while efonidipine increases dhea - s production in nci - h295r human adrenocortical carcinoma cells probably as a result of increased star expression (in figure 1). the reported actions of dihydropyridine ccbs on the expression of steroidogenic enzymes in in vitro studies are summarized in tables 1 and 2 [1825 ]. a previous study reported that a specific step in the steroidogenic pathway may be directly linked to agonist - induced increases in the cytosolic free ca concentration in intact isolated zona glomerulosa cells. stimulation by angiotensin ii or exposure to high extracellular potassium (which depolarized the cell membrane) elevates the intracellular ca and induces aldosterone production. this increase in intracellular ca can be blocked by the l - type ccb nifedipine, though not completely in the case of the angiotensin ii - induced intracellular ca elevation, with a concomitant decrease in aldosterone production. in addition, even verapamil and diltiazem, which are nondihydropyridine l - type ca channel antagonists, exerted inhibitory actions on aldosterone production in rat 's adrenal glands. these findings indicate that the production of aldosterone may be stimulated by processes other than by ca influx through the l - type calcium channel. for example, other studies on adrenal glomerulosa cells, in which t - type ca channel - specific antagonists (mibefradil, tetrandrine, and ni) were used, reported involvement of the t - type ca channel in aldosterone production in addition to the l - type ca channel [28, 3032 ]. these results indicate that adrenal aldosterone production requires the activation of t - type ca channels. thereafter, it was reported that a newly developed dihydropyridine ccb, efonidipine, which has inhibitory properties for both l- and t - type ca channels, suppressed aldosterone and cortisol production more potently than nifedipine by reducing the expression of cyp11b1 and cyp11b2 [18, 20 ]. interestingly, however, when used on nci - h295r human adrenocortical carcinoma cells, efonidipine increased the expression of star mrna and protein, possibly resulting in the increased production of dhea - s. it has also been reported that azelnidipine and benidipine, as well as efonidipine, have inhibitory actions on adrenal aldosterone production by decreasing the expression of cyp11b1 and cyp11b2 more potently than nifedipine [19, 20 ], and cilnidipine suppressed angiotensin ii - induced cyp11b2 mrna expression, but not cyp11b1. moreover, while azelnidipine, benidipine, and efonidipine have inhibitory properties against t - type ca channels (1h and 1 g), nifedipine has little effect on these t - type ca channel subtypes, indicating that the effects of ccbs on the t - type ca channel, in addition to that on the l - type ca channel, may be involved in their inhibitory actions on aldosterone production. recently, another group reported that cilnidipine, which has little or no effect on t - type ca channels but significant inhibitory actions against an n - type ca channel, also suppressed aldosterone production in nci - h295r human adrenocortical carcinoma cells. the n - type ca channel may be involved in aldosterone production, because -conotoxin giva, a specific n - type ca channel blocker, significantly suppressed aldosterone and cortisol secretions in nci - h295r human adrenocortical carcinoma cells without significantly influencing cyp11b2 or cyp11b1 mrna expression. given that aldosterone is a key factor in cardiac pathological stress, promoting processes such as fibrosis and oxidative stress, it is important to evaluate the effects of above - mentioned ccbs on aldosterone production in in vivo animal preparations and in patients with cardiovascular diseases. in this way, in vivo studies with benidipine and cilnidipine were found to reduce the plasma aldosterone concentration (pac) in stroke - prone spontaneously hypertensive rats, in the ischemia reperfusion mouse model, and in male shr / izm rats. in clinical studies, azelnidipine, benidipine, and efonidipine were shown to exert suppressive actions on pac in hypertensive patients with type 2 diabetes mellitus, in patients with mild - to - moderate stage chronic kidney disease with albuminuria, in patients with chronic glomerulonephritis, and in patients with essential hypertension. activation of mr by aldosterone is one of the important causes of arterial hypertension, and, due to the extrarenal effects of aldosterone, such as cardiac fibrosis and vascular inflammation, it is very important to antagonize the mr activities in such patients. dihydropyridine ccbs are widely recommended in the treatment of hypertension by several guidelines [4347 ] and may have more therapeutic potential in combination with antagonists for ras [4850 ]. combination therapies with ras inhibitors and ccbs are well - tolerated in hypertensive patients [5154 ], indicating that ccbs are the potentially important candidates as a concomitant drug with ras inhibitors. also, because monotherapy by ras inhibitors for hypertension may often cause diminished efficacy of treatment, so - called aldosterone breakthrough [8, 9 ], treatment of hypertensive patients with ras inhibitor alone is often required to add another antihypertensive drug to avoid aldosterone breakthrough. mras are often considered for such purpose, but concomitant usage of mras with ras inhibitors increases incidence of hyperkalemia, which is one of the life - threatening adverse effects. furthermore, recent data suggest that primary aldosteronism is present in approximately 10% of hypertensive patients, indicating that the suppressive property of adrenal aldosterone production without severe adverse effects may be a key element in treatment of hypertension. therefore, ccbs, which suppress adrenal aldosterone production and have tolerable property in concomitant usage with ras inhibitors, may be another useful choice to overcome aldosterone breakthrough and aldosterone - related hypertension without intolerable adverse effects, such as severe hyperkalemia. therefore, taken together with the recently reported data concerning the antagonistic properties of ccbs against mr activity [56, 57 ], such dihydropyridine ccbs may act as a new class of mras providing a therapeutic advantage for the treatment of aldosterone - related hypertensive patients. recent studies have revealed that dihydropyridine ccbs, such as azelnidipine, benidipine, cilnidipine, efonidipine, and nifedipine, have inhibitory actions on adrenal aldosterone biosynthesis in vitro. some studies have also shown that plasma aldosterone levels are decreased in the patients prescribed such dihydropyridine ccbs. based on accumulating evidence from in vitro and clinical studies of the actions of these drugs on aldosterone production, the clinical use of dihydropyridine ccbs may provide therapeutic advantages to combat aldosterone - related hypertension in affected patients. | aldosterone, a specific mineralocorticoid receptor (mr) agonist and a key player in the development of hypertension, is synthesized as a final product of renin - angiotensin - aldosterone system. hypertension can be generally treated by negating the effects of angiotensin ii through the use of angiotensin - converting enzyme inhibitors (ace - is) or angiotensin ii type 1 receptor antagonists (arbs). however, the efficacy of angiotensin ii blockade by such drugs is sometimes diminished by the so - called aldosterone breakthrough effect, by which ace - is or arbs (renin - angiotensin system (ras) inhibitors) gradually lose their effectiveness against hypertension due to the overproduction of aldosterone, known as primary aldosteronism. although mr antagonists are used to antagonize the effects of aldosterone, these drugs may, however, give rise to life - threatening adverse actions, such as hyperkalemia, particularly when used in conjunction with ras inhibitors. recently, several groups have reported that some dihydropyridine ca2 + channel blockers (ccbs) have inhibitory actions on aldosterone production in in vitro and in the clinical setting. therefore, the use of such dihydropyridine ccbs to treat aldosterone - related hypertension may prove beneficial to circumvent such therapeutic problems. in this paper, we discuss the mechanism of action of ccbs on aldosterone production and clinical perspectives for ccb use to inhibit mr activity in hypertensive patients. |
the physicochemical properties of nanoscale composite are a result of molecular interaction between materials of interest, such as a conducting polymer, promoting greater structural control of the formed films [13 ]. two of the most used methods to obtain nanostructured materials in the solid state are the langmuir - blodgett (lb) technique and the process of layer - by - layer (lbl) assembly. this last one emerges as a method of deposition of alternating layers formed through the electrostatic interaction between oppositely charged solutions, where the formation of the first monolayer on the substrate surface occurs initially through a process of adsorption. in addition to the possibility of controlling the structure formed at the supramolecular level, the lbl technique has the advantage of not requiring any sophisticated equipment or procedures such as the lb technique. the technological interest of several research groups in composites, and more recently in nanocomposites, comes mainly from the mechanical properties and biodegradability, which are both characteristic of natural polymers, allied to the conductive properties of some synthetic polymers, which provides a great versatility of applications in areas such as engineering, biotechnology, and medicine [68 ]. natural gums are macromolecules formed from units of sugars, monosaccharides, linked by glycosidic bonds resulting in natural polymers with long chains and high molecular weight. the gums may originate from plants exudates (e.g., arabic gum and cashew gum), seaweed extract (e.g., agar), animal (e.g., chitosan), seeds (e.g., guar gum), and others. the gum from exudates is produced in epithelial cells confined in the ducts of the trees that are released spontaneously or induced as a defense mechanism of the plant [10, 11 ]. the natural gums interact with water in two different ways : by retention of water molecules (thickness effect) or by building networks that enhance the connection areas (effect of gelation). because of these behaviors, the gums are also known as hydrocolloids [12, 13 ]. the cashew gum (anacardium occidentale l.) is particularly interesting because it is an exudate obtained from the cashew tree, very abundant in the northeast of brazil. it belongs to the same family of arabic gum, widely used in the food industry, and presents similarities in their composition and in their physicochemical properties [1214 ]. cashew gum (cg) is an acidic polysaccharide complex composed of a main chain of -galactose (13) with branches of -galactose (16), with the terminal residues glucuronic acid, arabinose, rhamnose, 4-o - methylglucuronic acid, xylose, glucose, and mannose. brazilian gum main constituents are galactose (73%), arabinose (5%), rhamnose (4%), glucose (11%), glucuronic acid (6.3%), and residues of other sugars (less than 2%) [12, 14 ] (scheme 1(a)). additionally, the terminal glucuronic acids in the structure of the gum are responsible for the anionic nature of the material when in aqueous solution. cashew gum has antimicrobial properties for therapeutic treatment, as well as thickening and emulsifying properties [15, 16 ] used in foods and drugs industry. these characteristics are due to its heterogeneous structure and high molecular weight of the polysaccharide chain, which interacts strongly with water, creating an effect of thickening or gelling in solution. the polyaniline (pani) belongs to the class of conducting polymers with high technological interest due to their potential applications as electroluminescent devices, corrosion protection, sensors, and biosensors [17, 18 ]. the versatility of pani is due to changes of its oxidation state, hence its electrical conductivity, which occurs rapidly and reversibly, and its chemical stability [19, 20 ]. the applications of polyaniline are limited by its poor solubility in aqueous media in its conductive form [17, 20 ]. a proposed solution for this limitation was given by geng. who prepared a water - soluble conductive polyaniline through the introduction of hydrophilic dopant such as phosphonic acid (pa) in the polymeric chain. the pa is a mixture of acids mono- and bihydroxyl phosphonate at 1 : 1 molar ratio (1). thus, the longer the hydrophilic chain of the conducting polymer, the higher its solubility in water, generating new applications : in this study, lbl films were produced with pani or pani - pa and the natural cashew gum (cg) in a bilayer fashion (pani / cg)n or (pani - pa / cg)n (where n is the number of bilayers). films containing a conventional anionic polyelectrolyte, for example, poly(vinylsulfonic acid) pvs, were compared to cashew gum in the (pani / pvs)n or (pani - pa / pvs)n films. we also investigated the ability of these nanocomposites to act as modified electrodes for dopamine sensing. cashew gum, collected in the state of cear (northeast region of brazil), was isolated and purified using sodium salt, as described by costa.. afterwards 0.25 ml of ethanol was added to 5.0 g of cashew gum, which was immediately dissolved in 100 ml of milli - q water under stirring for 12 h, followed by filtration. pani was synthesized by the oxidative polymerization of aniline doubly distilled in 1.0 mol l hcl solution containing a proper amount of ammonium persulfate ((nh4)2s2o8, vetec). the product was maintained in ammonia hydroxide (vetec) for 12 hours to obtain pani in the form of emeraldine base (eb). the mixture of mono- and bihydroxyl acids designated as pa, with molecular average weight of 896 g mol, was prepared as reported by geng.. for the processing of polyaniline solutions, 0.47 g pani - eb powder (with or without pa dopant) was dissolved in 25 ml dimethylacetamide (dmac, vetec) under stirring for 12 h. the solutions were filtered and slowly added to 26 ml of hcl solution, and the ph was adjusted at 2.8. poly(vinyl sulfonic acid) (pvs) was purchased from aldrich co. and used without previous purification in aqueous solutions at a concentration of 0.5 mg ml and ph 2.8. ultrapure water with a resistivity of 18.3 m cm (milli - q, millipore) was used for preparation of all solutions. nanostructured layered films were assembled in a bilayer fashion using pani or pani - pa as polycationic solutions in conjunction with cg or pvs as polyanionic solutions. the deposition of each layer consisted in the immersion of the substrate in the dipping solution for 5 min, followed by rinsing in the washing solution (hcl, ph 2.8) and drying in n2 flow. lbl films with four distinct architectures were investigated : (pani / pvs)n, (pani - pa / pvs)n, (pani / cg)n, and (pani - pa / cg)n where n is the number of bilayers. multilayer films with n = 2, 4,6, and 8 were obtained onto glass covered with indium tin oxide (ito), (scheme 1(b)). electrochemical measurements were carried out using a potentiostat autolab pgstat30 and a three - electrode electrochemical cell with 10 ml. a 1.0 cm platinum foil and saturated calomel electrode (sce) were used as auxiliary and reference electrodes, respectively. the lbl films onto ito (0.4 cm) were used as the working electrode. all the experiments were performed in inert n2 atmosphere at 22c in an electrolytic solution of 0.1 mol l hcl. pani - ap / gc lbl film containing 6 bilayers (n = 6) was subjected to dopamine (da) detection using cyclic voltammetry in electrolytic solution containing 10 to 230 mol l of da and sweep rate of 50 m vs. after the measurement the film tested was exhaustively washed with electrolytic solution and the reproducibility was investigated. furthermore, the effects of the interfering ascorbic acid (aa) in the presence of da were also studied using different proportions of aa and da. cyclic voltammograms of ito unmodified and modified electrodes with lbl film produced with poly(allylaminehydrochloride), pah, and the natural cashew gum, (pah / cg)6, were obtained in hcl 0.1 mol l and are shown in figure 1. under our experimental conditions, it is observed that the ito substrate has no electrochemical response to the potential range studied. however, the modified substrate with (pah / cg)6 shows that the presence of the lbl film activates the electrode surface increasing the double electrical layer of this system and therefore catalyzes the processes of oxygen evolution observed in the forward sweep and hydrogen evolution observed in the reverse sweep. the increase in current values is observed in the potential in which these processes occur. a previous study about the influence of the size and nature of this anion in the supporting electrolyte for the systems studied here was carried out in hcl or h2so4, both at 0.1 mol l, with scan rate of 50 mv s. the processes of oxidation and reduction, characteristic of conducting polymer, were shifted to more positive potentials in h2so4 media when compared to the profile obtained in hcl solution (data not shown). the potential difference observed for this redox process was 0.05 v. according to matveeva., both the processes of oxidation and reduction (e red and e ox) and the distance between the potential at which these transitions occur are dependent on the substrate used and the size and nature of the anion of the supporting electrolyte employed. the potential shift observed for the pani in h2so4 reflects a limitation in the processes of charge transfer across the interface between ito and polymeric film and also in the interface polymeric film and the electrolyte. probably this limitation of the charge transfer process could be related to differences in mobility and the steric hindrance originated from the anionic species present in both electrolytes studied, having hso4 and so4 for sulfuric acid and cl for hydrochloric acid, respectively. it was also noted that the degradation processes of pani were more intense and were best defined in h2so4 (data not shown). this observation is likely to be explained by the fact that h2so4 is a more oxidant acid than hcl intensifying the processes observed. thus, the whole study presented in this paper was performed in optimized conditions of hcl media. figure 2 shows the cyclic voltammograms obtained for the bilayers films containing the conductive polymer interspersed with cashew gum or pvs in hcl media. the electrochemical profile recorded for the lbl systems (pani / pvs)6 (pani - pa / pvs)6, (pani - gc)6, and (pani - pa / cg)6 (figure 2) indicates the presence of two redox processes characteristic of pani that correspond to interconversions in their states of oxidation. during the direct scan there was the transition from leucoemeraldine state to emeraldine pani, e pa1, with the expulsion of the proton, and the transition from emeraldine to pernigraniline, e pa2, was accompanied by the capture of the anion, cl. during the inverse sweep two reduction processes were observed ; pernigraniline to emeraldine, e pc2, accompanied by the expulsion of the anion and emeraldine to leucoemeraldine, e pc1, which was accompanied by proton uptake. an intermediate process between the transitions related above is defined as acidic degradation of pani with the formation of benzoquinone (oxidation) and hydroquinone (reduction) pair. the electrochemical behavior for the systems in figure 2 shows that the interactions between the pani or pani - pa with pvs and cg do not suppress the electroactive and electrochemical properties observed and described for polyaniline. the films of pani and pani - pa interspersed with pvs showed the three well - known oxidation processes of pani and a fourth oxidation process observed in the region of 0.86 v (figures 2(a) and 2(b)). this fourth oxidation was not observed for films in which pvs was replaced by cg in the multilayer structure (figures 2(c) and 2(d)). the oxidation process at 0.86 v can be explained as the result of an interaction between the cationic groups of pani with anionic groups of pvs. the phosphonic acid (pa) used in this study acts as both a modifying agent pani, increasing its solubility, and a dopant acid promoting a further increase in current values observed in the redox processes of pani - pa (figures 2(a) and 2(c)). this increase in current values contributes to providing an enhancement in selectivity of the pani - pa / pvs and pani - pa / cg systems compared to pani / pvs and pani / cg, and thus the presence of pa becomes an important factor to be considered in analytical determinations using electrochemical techniques. for the films pani - pa / pvs (figure 3(a)) and pani - pa / cg (figure 3(b)) with 2, 4, 6, and 8 bilayers, the cyclic voltammograms obtained at 50 mv s reveal that the increase in the number of bilayers is reflected in the increase of current values. this result indicates an increment in the amount of material adsorbed on the substrate as the number of bilayers increases. when pvs was replaced by cashew gum (figure 3(b)) the fourth oxidation process disappeared because the interaction between pani and pvs disappeared as well. the interaction between these pani and pvs can occur by two distinct mechanisms, similarly as proposed by raposo & oliveira for lbl films of poly(o - methoxyaniline) (poma) and pvs. this process can occur through the establishment of links between pani and psv in the presence of electrical charge (scheme 2(a)) and/or in the absence of electrical charge through the formation of networks of water molecules from the pani present in solution with pvs (scheme 2(b)). probably the adsorption processes proposed for the pani and pvs must be somehow related to the oxidation process observed in the region of 0.86 v in figure 3(a). in our studies we observed that the presence of cashew gum significantly decreases the degradation of the polymer in acid media, which is observed in films pani - pa / pvs around 0.43 v (shown in figures 2(a) and 3(a)). in figure 4 the cyclic voltammograms of the films with 6 bilayers of pani - pa / pvs (figure 4(a)) and pani - pa / cg (figure 4(b)) the 5th and the 20th successive cycles of scanning potential at 50 mv s are shown. the electrode polarization until 0.90 v led to a more visible degradation of the conducting polymer in system pani - pa / pvs. cyclic voltammograms for these films show the presence of intermediate redox process around 0.43 v, which is related to soluble products (radical benzoquinone / hydroquinone) formed during the acid degradation of polyaniline accompanied by a decrease of the current values in all the processes observed. on the other hand, the process for proton expulsion and anion uptake seems more stable for the pani - pa / cg film even after twenty successive cycles. therefore, the polyaniline suffers acid degradation, promoted by the high polarization potential and by electrolyte of hcl, and in the case in the pani - pa / pvs system this process is enhanced by the presence of pa modifier. on the other hand, when pvs was replaced by cg the degradation process of pani was reduced indicating that the cashew gum protects the film from the degradations processes mentioned above, presenting a greater stability during scanning in an acid medium and polarizations at 0.90 v compared to pani - pa / pvs or pani / pvs films studied. previous works from our group showed that lbl films of poma and gums have greater stability during scanning potential in an acid medium than films poma / pvs and that the gums as chich (sterculia striata) and angico (anadenanthera macrocarpa b.) act protecting the polymer film from this degradation. the process of charge transfer from the ito - modified electrode with pani - pa / cg film containing 6 bilayers was studied by varying the scan rate (v) in the range of 10 to 150 m v s (data not shown). in these conditions it was observed that the values of the anodic peak current (i pa1) increased linearly with scan rate for the film pani - pa / cg, according to the equation i pa1(a) = 1.92(0.76) + 0.30(0.010) v (mv s), with a linear correlation, r = 0.998. this behavior indicates a redox process of electroactive species that are strongly adsorbed on the ito surface, confirming that the electrochemical reaction is controlled by a kind of electron hopping mechanism of charge transfer on the electrode surface. the dependence on ph solution of the electrochemical behavior of pani - pa / cg film was studied and shown in figure 5. when increasing the ph from 1 to 2 an approximation of pani redox process occurs, and in ph 3.0 and 4.0 it is possible to observe the overlap of two redox processes at 0.30 v and 0.20 v for ph 3.0 and at 0.48 v and 0.10 v for ph 4. on the other hand at ph 5 the pani - pa / cg film no longer presents any electrochemical activity. however, when the same film was scanned again at ph 1.0 it presented the characteristic processes of electroactive polyaniline with potential and current values similar to the first scan done at ph 1.0 before variations in ph described above. this reversibility in the electrochemical behavior in function of the ph media is not observed in films of pani / pvs or pani - pa / pvs. thus, the results suggest that in the case of pani - pa / cg films, the cashew gum acts as a stabilizing element of the polyaniline. lbl films of cashew gum containing modified polyaniline with pa dopant are very interesting for applications in electrochemical sensors due to their high reproducibility and stability. thus, the self - assembled film of pani - pa / cg containing 6 bilayers was selected to be applied to detect dopamine (da), an important neurotransmitter in the central nervous system of mammals [34, 35 ]. figure 6(a) shows the cyclic voltammograms for the lbl film with 6-bilayers of pani - pa / cg in different concentrations of dopamine (da). in this figure a redox couple (e pa2/e pc) appears associated with oxidation of dopamine in dopamine quinone at 0.63 v and its reduction at 0.29 v [34, 35 ]. the anodic current peak (i pa2) increases linearly for concentrations of da between 0.01 and 0.23 mmol l for the modified electrode, as shown in the calibration curve in figure 6(b). the calibration equation obtained by linear regression is i pa2(a) = 0.15(0.043) + 23.10(0.37) [da]/mmol l with a correlation coefficient (r) equal to 0.998 (for n = 10) and sensibility of 23.10 a mmol l. the limit of detection (ld) of 1.5 10 mol l was estimated using 3/slope ratio, where is the standard deviation calculated from 10 blank samples and slope refers to the slope of the calibration curve, according to the iupac recommendations. the modified electrode presented reversibility after washing and a good repeatability for da determinations with relative standard deviation, rsd = 6% in five determinations in the presence of 0.23 mmol l da. table 1 summarizes the analytical performance on different modified electrodes for da detection by electrochemical process [2732 ]. the lowest ld obtained was observed for film from (pani - pa / cg)6, revealing itself as a competitive electrode for this analysis when compared to other modified electrodes. the relationship of peak current (i pa2) for da oxidation with the scan rate (v) has been investigated for 6 bilayers from pani - pa / cg film in 0.1 mol l hcl solution in the presence of 0.23 mmol l da (data not shown). under these conditions a linear relationship between anodic current peak (i pa2) and the square root of scan rate (v) for the pani - pa / cg film was found according to the equation i pa2(a) = 0.27(0.14) + 2.32(0.02) v (mv s), r = 0.999. this behavior indicates that the electrocatalytic process of electron transfer is controlled by dopamine diffusion from the solution to the redox sites of pani - pa / cg films. in order to investigate the selectivity of the pani - pa / cg modified electrode, we tested the simultaneous detection of 0.01 mmol l da in different ascorbic acid (aa) concentrations, the natural interfering of da. it is noted in figure 7 that when we increase the concentration of aa a proportional increase in the current values at 0.45 v is accompanied. moreover, in the oxidation potential of dopamine, 0.63 v, the increase of current with the addition of aa is minimal, showing that oxidation of dopamine at the surface of modified electrode is slightly affected by trace amounts of aa (figure 7). additionally it is important to observe that this new nanocomposite using cashew gum, which is a natural and biocompatible polymer, in the multilayer structure, gives rise to new applications as biosensor [38, 39 ]. additionally, this film could be used in wound repair because it has been shown that very small exogenously applied electrical currents produce a beneficial therapeutic result for wounds. in this work we propose a formation of electroactive nanocomposite, which can be a potential tool for wound repair when associated with the electrostimulation. the electrochemical profiles observed for the films studied showed the redox intrinsic characteristic transitions of polyaniline. the presence of cg increases the electrochemical stability of the film, suggesting that it acts by protecting the conductive polymer from acid degradation. lbl films of pani / pvs and pani - pa / pvs show an oxidation process around 0.86 v, which can be related to interaction between pani and pvs. detailed studies showed that the electrochemical reaction in the pani - pa / cg film is governed by a charge transfer mechanism at the surface electrode via electron hopping. the ito - modified electrode with pani - pa / cg film showed high reproducibility and stability, encouraging its use as a sensor of da. this modified electrode was able to detect electroactive molecule of da around 0.63 v in detection limits consistent with the pharmaceuticals formulations. | we take advantage of polyelectrolyte feature exhibited by natural cashew gum (anacardium occidentale l.) (cg), found in northeast brazil, to employ it in the formation of electroactive nanocomposites prepared by layer - by - layer (lbl) technique. we used polyaniline unmodified (pani) or modified with phosphonic acid (pa), pani - pa as cationic polyelectrolyte. on the other hand, the cg or polyvinyl sulfonic (pvs) acids were used as anionic polyelectrolytes. the films were prepared with pani or pani - pa intercalated with cg or with pvs alternately resulting in four films with different sequences : pani / cg pani - pa / cg, pani / pvs and pani - pa / pvs, respectively. analysis by cyclic voltammetry (cv) of the films showed that the presence of gum increases the stability of the films in acidic medium. the performance of the modified electrode of pani - pa / cg was evaluated in electro analytical determination of dopamine (da). the tests showed great sensitivity of the film for this analyte that was detected at 105 mol l1. |
cryocrystalglobulinaemia is one the rarest manifestations of monoclonal gammopathy characterized by the reversible crystallization of monoclonal immunoglobulins composed of both light and heavy chains below 37c. these crystals can be intracellular, often in plasma cells, or extracellular in various organs and tissues. initially described in patients with multiple myeloma, cryocrystalglobulinaemia has since been reported with various b - cell lymphoproliferative disorders. patients typically present with a systemic necrotizing vasculitis manifested by cutaneous purpura and ulcers, mucosal ulcers, erosive polyarthropathy and renal dysfunction. a 51-year - old male previously in excellent health developed increasing fatigue and decreased exercise tolerance in 2002. other symptoms included recurrent sinusitis, transient cough, arthralgias, hives and swollen tender feet. he particularly noted that his fatigue worsened with exposure to the cold weather. in september 2003,, the patient was hospitalized for extreme shortness of breath and a serum creatinine (scr) of 4.1 mg/ dl (362 mol / l). a renal biopsy was performed. the renal biopsy (figure 1) showed eosinophilic immunoglobulin thrombi within many of the arterioles and glomerular capillaries, including the vascular pole of the glomerulus. this material was variably pale to somewhat positive on a pas stain and was negative on a congo red stain. the glomeruli did not show mesangial hypercellularity or mesangial matrix expansion, no glomerular basement membrane thickening or duplication was identified and no crescents were present., the immunoglobulin thrombi showed bright staining for igg, c3, fibrinogen and kappa light chain, lesser staining for c1q and no staining for igm or lambda light chain. (top left) several immunoglobulin thrombi are seen within glomerular capillary lumens and within arteriolar lumens (arrows) (haematoxylin and eosin). by immunofluorescence, these areas stained for kappa light chain (inset) and fibrinogen but not for lambda light chain (immunofluorescence). (top right) an arteriole contains an immunoglobulin thrombus with a crystalline structure (haematoxylin and eosin). (bottom left) by electron microscopy, crystalline immunoglobulin thrombi fill a glomerular capillary lumen. (bottom right) on higher magnification, crystalline immunoglobulin thrombi show a fine periodicity. serum protein electrophoresis showed a small monoclonal band in the gamma region, but immunofixation was not performed. a bone marrow biopsy at that time showed no definite evidence of a plasma cell disorder. tests for hiv, hepatitis b and c infection were negative. testing for hypercoagulable states was not performed. renal function also improved [scr = 1.9 mg / dl (168 mol / l) ], but he continued to have fatigue. in september 2004, another leg ulcer developed. he was treated with azathioprine 50100 mg / day and prednisone 50 mg / day that was tapered to 5 mg twice a day over 6 months. in february 2006 his blood pressure was 200/100 mmhg and a scr was 8.2 mg / dl (725 mol / l). the ldh was elevated at 1253 u / l, but creatinine kinase was normal. laboratory evaluation including cryoglobulins, ana, anca and ena were negative, and serum c3 and c4 were normal. g / l) with wbc of 8.5 10/l, but the platelet count was 12 10/l. the patient 's thrombocytopaenia prevented a renal biopsy at the time and high - dose intravenous methylprednisolone was started empirically. no other causes of acute renal failure, such as initiation of a new drug, were identified. the patient remained anuric, and a renal angiogram (figure 2) was performed which showed abnormalities of the main, tertiary and quaternary branches with no cortical perfusion. thrombophilia testing for factor v leiden, g20210a prothrombin gene variant and anti - phospholipid antibodies was not performed. renal angiogram : decreased flow bilaterally with abnormalities of the main branch of the left renal artery. these abnormalities persisted after 12 h of intra - arterial tpa infusion. in september 2007, patient presented for a second opinion. his total urinary protein level was 133 mg / day in 182 ml of urine a day. serum immunofixation did reveal a monoclonal igg in the serum with an m - spike of 0.4 g / dl (4 his serum iga and igm were suppressed at 35 mg / dl (normal range, 50400 mg / dl) [0.35 g / l (normal range, 0.050.40 g / dl) ] and 48 mg / dl (50300 mg / dl) [0.48 the serum total igg was normal at 837 mg / dl (normal range, 6001500 mg / dl (8.37 g / l (615 g / l)) ]. the kappa free light chain was 33.9 mg / l (normal range, 3.319.4 mg / l) and lambda was 42.7 mg / l (5.726.3 mg / l) with a ratio of 0.79 (0.261.65). the previous renal biopsy was reviewed and additional staining was performed on paraffin - embedded tissue. in addition to positive staining for fibrinogen by immunofluorescence, the immunoglobulin thrombi were also positive on a phosphotungstic acid - haematoxylin (ptah) stain. no linear staining of glomerular or tubular basement membranes for a monoclonal immunoglobulin was present. electron microscopy (em) revealed the immunoglobulin thrombi to be composed of crystalline structures with an organized substructure showing a periodicity of 20 nm. in areas of the immunoglobulin thrombi, the endothelial cells appeared reactive, with enlargement of the cells and loss of fenestrations. no finely granular deposits suggestive of monoclonal immunoglobulin deposition disease were identified within basement membranes. because the renal biopsy showed co - localized monotypic immunoglobulin and fibrinogen, the patient 's serum was evaluated by immunoprecipitation for the presence of fibrinogen and was found to be negative. a repeat bone marrow biopsy and aspirate showed minimal involvement by a kappa light chain - restricted plasma cell proliferative disorder based on flow cytometry, with 1% plasma cells in the marrow. cryocrystalglobulins may precipitate in vivo and in various tissues and organs, particularly in arterial walls causing vasculitis and vessel occlusion. clinically, cryocrystalglobulinaemia usually presents as skin lesions, with recurrent purpura, petechiae and skin ulcers. cryocrystalglobulinaemia may also present as an arthropathy, with crystal deposition in joint fluid [810 ]. renal and intestinal small vessel involvement, including a clinical picture of systemic vasculitis, has also been described [5,912 ]. cryocrystalglobulinaemia is similar to type i cryoglobulinaemia in which the precipitated protein forms crystalline structures. crystals of various shapes, from fusiform to diamond, have been observed. cryocrystals are usually composed of a monoclonal igg although a rare light chain only variant has also been described [25,8,10,1215 ]. while purpuric rashes, skin ulcers and raynaud 's phenomenon are common to type i cryoglobulinaemia, renal manifestations are rare. cryocrystalglobulinaemia on the other hand also presents with purpura and ulcers, but raynaud 's phenomenon is notably absent. the true incidence of cryocrystalglobulinaemia is unknown. a review of the literature found 50 cases, but it is important to distinguish cryocrystalglobulinaemia and the other immunoglobulin crystalline nephropathies (crystal - storing histiocytosis, light chain fanconi syndrome or light chain crystal deposition disease) which do not show crystal formation upon cooling [25,8,10,1215,1822 ]. in addition, the cutaneous and arthropathic features that are often found in cryocrystalglobulinaemia are absent in these entities. patients with so - called essential cryocrystalglobulinaemia (without myeloma) tend to do better with survival often measured in years. treatment with chemotherapy has produced mixed results. while some patients respond to steroids alone, others have failed cyclophosphamide and melphalan. plasma exchange can rapidly improve symptoms, but multiple courses may be needed and durable response must be maintained with effective chemotherapy. more recently, improvement in ulcers and renal function was reported with thalidomide and dexamethasone in one patient. although there is no standard therapy, most authors advocate the importance of early recognition and rapid initiation of effective therapy as the key to better outcomes. the presentation of bilateral renal arterial occlusion in our patient is unusual, if not unique, in cryocrystalglobulinaemia. first, while renal involvement is common, the complete occlusion of the arterial supply is unusual. complete vascular occlusion is typically limited to small peripheral vessels although focal visceral ischaemia has been reported. precipitation and crystallization are thought to be less likely to occur with the warmer blood in the visceral circulation. even in the periphery, vascular occlusion had only been reported in patients with advanced myeloma. in those cases, our patient, however, did not have myeloma (1% monoclonal restricted plasma cells in the bone marrow) and is still alive 20 months after bilateral renal artery occlusion. the cause of the sudden flare resulting in renal artery occlusion and then disease quiescence despite the persistent presence of the monoclonal igg remains unexplained. one particularly unusual aspect of this case is the co - localization of the monoclonal immunoglobulin thrombi with fibrinogen, as demonstrated by immunofluorescence and by light microscopy by ptah staining. a somewhat similar case of microvascular thrombosis with a plasma cryoprecipitate composed of fibrinogen and monoclonal igg- has been reported ; this patient developed skin ulcers upon exposure to the cold, and laboratory evaluation revealed a monoclonal igg- in the serum and negative tests for cryoglobulins in the serum. we hypothesize that in the current case the igg- paraprotein, unlike other monoclonal immunoglobulins, is particularly thrombogenic, as evidenced by its co - localization with fibrinogen in tissue sections. as demonstrated by this case, the renal manifestations of monoclonal gammopathy can be vast and diverse. a renal biopsy should be performed in all patients with monoclonal gammopathy and evidence of renal involvement. | cryocrystalglobulinaemia is an extremely rare complication of monoclonal gammopathy. its presentation has features of both type i and ii cryoglobulinaemia. although peripheral and digital ischaemia is common, visceral ischaemia is rare. when it does occur, it is usually associated with multiple myeloma and has an extremely poor prognosis. we present a case of bilateral renal artery thrombosis associated with cryocrystalglobulinaemia in a patient without myeloma. more unusual, the cryocrystal protein in this case was associated with fibrinogen, which may have led to increased propensity towards thrombosis. although the patient was unable to recover his kidney function, he remained alive on dialysis 2 years after the incident. the patient did not have any further ischaemic event despite no definitive therapy. this case represents an unusual presentation for this rare disease. |
although breast cancer incidence is higher overall in women of european descent than in women of african ancestry, african - american (aa) women are more likely than european - american (ea) women to be diagnosed before age 40 and to have breast tumors with more aggressive features, including high - grade and negative estrogen receptor (er) status (reviewed in). there are no facile explanations for these differences in the epidemiology of breast cancer by ancestry. there have been several studies of breast cancer risk that include both aa and ea women, such as the carolina breast cancer study, the care study, and the black women 's health study ; however, none were specifically designed and powered to evaluate numerous risk factors for early / aggressive breast cancer and to evaluate the distribution of these risk factors within and across racial / ethnic groups. because of the large, racially mixed population of women in metropolitan new york city (nyc) and eastern new jersey (nj), we are currently conducting a case - control study, the women 's circle of health study (wchs), with the goal of accruing 1200 aa and 1200 ea women with breast cancer and an equal number of controls, to specifically address these questions. initial funding for this study was through a center of excellence for biobehavioral breast cancer research (bovbjerg, pi) focusing on aa women, funded by the department of defense (dod). additional r01 funding (ambrosone, pi) from the national cancer institute (nci) was subsequently obtained which allowed us to increase the sample size and to extend the study to ea women. as illustrated in figure 1, the study has included two bases for recruitment and interviewing, one in nyc, based at mount sinai school of medicine (mssm), and one in nj, based at the cancer institute of new jersey (cinj), with data and biospecimens sent to roswell park cancer institute (rpci) in buffalo, ny, for processing and storage. in the nyc metropolitan region, there are more than 60 hospitals where surgery for breast cancer is performed. when this study began in 2003, to maximize efficiency, we targeted the hospitals that had the greatest referral patterns for aa women in the boroughs of manhattan, brooklyn, queens, and the bronx. our initial plan was to employ the approach commonly used in case - control studies, such as the carolina breast cancer study and the long island breast cancer study project, wherein rapid case ascertainment is used to identify women newly diagnosed with breast cancer through periodic review of pathology reports in the targeted hospitals. when women with breast cancer are identified, a letter is sent to the treating physician, notifying them that unless they object, the patient will be contacted to describe the study and assess interest in participation. we were unable to use this approach, however, due to the implementation of the health insurance portability and accountability act (hipaa) privacy rule in 2003, while we were establishing the infrastructure for the study. this extension of the hipaa regulation prevents the release of private health information (phi) without consent from the patient. for our research purposes, this act prevented the identification of eligible cases without the patients ' prior permission given to their doctors. although there may be situations in which an hipaa waiver can be obtained to circumvent the need to obtain patient permission for release of identifying information to researchers [4, 5 ], the several participating hospitals and their institutional review boards (irb), many not extensively familiar with epidemiological research, would not grant these waivers to allow patient identification. thus, we developed a procedure for patient ascertainment and contact that complied with the regulations of hipaa. as an alternative strategy, we expanded our catchment area to include eastern nj, by partnering with cinj and the nj state cancer registry, a surveillance, epidemiology and end results program (seer) site, housed at the nj state department of health and senior services (njdhss). the study has been approved by the irb at rpci, robert wood johnson medical school (for the cinj), mssm, the individual hospitals in nyc, and the njdhss. in this paper we report on both of our approaches to case ascertainment and consenting, discussing effort and costs associated with each methodology. currently, recruitment efforts are focused only in nj, and accrual has been discontinued in ny. we also present an overview of the study design, report on distributions of demographic and selected breast cancer risk factors among both cases and controls by race / ethnicity, and compare clinical breast cancer characteristics between groups in a subset of the population enrolled to date. aa and ea women, 20 to 65 years of age, with no previous history of cancer other than nonmelanoma skin cancer, diagnosed within 9 months with primary, histologically confirmed invasive breast cancer or ductal carcinoma in situ who speak english were eligible for participation in the study. they were ascertained from designated hospitals that have large referral patterns for aa women in the nyc boroughs (manhattan, bronx, brooklyn, and queens ; due to few aa breast cancer patients, staten island was not included). to maintain comparability between cases and controls, women with breast cancer must have had a residential telephone given that controls were ascertained using random digit dialing (rdd). this eligibility criterion has now been expanded to cell phone usage, however, with rdd also covering cell phones for control ascertainment. to address hipaa regulations that prohibit identification of women with breast cancer using pathology reports, tumor registry data, or medical records, we worked to develop collaborative relationships with physicians, research nurses, and patient navigators at each of the participating hospitals. our research assistants initiated frequent visits to each site, particularly on clinic days, and became well known by staff and clinic personnel. as we began working with physicians at each site, clinicians reviewed their records for retrospective ascertainment and identified women who were eligible to be in the study (e.g., had been diagnosed within the last 9 months). at each of the participating hospitals, physicians telephoned women who were not returning for followup and would not be seen at subsequent visits, asking if wchs staff could contact them regarding the study. those scheduled for routine followup appointments within the 9-month interval were seen and asked if they were willing to be contacted for this study. for contemporaneous recruitment, our study staff was present in the offices on breast clinic days and was informed by the physicians or research nurses at that time of patients scheduled on those days who were eligible for the study. study materials were placed in the charts of the eligible patients as a reminder for the clinician to discuss the study. if in agreement, the patient was then referred to our waiting study staff. a number of patients participated in the informed consent procedures at the time that they were first approached and a pretreatment blood specimen was obtained. other women preferred to be contacted at a later date by the research assistant (ra)/study interviewer, to schedule a date to obtain consent and conduct the in - person interview. to strive for complete case ascertainment, we periodically requested that physicians review their records to confirm that we had not missed potential cases, and that they follow the procedures described above if there were women who were not previously approached to participate in the study. it was our intent that this periodic review would allow us to estimate a denominator, to some extent, and to keep track of women who refused to be contacted so that selection bias could be examined. however, these data were not easily obtained with our inability to access records of women diagnosed who had not been approached, and competing priorities of busy surgeons. this approach to case ascertainment and contact yielded good participation rates for both aa and ea cases but was extremely labor intensive, requiring frequent communications between our research staff and clinical personnel as well as the presence of ras at the hospitals on clinic days. besides being costly in personnel time, this methodology required a good deal of dedication and commitment on the part of physicians, with frequent reminders from study staff for them to check their appointment ledgers and contact patients who may have been missed on clinic days. because of all of the limitations of this approach, in 2006 we established collaboration with the new jersey state cancer registry, based at the njdhss for rapid case ascertainment, and phased out recruitment in metropolitan new york, ending in december 2008. in nj, cases are actively being identified at all major hospitals in passaic, bergen, hudson, essex, union, middlesex, and mercer counties through rapid case ascertainment. in addition, njdhss study staff routinely check the new jersey state cancer registry (njscr) database for eligible cases who reside in the target counties but are reported by hospitals outside of those seven counties or out - of - state. all aa women less than 65 years of age who are newly diagnosed with incident breast cancer are identified as potential participants. for each aa case, an ea woman with breast cancer is randomly selected, matching on age (5 years) and county of residence. njdhss study staff review pathology reports of potential cases, contact doctors ' offices, and hospitals to verify patients ' race and demographics and check the njscr database for prior diagnoses of cancer. after contact with clinicians by njdhss staff for passive consent (e.g., contact from physician only in the event that they do not give permission to contact their patients), eligible women are telephoned by njdhss staff to obtain verbal consent to release names and contact information to wchs research staff at cinj. patients who agree to be contacted by wchs study staff are then telephoned by one of our interviewers, and appointments are scheduled for in - person interviews at home or at another mutually convenient location. aa and ea women 20 to 65 years of age without a history of any cancer diagnosis other than non - melanoma skin cancer are eligible to be controls. the choice of a proper control group is a difficult issue in epidemiology today, particularly for a study that is not population - based. when planning for the wchs, we evaluated several potential sources of control groups, weighing the strengths and weaknesses for each. while we considered using hospital controls in nyc, we felt that they would not necessarily represent the same populations from which the cases were derived. for example, many of the treating physicians at mssm have private surgical practices ; there is no indication that clinic patients from the hospital would be similar to those being treated by private physicians. furthermore, there are well - recognized potential biases associated with the use of hospital controls. in theory, the generalizability of study results is likely to be greater in studies using community controls rather than those using friend or hospital controls. yet, in contrast to the western european national health care records, none of the available united states (us) lists, such as that of licensed drivers, municipal tax roles, voter registration, and listed phone numbers, provide complete source population enumeration. population coverage, access to this information, and the quality of contact information vary geographically in the us. of nyc residents, it is estimated that only 52.1% have drivers licenses, only 30.2% pay residential taxes, and only 56.2% are registered voters. these examples typify the acknowledged weaknesses of us and nyc sampling frames. for generating a control group of adults under 65 years of age we used random digit dialing (rdd) because unlisted numbers can be reached by this method, thereby avoiding possible selection bias (nyc study found that 27% of rdd controls had unlisted numbers). thus, rdd provides an ideal source when phone coverage is near complete ; 93% of nyc residences have phones. high phone coverage makes rdd one of the best sources for generating a sampling frame for controls of nyc area women under 65 years of age. even when the source population is not solely defined by geography, a modified version of rdd is available that creates a control sampling frame using the cases ' telephone numbers [10, 12 ]. rdd controls have been compared to a privately conducted census population as well as to area survey controls, and both comparisons found that rdd controls were similar to those from other sources. most importantly, high response rates within a minority community were demonstrated using the modified waksberg rdd method, and in the wchs, response rates among minorities are similar to those among ea women. the elimination of household landline phones in favor of cell phones represents a challenge for telephone surveys based on rdd to landline telephones [16, 17 ]. however, because the percentage of households without landlines remains low, any potential bias associated with this issue is likely to be small. furthermore, once subjects agree to participate in the study, cell phones tend to facilitate scheduling interviews and completing study materials because the calls go directly to the participants and are not screened by other household members. for rdd in nyc, the telephone exchanges (area code plus three - digit prefixes) of the breast cancer cases who received medical care at the participating hospitals in previous years were used for sampling. we frequency matched controls to cases on the expected breast cancer case distribution (based on 19941998 data from the nys tumor registry) by 5-year age groups and race. the age distribution of targeted controls was periodically modified based upon the actual distributions of age among the cases. controls were identified, recruited, and interviewed in the same manner and during the same time period as the cases to eliminate any bias related to secular trends or changes over the interviewing period. in nj, the same methodology is used for ascertainment of eligible controls ; however, rather than using telephone numbers from participating hospitals, the entire county is sampled, because cases include those from all hospitals in the seven targeted counties. controls, once identified, are contacted to schedule an in - person interview ; interviews are conducted either at the participant 's home or at another convenient location. for both cases and controls in nyc and nj who decline participation, we request that they complete a short telephone interview (510 minutes) to obtain basic information on demographic and exposure factors. in the final analysis, data from women who refused study participation will be compared to data from women who completed an interview to evaluate potential bias related to non - participation. women who complete the study are offered a $ 50 gift certificate to one of several local stores as incentive for participation. we had initially offered $ 25 at the beginning of the study, but later increased the amount due to inflation and efforts to increase participation. the in - person interview consists of the informed consent process, an in - depth in - person interview, completion of several behavioral questionnaires including a food frequency questionnaire (ffq), collection of biospecimens, and body measurements. for cases, we also request a release for access to medical records, pathology data and for tumor tissue, as well as permission to conduct followup. the survey instrument is an adaptation of several questionnaires, including validated surveys from the women 's health initiative and the western new york diet study. developmental history questions were taken from the women 's interview study of health (wish), and lifetime physical activity is assessed using a modified version of friedenreich 's validated questionnaire. information on medical history, family history of cancer, lifestyle factors including smoking, alcohol consumption, and use of hair products is also collected. the most recent version of the ffq developed at fred hutchinson cancer center and validated in the nci / swog prostate cancer prevention trial is used for dietary assessment. this ffq has been validated for use in an aa population. at the end of the visit participants are asked to wear light clothing, as weight, standing height, and waist, and hip circumferences are measured. body composition (lean and fat mass) is measured using a bioelectrical impedance analysis scale (tanita scale). questionnaires are coded by two separate ras, and double data entry is performed by two separate clerks, with data managed at rpci. we initially collected blood samples which were processed and stored in the laboratory at mssm. in 2007, to reduce costs and to facilitate participation, we transitioned to collection of saliva using oragene kits (dna genotek, inc, ottawa, on, canada) for dna extraction. these collection kits yield large quantities of high - quality dna, comparable to that obtained from whole blood [20, 21 ]. protocol for dna extraction from saliva or the flexigene method (qiagen inc, valencia, ca) for whole blood or buffy coat. dna is evaluated for purity and concentration using a nanodrop uv spectrophotometer to obtain a230, a260, and a280 readings, and double stranded dna is quantitated using a picogreen - based fluorometric assay (molecular probes, invitrogen inc, carlsbad, ca). saliva specimens have been stored at room temperature until extraction, and dna samples are stored at 80c at rpci. formalin - fixed paraffin - embedded blocks and corresponding pathology reports from patients who signed the pathology and tissue release have been retrieved from hospitals on an ongoing basis. to date, 1193 patients have agreed for release of their tumor tissue (91%), and this proportion does not vary between nj and ny. pathology reports are reviewed in order to identify a representative tumor block used to make the primary breast cancer diagnosis for each case. the tumor blocks are shipped to rpci, where they are labeled and entered into the tracking database. hematoxylin and eosin (h&e) slides are cut and reviewed by the study pathologist (hh) to determine the locations from which cores should be taken for construction of tissue microarrays (tmas), taking punches from both tumor and normal tissues and for consistent determination of grade by one pathologist. representative tumor tissue is also labeled and punches taken to be stored for future dna extraction and analysis. pathology departments that do not release blocks have instead been asked to process and cut the requested number of slides (eleven unstained 5 slides and six unstained 10 slides), which are then sent to the laboratory at rpci. tissue blocks and pathology reports are collected in tandem and include the abstraction of medical record data. because the consent process includes a tissue block and medical record release form, and blocks are being requested in real time, there has been little resistance on the part of the hospitals to provide tissue. in establishing the infrastructure for this study, and making efforts to conduct a study based in community hospitals in the face of stringent hipaa and confidentiality requirements, our group brainstormed and adapted to achieve maximum case ascertainment, contact of patients, and recruitment into the study. with the help of committed and dedicated clinicians, this approach was successful at some hospitals, but not all. clearly, it places a burden on already busy clinical practices, and it is likely that a complete denominator was not available, due to patients overlooked or deemed not suitable for participation in the study by their physician. in our experience, this is not a practical way to conduct a study and, unless one can ascertain cases through pathology reports or medical records, the costs of such efforts through local hospitals may not justify the numbers of cases able to be accrued. in contrast, by working through the njdhss, an nci seer site, we capture all cases diagnosed within a circumscribed area and truly know the denominator of the study for calculation of response rates. an additional advantage is that information on tumor characteristics is available for non - participating cases. when women were personally apprised of the study by their physician, response rates were relatively high, with 75% of ea and 75% of aa women completing interviews and providing blood or saliva samples. however, we have no data on the number of women who were eligible for the study and were not approached by their physician, or those who requested not to be contacted by our study staff. when contacted by the njdhss, 73% agreed to be contacted by an interviewer, and 93% of those women were interviewed and provided a saliva sample, for a total participation rate of 68%. participation was poorer for aa women in nj ; 60% agreed to be contacted by an interviewer when telephoned by staff from the njdhss, and of those, 90% were enrolled into the study, for a total participation rate of 54%. we have met approximately half of our accrual goal, to date, and efforts are constantly made to improve response rates. in nj, newly diagnosed patients from all hospitals in the 7 targeted counties are ascertained and contacted by the njdhss., we focused on those hospitals with the highest referral patterns for aas in the 5 boroughs excluding staten island, and it is clear that coverage was not complete. while an average of 1273 cases per year are reported in aa women in the boroughs, we were only able to ascertain approximately 67 per year through working with clinicians in selected hospitals. we expect that the control sampling frame in ny results in a representative population, nonetheless, because the first three numbers of breast cancer patients seen in previous years at each hospital were used to obtain women in the same residential areas. when confronted with difficulties in case ascertainment in nyc, we sought ways to expand eligibility criteria without compromising the integrity of the study. we initially limited eligibility for case participants to those between the ages of 20 to 64 years, primarily because of the low response rates using rdd for controls 65 years and older. in 2007, we extended the upper limit of age eligibility to 75 years for cases, but not controls. although these older women can not be used in case - control comparisons, they will allow for case - case analysis of younger versus older age at onset of breast cancer, in which age of the patient is the dependent variable. this will allow us to explore possible differences in study variables (e.g., aggressive versus non aggressive disease characteristics) between older breast cancer patients and younger breast cancer patients. we will also explore the possibility that such differences might differ by race / ethnicity groups and by other disease characteristics defined by pathology. we had initially trained wchs interviewers in phlebotomy and made consent for specimen collection a requirement of the study. three tubes of blood were collected and processed, with straws stored with plasma, serum, red blood cells (rbc), and buffy coat for dna extraction. our intent was, when possible, to collect pretreatment blood samples to be able to compare biomarkers in cases and controls and for use later in studies of breast cancer prognosis. because of the difficulties in accrual in nyc, and in planning approaches in nj where we knew that we would not be able to coordinate specimen collection prior to initiation of cancer therapy, we decided to collect saliva as a source of dna only, using oragene saliva dna self - collection kits when we began recruitment in nj. again, our ideal approach would be to have pretreatment blood specimens on all cases, but in the interests of cost and feasibility and what was viewed as long term utility of samples other than dna, compromises had to be made. to date, we have serum, plasma, and rbcs banked on 261 aa and 197 ea controls as well as 198 and 147 aa and ea cases, respectively, which should provide us with capabilities to investigate, in a limited sample set, differences in biomarkers among controls only, and case control evaluations for markers that are not likely to be affected by surgery or adjuvant therapy. all other cases and controls provided saliva samples, and there are no participants in the study for whom a source of dna is not available. as noted above, case ascertainment and accrual in nyc was terminated in 2008, and all efforts are now ongoing and focused on enrollment in nj. table 1 shows current recruitment numbers for cases and controls, by race, in nyc and in nj. for the scope of this paper, we are reporting data on the subset of cases and controls who have questionnaire data which have been processed and verified through double data entry, which includes 858 controls and 1119 cases. in examining preliminary data through february 2009 because we are still in data collection phase, we have made limited comparisons between cases and controls in this report. rather, we have contrasted demographic and tumor characteristics among aa and ea women in our study samples. among controls (table 2), there are differences in country of birth, with more aas born in the caribbean. eas are more likely to be married, to have graduated college, and to have employer - provided health insurance. higher proportions of ea women have incomes above $ 90,000 per year and ea women have fewer pregnancies and at a later age than aas. rates of screening mammography are similar between aa and ea women without breast cancer (86% and 87%, resp.). notably, aa controls are more likely to be overweight than eas (30% versus 25%) or obese (52% versus 26%) but are less likely to use hormone replacement therapy (hrt) than eas (15% versus 24%). demographic characteristics of cases (table 3) and differences by race / ancestry are, for the most part, similar to distributions for controls in terms of birthplace, marital status, education, health insurance, and income. twenty percent of aa women with breast cancer in our study either do not have health insurance (17%) or pay for insurance out of pocket (3%), compared to 12% of ea cases (4% with no insurance, 8% self - purchased). in contrast to controls, where use of mammography is similar by race / ancestry, only 78% of aa cases ever had a screening mammography, compared to 88% of ea women, and 51% of ea cases had their breast cancer discovered by mammography versus only 36% of aa women. there also appear to be greater differences by race / ancestry for hormonal and reproductive factors among cases than among controls. twenty - nine percent of aa cases experienced menarche at or below age 12, compared to only 24% of ea women ; these differences are not as notable among controls (27% versus 25%). african american cases also tend to have more children and at an earlier age than ea cases, similar to patterns observed among controls. as observed for controls, aa women with breast cancer are also more likely to be overweight (31%) or obese (53%) than ea cases (26% and 26%, resp.) and are less likely to use hrt than eas (15% versus 27%). of the pathology reports abstracted to date, the characteristics of tumors of women in our study are similar to those noted in literature. african - american women are more likely than ea to have high - grade tumors (52% versus 32%) with er negative (34% versus 22%) and pr negative (48% versus 34%) status. it is possible that differing methods of ascertainment and accrual could result in selection bias. we compared clinical and some epidemiological data between participants in ny and those in nj. as shown in table 4, aa cases from ny are more likely to have less than 11th grade education (22% versus 9%), more likely not to have health insurance (23% versus 9%), or be receiving medicaid (21% versus 8%). cases in ny had a lower incidence of dcis (21% versus 13%), with invasive cancers being slightly higher (87% versus 79%). these differences may be due to the fact that, in new york, the majority of aa cases were ascertained at kings county hospital in brooklyn which serves a large caribbean community, many with low socioeconomic status, or because participation rates were higher in ny, resulting in some selection bias among those who agreed to be contacted in nj. for ea patients (table 5), ny cases were more likely to be postgraduates (36% versus 22%) and but were less likely to have insurance (5% versus 2%) and receive medicaid (4% versus 0%). cases in ny were less likely to be obese (32% versus 22%) and had an older age at menarche (52% versus 42%). differences between controls in ny and nj (tables 6 and 7) showed some similar patterns as those for cases. ny aa controls were more likely to be on medicaid (18% versus 10%) and were more likely to be obese (55% versus 34%). it is difficult to ascertain the representativeness of our participants in relation to the underlying populations they were derived from. however, we did ask those who refused to be interviewed to complete a short telephone interview. in ny, cases who refused tended to be older > 49, insured, either through medicaid, medicare, or employee - based insurance, have never taken hormone replacement therapy, and have had screening mammograms. similar differences were noted for cases in nj and for controls (insured, no hrt, and higher prevalence of screening mammograms). for controls, those who refused were more likely to have employer - provided insurance. the higher participation rates of cases in ny suggest that there would be less selection bias than in nj, particularly for aa cases, because of lower participation rates in nj. on the other hand, the population of cases in ny is somewhat skewed towards those treated at the county hospital, where there is a large caribbean population. when embarking on the conduct of a case - control study, a number of factors should be considered with respect to methodology. uppermost in importance is feasibility, which is often overlooked by young, eager investigators. although we recruited and interviewed over 500 cases through hospitals in nyc, the approach was often a struggle, and there is no question that case ascertainment through collaboration with a state seer cancer registry is much more efficient. using this approach, we are currently interviewing over 60 women per month, with numbers expected to rise with additional interviewers hired. we are confident that we will reach our accrual goals within the next 24 to 36 months, with ample power to evaluate our main study hypotheses, yielding important information regarding the etiology of aggressive breast cancers among aa as well as ea women. since initiating the study, scientific knowledge has advanced, and while our earlier aims were to categorize women according to age at onset, tumor grade, and er status, we are currently reclassifying tumor grade based on readings from one pathologist and building tmas with funding from the breast cancer research foundation to stain and read all tissue for er, pr, and her2 for assessment of triple negative breast cancers as well as cytokeratins 5 and 6 and her1 to help classify basal - like breast cancers. the successful enrollment of cases and controls, and collection of tissue blocks, has also facilitated numerous collaborations for pooled studies to conduct genomewide association studies and to determine the extent of african admixture in relation to tumor characteristics. with tumor tissue dna as well as tmas in addition to the epidemiologic data and biospecimens, we will have numerous opportunities not only to address our primary hypotheses but also to address novel hypotheses regarding ethnic / racial disparities in breast cancer incidence and mortality. epidemiological research has become increasingly difficult with the growing concerns regarding privacy and legal issues. to be able to address pressing issues in breast cancer research, particularly causal factors for the more aggressive breast cancers in aa women, creative strategies are required to conduct hospital and population - based studies. partnership with seer site is one approach for successful and complete case ascertainment and can facilitate the needed research in breast cancer disparities. | breast cancer in african - american (aa) women occurs at an earlier age than in european - american (ea) women and is more likely to have aggressive features associated with poorer prognosis, such as high - grade and negative estrogen receptor (er) status. the mechanisms underlying these differences are unknown. to address this, we conducted a case - control study to evaluate risk factors for high - grade er- disease in both aa and ea women. with the onset of the health insurance portability and accountability act of 1996, creative measures were needed to adapt case ascertainment and contact procedures to this new environment of patient privacy. in this paper, we report on our approach to establishing a multicenter study of breast cancer in new york and new jersey, provide preliminary distributions of demographic and pathologic characteristics among case and control participants by race, and contrast participation rates by approaches to case ascertainment, with discussion of strengths and weaknesses. |
the major growth factors for eosinophils are interleukin (il)-3, il-5, and granulocyte monocyte - colony stimulating factor (gm - csf). these regulators are produced by activated t lymphocytes, mast cells, and tissue stroma cells. peripheral blood eosinophilia is defined as an absolute eosinophil count (aec)0.510/l. the degree of blood eosinophilia is classified as either mild (aec, 0.510/l to 1.510/l), moderate (aec, 1.510/l to 5.010/l) or severe (aec,>5.010/l). in general, eosinophilia is divided into either familial or acquired types. acquired eosinophilia is classified as primary or clonal eosinophilia (based on the presence of histological, cytogenetic or molecular markers of a myeloid malignancy in the bone marrow), secondary eosinophilia (a cytokine - driven reactive phenomenon), and idiopathic eosinophilia (neither secondary nor clonal), which includes idiopathic hypereosinophilic syndrome (he s). the causes of secondary eosinophilia are infections (mostly helminthic), drugs, pulmonary eosinophilia, autoimmune diseases, inflammation, endocrinopathies, and malignancies [2 - 4 ].. however, there have been few reports on the association between multiple myeloma (mm) or plasma cell disorders and eosinophilia, and the few cases that have been reported have found outcomes after chemotherapy to be poor [6 - 8 ]. we now report a case of a 31-year - old male with mm who had marked absolute peripheral blood and bone marrow eosinophilia, leading to multiple organ dysfunction with eosinophil infiltration. after chemotherapy followed by autologous stem cell transplantation (asct), the patient achieved complete remission (cr). a 31-year - old man was hospitalized for fever, fatigue, and abdominal discomfort. he reported no recent travel history, there was no history of urticarial or other allergic symptoms, and he had no history of alcoholism. vital signs at the time of admission were as follows : temperature, 38.3 ; pulse, 92 beats per minute ; blood pressure, 125/75 mm hg ; and respiratory rate, 18 breaths per minute. the initial physical examination revealed hepatosplenomegaly, a distended abdomen with ascites and significant pitting edema in both lower limbs. an initial complete blood count (cbc) found a hemoglobin (hb) level of 8.3 g / dl, a white blood cell count (wbc) of 12,300/l and a platelet count of 77,000/l. a differential wbc showed 36% neutrophils (4,430/l), 28% lymphocytes (3,440/l), and 35% eosinophils (4,310/l), but did not include circulating myeloid precursors or other left - shifted neutrophils. the patient had also suffered from eosinophilia eight months previous (hb, 13.4 g / dl ; wbc, 8,590/l [eosinophils, 2,233/l ] ; platelet count, 203,000/l). other laboratory findings were as follows : lactate dehydrogenase, 496 iu / l ; protein, 12.7 g / dl ; albumin, 2.2 g / dl ; ige,>3,000 iu / ml ; igg,>5,000 mg / dl ; iga, 152.7 mg / dl ; igm, 162.8 mg / dl ; bence - jones protein (+) ; 2-microglobulin, 4.70 mg / l ; serum vitamin b12 level, 1,428 pg / ml ; folic acid level, 2.91 ng / ml ; serum free kappa light chain, 566.0 mg / ml ; serum free lambda light chain, 765.0 mg / ml ; anti - nuclear antibody (-) ; anti - double strand dna antibody (-) ; anti - mitochondrial antibody (-) ; hepatitis b virus surface antigen (-) ; and anti - hepatitis c virus antibody (-) (serum tryptase was unavailable for testing). bone marrow aspiration and biopsy showed hypercellular marrow with high levels of eosinophilic (19.1%) and plasma (22.9%) cells with no increases in other matured myeloid precursors (fig. a chromosomal study was normal (46, xy), and fluorescence in situ hybridization (fish) analyses for platelet - derived growth factor receptor (pdgfr) and bcr / abl were negative. our diagnostic evaluation of the eosinophilia included a stool specimen negative for parasitic infection and a negative multiple antigen simultaneous test (mast). a computed tomography (ct) scan of the chest showed effusion of the right pleura (fig. abdominal ct revealed hepatosplenomegaly of both hepatic lobes without a definite focal lesion, fluid in the perihepatic space and lymph node enlargement in the peripancreatic, mesenteric, and aortocaval areas (fig. an echocardiogram showed minimal pericardial effusion, septal hypertrophy and mild global hypokinesia of the left ventricle (fig. endoscopy found diffuse - fashioned erythema of the stomach mucosa in the antrum (fig. we could not perform a biopsy of the heart or stomach because the patient was prone to complications from invasive procedures (e.g., excessive bleeding and pleural effusion). a liver biopsy showed parenchymal infiltration of numerous eosinophils and plasma cells on the hemorrhage focus (fig. plasma cell markers were positive in the peritoneal (cd138, 60.12% ; cd38, 96.2%) and pleural (cd138, 96.18% ; cd38, 99.8%) fluids (fig. the cytology results of the ascites and pleural fluids showed plasma cells and eosinophils (fig. 3c and d). based on these results, eosinophilia involving the bone marrow, liver, and lungs was confirmed, and eosinophilia involving the peritoneal cavity, heart, and stomach were considered possibilities. a monoclonal peak was observed in the gammaglobulin fraction from serum protein electrophoresis (fig. 5a), and immunofixation electrophoresis revealed igg, kappa and lambda paraproteins (fig. three weeks after starting dexamethasone, the patient was treated with a bortezomib (velcade, ben venue laboratories, inc., bedford, oh) plus dexamethasone (vd) regimen every three weeks. four months following the start of the vd regimen, the patient 's laboratory findings improved. his treatment was maintained (with thalidomide plus dexamethasone) for six months after asct. currently, he has finished treatment and has been followed - up with laboratory tests for nine months. a recent cbc revealed an hb level of 14.1 g / dl, a wbc of 4,100/l with a differential cell count of 10% eosinophils (410/l) and a platelet count of 127,000/l. the eosinophil count of the patient did not exceed 1.510/l on consecutive readings after initiation of asct. other laboratory findings included an igg concentration of 1,085 mg / dl, serum free kappa / lambda light chain of 16.60/17.60 mg / ml, a kappa / lambda ratio of 0.94, and negative immunofixation and 2-microglobulin of 1.70 mg / l. his liver and spleen have decreased in size, and they and other organs have recovered their functionalities. a wide variety of solid tumors (predominantly carcinomas of the lung, head and neck, uterus, breast, pancreas, colon and rectum, thyroid gland, adrenal glands, and the biliary tract) may be associated with eosinophilia. a number of lymphoid neoplasms may be accompanied by eosinophilia, including hodgkin 's disease and t - cell lymphomas and, less frequently, b - cell non - hodgkin 's lymphomas, natural killer cell neoplasms, and other b cell malignancies. however, there have been few reports on eosinophilia with mm or plasma cell disorders. in korea, only one case has been diagnosed as chronic eosinophilic leukemia with mm. in our case, primary and clonal eosinophilia were excluded based on the results of fish and chromosomal studies, and myeloproliferative disease was ruled out based on the absence of circulating myeloid precursors and normal chromosomal and bone marrow findings, without a left shift in maturation except for eosinophils and plasma cells. in japan, a case of mm and chronic hepatitis c has been reported. according to the laboratory findings of our case, the patient did not have a chronic liver disease. we diagnosed secondary eosinophilia associated with mm because the patient had an eosinophilia - concomitant hematologic malignancy. there is no acceptable mechanism to explain the occurrence of peripheral blood eosinophilia in patients with plasma cell disorders. first, the release of a protein from necrotic tumor cells may result in eosinophilia. second, the presence of a metastatic tumor in the bone marrow may stimulate eosinophilopoiesis. finally, neoplastic cells may actively secrete substances that either directly or indirectly (via secretion by other cells) result in eosinophilia. according to stefanini., il-3, il-5 and gm - csf promote eosinophilopoiesis in patients with hematologic malignancies. however, we could not study these relationships in our case, because il-3, il-5, and gm - csf levels were not measured in our patient. in our case, biclonal gammopathy there have been few reports on mm with biclonal gammopathy, and its incidence is about 1% of all monoclonal gammopathies. imatinib is the standard treatment in patients with clonal eosinophilia including pdgfr mutations, and cytoreductive therapy or steroids are the treatments of choice with he s. the main therapy for plasma cell disorders with eosinophilia has been melphalan, vincristine, adriamycin and dexamethasone (vad) or cyclophosphamide. the majority of these published cases did not achieve sustained responses, and plasma cells or eosinophils increased in the bone marrow or the peripheral blood of these patients shortly after termination of the treatment., there is no standard treatment for patients with mm and eosinophilia despite the poor response and prognosis. even though there are no reports that suggest treatment via hematopoietic stem cell transplantation in cases similar to ours, we performed asct after induction chemotherapy with a vd regimen. through this treatment, our patient achieved cr of at least six months without other therapies. | eosinophils are derived from hematopoietic stem cells. peripheral blood eosinophilia is defined as an absolute eosinophil count of 0.5109/l. eosinophilia is classified into primary or clonal eosinophilia, secondary eosinophilia, and idiopathic categories including idiopathic hypereosinophilic syndrome. both hematopoietic and solid neoplasms may be associated with peripheral blood eosinophilia, but multiple myeloma is rarely associated with eosinophilia. we now report the case of a 31-year - old man with multiple myeloma associated with marked eosinophilia who developed multiple organ dysfunction with infiltration of eosinophils. he recovered after treatment with chemotherapy followed by autologous stem cell transplantation. |
foreign body aspiration can be a life - threatening condition if the aspirated object is large enough to cause complete airway obstruction necessitating prompt intervention (1). however, the diagnosis may be delayed when the history is atypical or when clinical and radiologic findings are misleading or overlooked by the physician. in spite of that, nearly all pharyngeal and airway foreign bodies are considered as medical emergencies (2, 3). we are presenting an old man who aspirated a large foreign body and misdiagnosed as a case of aspiration pneumonia. a 90-year - old man was brought to the emergency room due to acute onset of severe generalized weakness, sudden onset of dyspnea, pleasure whistling in respiration, acute dysphagia to both liquids and specially solids and inability to speak, from 2 days ago. he had medical history of achalasia, recurrent pneumonia in last months and head trauma 2 months ago with sub - arachnoids hemorrhage and several convulsions. on arrival to hospital, his vital sign were as oral temperature : 37.8c, heart rate : 100/minute, respiratory rate : 16/minute, blood pressure : 100/80 mmhg with 95% oxygen saturation on room air. in physical exam except for severe cachexia and superficial ulcers on two forearms, there was no other finding. none of examiners noticed to his respiration with pleasure whistling and the history of losing his upper dental prosthesis from two days ago. posterior - anterior chest x - ray revealed bilateral consolidations with the most prominence in the right middle lobe (figure 1). chest computed tomography (ct) scan revealed the same finding as well as bilateral pleural effusion (figure 2). he was admitted to the infectious disease ward with diagnosis of aspiration pneumonia. in next day, his dyspnea guts worst so made the corresponding physician to take a lateral neck radiography (figure 3). a large foreign body was lodged above epiglottis between oropharynx and hypopharynx. on physical examination, his lost upper complete upper denture with 5 x 5 centimeter size his three days dyspnea, whistling, inability to talk and dysphagia was dramatically improved. he was treated for aspiration pneumonia due to last convulsions and was discharged in a stable condition after one week. in third visit after two months, he had no alimentary and respiratory sign and symptoms with good condition. posterior - anterior chest x - ray of patient axial chest computed tomography scan of patient lateral neck x - ray of patient, white arrow shows aspirated dentures adults account for only about 20% of the reported cases of foreign body aspiration and most of them are seen after the sixth or seventh decade of life, when airway protective mechanisms lose their proper function (4, 5). the clinical features of foreign - body aspiration in geriatric patients are usually more obscure than in non - geriatric adults, which may lead to long delay in diagnosis ranged from 1 month to 3 years (6). in the adult population, such aspiration is most commonly secondary to unconscious accidental ingestion during general anesthesia, sedation, intoxication, seizures or neurologic disorders affecting the oropharynx. foreign bodies have a tendency to lodge in the right main stem bronchus as it is more vertical and larger in diameter than the left main stem bronchus (3). clinical management of an aspirated foreign body includes early recognition, acute emergency interventions, supportive care, and preventive anticipatory guidance. the diagnosis of foreign body aspiration can be difficult, especially if the patient does not recall an aspiration episode. foreign body aspiration has variable clinical manifestations, ranging from trivial symptoms to irreversible lung damage and life - threatening infection, atelectasis, and massive hemoptysis. patients may present with a history of fever, breathlessness, or wheezing or with features of a non - resolving pneumonia. on physical examination, these patients may have decreased breath sounds on the side with the foreign body or localized wheezing, or they may be asymptomatic. the clinical triad consists of wheezing, coughing, and diminished or absent breath sounds that is considered to be diagnostic of foreign body aspiration can be present based on the size and site of foreign body (6, 7). although plain films may be interpreted as normal, radiopaque foreign bodies may be seen. on chest x - ray, unilateral hyperinflation, lobar or segmental atelectasis, and mediastinal shift, or pneumomediastinum many foreign bodies are incidentally seen on radiographic imaging ordered for symptoms mistakenly attributed to other medical conditions including asthma and unresolving recurrent pneumonia (9). if a diagnosis of foreign body aspiration is delayed, a retained foreign body may result in unresolving pneumonia, lung abscess, and bronchiectasis. also, formation of granulation tissue around the foreign body may occur and may resemble bronchogenic carcinoma (10). ct scans can depict a foreign body within the lumen of the tracheobronchial tree and the 3-dimensional position of the foreign body within the thorax (7). because foreign body aspiration can mimic other respiratory conditions, a high index of suspicion is necessary in all patients with pneumonia, atelectasis, or wheezing with an atypical course, especially in patients who are unresponsive to medical therapy (11). this case is one of the unusual cases of such a large foreign body aspiration that the diagnosis was delayed for about three days. he was too debilitated to say about his denture in pharynx and chest x - ray could not reveal the plate because it was made from a plastic material and had no radiopaque metal. diagnosis of foreign body aspiration may be retarded because of nonspecific signs specially if there is no recollection of the episode. the physician may be suspicious of this condition, even if the patient history and imaging obscure the clinical picture and pay attention to present history well and do a complete physical examination. all authors passed four criteria for authorship contribution based on recommendations of the international committee of medical journal editors. | foreign body aspiration is unusual in adults, except those who are debilitated or have neuropsychiatric disorders. it can be a life - threatening situation and it often requires a high index of suspicion, because the diagnosis can be obscure. prompt diagnosis and intervention through foreign body retrieval are critical to prevent significant morbidity and mortality. we present a case of denture aspiration by a debilitated 90 years old man. he had aspirated his complete upper denture to pharynx causing incomplete obstruction with pleasure whistling respiratory sound, dyspnea, dysphagia and dysphonia. he underwent successful retrieval of the dental plate manually by fingers with complete resolution of symptoms. |
radiofrequency catheter ablation techniques have developed rapidly since 19901, 2 and have been deemed efficient therapy for the treatment of sustained supraventricular tachycardia and ventricular arrhythmias. nevertheless, a reliable noninvasive approach for the localization of abnormal conduction and accessory pathways (aps) such as those in wolff - parkinson - white (wpw) syndrome has not been completely studied yet. such a technique could be used to guide the radiofrequency ablation electrode to the proper cardiac chamber and target tissue and could help recognize the effectiveness of the ablation procedure. currently, intracardiac electrophysiology and fluoroscopy are utilized to localize the site of abnormal ventricular stimulation, as is the case in the aps of wpw syndrome and ventricular arrhythmias. this time - consuming technique is, however, invasive and requires radiation exposure and does not provide spatial resolution of cardiac structures.3, 4 recently, it has been determined that tissue doppler imaging (tdi) can display the distribution, direction, and amount of myocardial velocity and acceleration within the myocardium during the cardiac cycle.57 the site of the initial contraction in wpw syndrome (or a ventricular arrhythmia) is recognized as the position of the initial velocity.8 in this study, we sought to examine the diagnostic accuracy of the noninvasive prediction of the ap localization in patients with wpw syndrome using strain imaging as a new noninvasive mapping procedure. twenty - five patients (mean age = 32 17 years, 58.3% men) who had recurrent supraventricular tachycardia with the evidence of pre - excitation on electrocardiography (ecg) were prospectively studied. all the patients underwent electrophysiological endocardial mapping for the radiofrequency ablation of the suspected accessory bypass tract. all the subjects were in sinus rhythm and had a normal left ventricular (lv) function under resting conditions. all the examinations and measurements were made by a single investigator, who was unaware of the electrocardiographic and electrophysiology study (eps) data. all the patients underwent color - coded tissue doppler study using a vivid 7 digital ultrasound scanner (ge, milwaukee, wisconsin, usa). color - coded tissue doppler cine loops from three consecutive beats with optimal image quality were recorded. the images were obtained from the apical four - chamber, two - chamber, and apical long - axis views using a 3.5-mhz transducer at a depth of 16 4 cm. frame rate was more than 120/sec., and sweep speed was set at 100 mm / sec. the timing of the aortic valve opening and closure was determined from the pulsed - wave doppler of the lv outflow tract and was superimposed on the tissue velocity and strain waveforms. the regions of interest were defined as a 6 6-mm area with a circular shape for tissue velocity measurements and as a 6 10-mm area with an oval shape for strain measurement, and they were placed in the basal and mid segments of the lv and right ventricular (rv) walls to measure the time interval from the onset of delta wave () on the surface ecg to the time to onset and time to peak systolic velocity of tdi and time to peak systolic strain of strain imaging. all the intervals were corrected for heart rate (corrected interval = measured interval / [rr interval ]). the patients were examined in the left lateral decubitus position with a conventional ultrasound system (vivid 7) equipped with tdi, strain, and strain rate imaging. apical four - chamber, two - chamber, long - axis, and rv inflow views were obtained : the apical four - chamber view to examine the rv lateral wall and the lv septal and lateral walls ; the apical two - chamber view to examine the lv anterior and inferior walls ; the apical long - axis view to examine the lv anteroseptal and posterior walls ; and the rv inflow view to examine the rv posteroseptal and anterior walls. the bipolar limb lead electrocardiogram (i, ii, or iii) that demonstrated the clearest onset of delta wave was selected and displayed on the ultrasound monitor. this electromechanical interval was defined as the time difference between the onset of electrocardiographic delta wave and the onset of regional myocardial contraction. localization of the aps was defined as the myocardial region with the shortest electromechanical time interval. the following data were obtained (figure 1) : 1) time difference between the onset of delta wave () and the onset of regional myocardial contraction (-so) ; 2) time difference between the onset of delta wave and peak systolic motion (-sm) ; and 3) time difference between the onset of delta wave and onset of strain (), peak strain (p), and peak strain rate (srp). prediction of the localization of the aps from a standard twelve - lead ecg was made separately by an expert observer using three previously described algorithms. the electrophysiologist who performed the endocardial mapping for the ap localization was blinded to echocardiographic data. the site of successful radiofrequency ablation in one out of the five regions of left lateral (ll), left posteroseptal (lps), right posteroseptal (rps), right lateral (rl), and right anteroseptal (ras) was used as the reference location for comparison with echocardiographic and electrocardiographic localization techniques. statistical analysis was conducted with spss (statistical package for the social sciences version 15.0, spss inc. the qualitative variables are expressed using percentages, while the quantitative data are defined using mean, standard deviation, and/or confidence intervals. the analysis of variance (anova), the paired - sample t - test, and bivariate correlation tests were employed for inferential statistics. a normal distribution of the quantitative data was checked using the kolmogorov - smirnov test. localization of the aps via the ecg and eps and the frequency of the aps via eps are depicted in tables 1. table 2 shows the frequency of the location of the aps using different tdi variables versus the ecg. the ecg had the highest positive predictive value (ppv) for the localization of the aps in the right ras and lps (100%), rps (75%), and ll (83.3%). time difference between the onset of delta wave and the onset of regional myocardial contraction (-so) had the highest ppv for the localization of the aps in the lps (100%) and ll (91.6%). the ppv of (-) for the localization of the aps in the lps, ras, rps, and rl was 100%, as opposed to 66.6% in the ll. there was a significant difference between the time to onset of delta wave to the onset of peak systolic motion (mean sd) in the ap location (a) and normal segments (b) versus that in the normal volunteers (c) [a : (57.08 23.88 msec) vs. b : (75.20 14.75) vs. c : (72.9 0 11.16) ; p value (a vs. b) = 0.004 and p value (a vs. c) = 0.18 ] and [a : (49.17 35.79) vs. b : (67.60 14.51) vs. c : (67.40 6.06 msec) ; p value (a vs. b) = 0.000 and p value (a vs. c) = 0.12, respectively ]. there was no significant difference in the strain and strain rate (mean sd) of all the myocardial segments between the patients and the normal subjects [a : (17.29 3.34%) vs. b : (18.52 2.56%) vs. c : (18.63 1.91) ; p value (a vs. b) = 0.57 and p value (a vs. c) = 0.22 ] and [a : (1.33 0.33) vs. b : (1.36 0.29) vs. c : (1.45 0.48 s) ; p value (a vs. b) = 0.17 and p value (a vs. c) = 0.52, respectively ]. there was a ppv of 100% for (-strain) for the localization of the aps in the lps, ras, rps, and rl. the ppv of (-strain) for the localization of the aps in the ll was 66.6%. the highest ppv of (-so) for the localization of the aps was seen in the lps (100%) and ll (91.6%) and the lowest ppv of (-so) for the localization of the aps was observed in the rl and ras (50%) and rps (0%). in general, a ppv of 56% for tdi (-so) and a ppv of 84% for strain imaging (-strain) were found for an accurate localization of the aps in the patients with wpw. our study showed that strain imaging is superior to the ecg in the localization of the aps (84% vs. 76%). (-sm) had the lowest ppv for the detection of the location of the aps (44%). m - mode echocardiography was the first modality to be employed for the localization of the aps in wpw syndrome and has been used since the mid-1970s.9 however, because of its inherent limitation (unidimensional gray scale technique), the location of the ectopic excitation and pre - excitation induced by the aps could not be exactly defined.9, 10 the two - dimensional transthoracic and transesophageal echocardiographic phase analysis techniques have been drawn upon to localize the aps in wpw syndrome by evaluating the ventricular motion at early systole. nonetheless, it is still difficult to localize an intramural focus of the earliest ventricular contraction. the phase angle in phase images presents the thickening of the ventricular wall and motion of the ventricular endocardium with lower temporal resolution (16 frames / s).11, 12 some previous studies have revealed that even without the classic delta wave or wide qrs complex, tissue doppler velocity imaging can localize the majority of the aps in wpw syndrome.8 the feasibility of tdi for evaluating the early contraction sites in wpw syndrome was examined for the first time by nakayama. in 1998.13 they analyzed the time - sequential changes in ventricular wall motion in wpw syndrome with tdi, which demonstrated that the early contraction sites were well coincided with the sites of the aps, as determined by the electrophysiological examination (p value < 0.01). the authors suggested that tdi was helpful in localizing the aps and in evaluating the results after radiofrequency ablation. in a study by yin.14 to validate the use of tissue doppler acceleration imaging for the evaluation of the onset of ventricular contraction, the researchers concluded that this imaging modality was a useful noninvasive method that was successful in visualizing the intramural site of origin of ventricular mechanical contraction (agreement of 90% [18 of 20 ] for tissue doppler acceleration imaging with the position of the aps localized from intracardiac electrophysiology testing). in another study by tuchnitz.15 for a noninvasive prediction of the ap localization in patients with wpw syndrome using myocardial doppler imaging, the investigators claimed that this imaging modality was superior to electrocardiographic algorithms for the localization of the ap (84% vs. 4860%, respectively). we found a lower ppv (56%) for the time difference between the onset of delta wave and the onset of regional myocardial contraction (-so) in our patients. in the article by tada h.,16 tissue tracking imaging was performed in 33 patients with idiopathic ventricular arrhythmias before radiofrequency catheter ablation. in that study, tissue tracking imaging provided detailed and accurate information on the arrhythmia origin, especially in the outflow tract, and facilitated the catheter ablation of idiopathic ventricular arrhythmias. be that as it may, tissue tracking imaging can not be considered as the modality of choice because of some limitations such as translational, rotational, and deformational movements. in addition, if the initial activation occurs in a plane perpendicular to the interrogating plane, tissue tracking may show tissue displacement from the adjacent areas due to tethering movements.17 myocardial deformation, however, can be assessed from the b mode images by speckle tracking. it has the advantage of angle independence and may be superior to tissue doppler - based techniques for strain estimation.17 the first report on the use of two - dimensional strain imaging technique to assess the origin of an accessory bypass tract in ventricular pre - excitation in a 51-year - old man with drug refractory wpw syndrome was published by de boeck.18 using strain curves, the authors localized the site of the first ventricular contraction in the inferior wall, and invasive electrophysiological mapping confirmed a left - sided bypass with an inferoseptal ventricular insertion. in some of our patients, it was difficult to define the precise beginning of delta wave. doppler - based strain data are influenced by doppler angle ; nonetheless, particular attention was paid to make the segments as parallel to the ultrasonic beam as possible in our study. it is also deserving of note that only longitudinal strain and sr were measured in the present study. the accuracy of strain and sr would have been enhanced if circumferential, radial, and longitudinal strains had been measured simultaneously. a precise detection of the diagnostic yield of strain imaging for the noninvasive localization of the aps requires another study with a large sample size of all the known sites of the aps. our results suggest that strain imaging can be used to accurately predict single ap locations in patients with wpw syndrome. strain imaging is a readily available and noninvasive procedure. despite advanced mapping technology in the ablation procedures of wpw syndrome, there are still cases in which the exact localization of the right versus left lateral and right versus left posteroseptal pathways may be difficult. considering the different catheter approach in both of these localizations, tdi and strain data may provide important information for the electrophysiologist. tdi - derived parameters such as onset of systolic velocity (-so) and strain (-strain) have better diagnostic yields than the ecg for the noninvasive localization of the aps in patients with wpw syndrome. consequently, we would suggest that in some cases, the use of these parameters for a noninvasive prediction of the ap location may result in shorter invasive mapping durations and especially shorter x - ray exposure times. | background : noninvasive techniques for the localization of the accessory pathways (aps) might help guide mapping procedures and ablation techniques. we sought to examine the diagnostic accuracy of strain imaging for the localization of the aps in wolff - parkinson - white syndrome.methods:we prospectively studied 25 patients (mean age = 32 17 years, 58.3% men) with evidence of pre - excitation on electrocardiography (ecg). electromechanical interval was defined as the time difference between the onset of delta wave and the onset of regional myocardial contraction. time differences between the onset of delta wave () and the onset of regional myocardial contraction (-so), peak systolic motion (-sm), regional strain (-), peak strain (-p), and peak strain rate (-srp) were measured.results:there was a significant difference between time to onset of delta wave to onset of peak systolic motion (mean sd) in the ap location (a) and normal segments (b) versus that in the normal volunteers (c) [a : (57.08 23.88 msec) vs. b : (75.20 14.75) vs. c : (72.9 0 11.16) ; p value (a vs. b) = 0.004 and p value (a vs. c) = 0.18 ] and [a : (49.17 35.79) vs. b : (67.60 14.51) vs. c : (67.40 6.06 msec) ; p value (a vs. b) < 0.001 and p value (a vs. c) = 0.12, respectively].conclusion : our study showed that strain imaging parameters [(-so) and (-strain) ] are superior to the ecg in the localization of the aps (84% vs. 76%). |
type 2 diabetes mellitus (dm) is a major risk factor for alzheimer disease and vascular dementia. associated brain atrophy is widespread and generalized, advancing brain age (1) and accelerating cognitive decline in older dm populations (24). although the underlying pathophysiology of gray matter atrophy is complicated, hyperglycemia - induced small - vessel disease is a potential pathway for altered neurovascular coupling, impaired vasoreactivity and regional hypoperfusion (57), and neurotoxicity (8). typically, vasodilatatory responses to hypercapnia or cognitive task performance are diminished in multiple brain regions (1,6). central insulin receptors are abundant and yet are mostly dependent upon insulin transport through the blood - brain barrier. therefore, inadequate insulin delivery may affect perfusion and cortical activity in associative regions with high - energy demands, such as cognitive networks (9). clinical studies suggest that augmenting cerebral insulin may enhance cognitive function and memory in healthy young and older adults and in cognitively impaired non - dm people with both acute and chronic intranasal administration (1012). intranasal administration of insulin delivers the compound to the brain, thus bypassing the blood - brain barrier and avoiding systemic effects (13). intranasal insulin increases rapidly in cerebrospinal fluid and binds to receptors along trigeminal and autonomic pathways in the frontal lobe, limbic system, hypothalamus, and other areas (14,15). we aimed to determine the acute effects of intranasal insulin on regional perfusion, vasoreactivity, and cognition in older adults with and without type 2 dm in a proof - of - concept, double - blind, placebo - controlled, crossover study. we hypothesized that intranasal insulin acutely improves regional perfusion and that improvement of cognition may be dependent upon regional vasoreactivity in older dm adults compared with non - dm adults and compared with placebo treatment. this was a single - center, randomized, double - blind, placebo - controlled safety and efficacy pilot intervention with crossover assignment [food and drug administration investigational new drug application (fda - ind) 107690 ] to evaluate acute effects of intranasal insulin on regional vasoreactivity and cognition in older dm and non - dm adults. primary end points were insulin - related changes in regional perfusion, vasoreactivity to co2 challenges, and cognitive exam scores in the dm group compared with placebo and with the control group. as no preliminary data on the effects of intranasal insulin on these end points in dm subjects were available at the time of study design, we based our vasoreactivity estimates on perfusion response to hypoglycemia (16) and our cognitive outcome estimates on intranasal insulin studies in non - dm subjects (10,11,17). we estimated that a total of 60 subjects would be needed to detect a 10% improvement in cognitive performance with 81% power, = 0.05. studies were conducted at the syncope and falls in the elderly laboratory, the center for advanced mr imaging, and the clinical research center (crc) at beth israel deaconess medical center (bidmc). of 262 participants screened over the phone, 94 were eligible and 64 completed a screening visit and provided written informed consent. of these, 29 (15 dm and 14 control subjects) completed the protocol (table 1), 28 were excluded, and 7 withdrew consent. demographic characteristics of the dm and control groups dm participants were included if they were diagnosed with type 2 dm for > 5 years and treated with oral anti - dm agents. control subjects were required to be normotensive, have fasting blood glucose 10. vasodilatation was calculated as a change in perfusion between baseline and hypercapnia divided by change of co2 ; vasoconstriction was calculated as a change in perfusion between baseline and hypocapnia, and vasoreactivity rate was calculated as a slope of regression between baseline, hypocapnia, and hypercapnia for each subject within brain regions of interest (6,21). baseline assessment included measures of verbal learning (hopkins verbal learning test - revised), executive function (trail - making tests a and b ; digit span), visual memory (rey - osterrieth complex figure test), and mmse. testing on insulin versus placebo (day 2 and day 3) had to be completed within a short time - frame of 2 h after insulin administration because of insulin pharmacokinetics (10,11,22). therefore, we selected a brief battery of parallel versions of the brief visuospatial memory test - revised (bvmt) and the verbal fluency measures (fas, category, and switching conditions) of the delis - kaplan executive function system assessment, which have previously shown sensitivity to cognitive changes in similar populations (23,24). all variables were summarized using descriptive statistics and compared between groups using one - way anova, nonparametric tests, and the least square (ls) models. insulin and placebo conditions were compared within each group and within the entire cohort using a paired t test. dependent bvmt variables reported as age - adjusted t scores were performances on each of the three immediate recall trials (t1, t2, and t3), the total learning score across the three immediate recall trials (total recall), delayed recall, and the change in performance from immediate recall to delayed recall trials (learning). performances on the fas, category, and switching verbal fluency trials were also reported as age- and education - adjusted t scores. a composite verbal fluency score was created by averaging the t scores of the three trials (jmp pro, 10.0.0 ; sas institute, cary nc). ls models were also used to evaluate the relationships among perfusion, vasoreactivity, and cognition. ls models were calculated separately within group and condition (e.g., dm group on insulin) for each variable to minimize multiple - comparison effects. bvmt and verbal fluency t scores were included as dependent variables, and model effects included age, sex, and regional perfusion or vasoreactivity. conservatively, we selected models with r > 0.25, and p 10. vasodilatation was calculated as a change in perfusion between baseline and hypercapnia divided by change of co2 ; vasoconstriction was calculated as a change in perfusion between baseline and hypocapnia, and vasoreactivity rate was calculated as a slope of regression between baseline, hypocapnia, and hypercapnia for each subject within brain regions of interest (6,21). baseline assessment included measures of verbal learning (hopkins verbal learning test - revised), executive function (trail - making tests a and b ; digit span), visual memory (rey - osterrieth complex figure test), and mmse. testing on insulin versus placebo (day 2 and day 3) had to be completed within a short time - frame of 2 h after insulin administration because of insulin pharmacokinetics (10,11,22). therefore, we selected a brief battery of parallel versions of the brief visuospatial memory test - revised (bvmt) and the verbal fluency measures (fas, category, and switching conditions) of the delis - kaplan executive function system assessment, which have previously shown sensitivity to cognitive changes in similar populations (23,24). all variables were summarized using descriptive statistics and compared between groups using one - way anova, nonparametric tests, and the least square (ls) models. insulin and placebo conditions were compared within each group and within the entire cohort using a paired t test. dependent bvmt variables reported as age - adjusted t scores were performances on each of the three immediate recall trials (t1, t2, and t3), the total learning score across the three immediate recall trials (total recall), delayed recall, and the change in performance from immediate recall to delayed recall trials (learning). performances on the fas, category, and switching verbal fluency trials were also reported as age- and education - adjusted t scores. a composite verbal fluency score was created by averaging the t scores of the three trials (jmp pro, 10.0.0 ; sas institute, cary nc). ls models were also used to evaluate the relationships among perfusion, vasoreactivity, and cognition. ls models were calculated separately within group and condition (e.g., dm group on insulin) for each variable to minimize multiple - comparison effects. bvmt and verbal fluency t scores were included as dependent variables, and model effects included age, sex, and regional perfusion or vasoreactivity. conservatively, we selected models with r > 0.25, and p < 0.05. here, we present radjusted (adjusted for model covariates). nominal observed p values are reported without adjustment for multiple testing in this small proof - of - concept study. baseline cognitive testing conducted on day 1 showed that the dm group performed worse than the control group on verbal learning measures (hopkins verbal learning test - revised learning was borderline, p = 0.052 ; delayed recall, radjusted = 0.31, p = 0.008 ; retention, radjusted = 0.21, p = 0.046, and radjusted = 0.1 recognition, p = 0.038), processing speed (trail making test a, radjusted = 0.2, p = 0.01) and executive function (trail making test b, radjusted = 24, p = 0.005) (ls models adjusted for education years) and had fewer years of education (p = 0.04) and lower global gray matter volume (p = 0.02). table 2 summarizes the time course of glucose (intravenous and finger stick) and cardiovascular vital signs between insulin versus placebo conditions, which were similar within each group. glucose levels and vital signs were stable and similar across insulin and placebo conditions in both groups. blood sample collection times and cognitive testing administration times did not differ between insulin and placebo. blood flow velocities (bfvs) in the aca and mca, measured by tcd, declined during administration in both insulin and placebo conditions for control and dm subjects by 9% (p = 0.050.001) but returned to baseline within 5 min after administration. bvmt performances after insulin administration tended to be higher than on - placebo performances, and control subjects performed better than dm subjects. overall, control subjects on insulin performed better than the dm group on insulin and on placebo on measures of immediate recall trials 2 and 3 (t2 and t3) and total learning (total recall) (fig. control subjects on insulin were the highest - scoring subgroup, while dm subjects on placebo scored the lowest. this relationship was observed for immediate recall t2 (ls model adjusted for age radjusted = 0.14, p = 0.029 ; control subjects on insulin compared with dm group on placebo p < 0.01), t3 (radjusted = 0.14, p = 0.026), and total recall (radjusted = 0.18, p = 0.02). brief visual memory scores for immediate recall trials 13 (t1t3) and total recall for the dm and control groups. overall, control subjects on insulin performed better than the dm group on insulin and on placebo ; p < 0.03 and p < 0.01 control subjects on insulin vs. dm group on placebo (ls models adjusted for age). for the whole cohort, performance on insulin improved compared with placebo for t2, p = 0.04, and was borderline for total recall, p = 0.052 (paired t test). these effects remained similar after adjustment for potential confounding effects of education on immediate recall t2 (radjusted = 0.12, p = 0.017) and t3 (radjusted = 0.1, p = 0.029) (ls model age, education adjusted). for the whole cohort, the performance on insulin improved compared with placebo on t2 (p = 0.04) and was borderline for total recall (paired t test, p = 0.052). in both groups, subjects were also better able to correctly identify target figures on insulin than on placebo (paired t test, raw scores, p = 0.02) and registered fewer false alarms (paired t test, raw scores, p = 0.05), though normative data for these measures was highly skewed in the test population and no t scores were available. verbal fluency performances after insulin administration tended to be higher than on - placebo performances. control subjects on insulin performed better than dm subjects on insulin on fas (ls model adjusted for age radjusted = 0.26, p = 0.0045 ; ls model adjusted for age and education radjusted = 0.25, p = 0.018), switching (radjusted = 0.2, p = 0.006 ; radjusted = 0.17, p = 0.012), and composite verbal fluency (radjusted = 0.12, p = 0.02 ; radjusted = 0.11, p = 0.049). on placebo, control subjects were better only on fas not other verbal fluency measures (ls model adjusted for age and education, radjusted = 0.27, p = 0.019). regionally, changes in perfusion and vasoreactivity after insulin administration were observed in the mca territory, which contains the insular cortex and integrative areas for learning, memory, and language within the temporal and parietal lobes. baseline perfusion was lower in the dm group in the insular cortex (p = 0.039) as compared with control subjects (table 2). in the dm group, perfusion in the right insular cortex increased after insulin administration (p = 0.001) compared with placebo. voxel - based analyses have shown that increase of perfusion on insulin was greater in the dm group compared with the control group (p = 0.0003) (fig. voxel - based analysis demonstrates that within the dm group, intranasal administration of insulin induced more increased perfusion compared with placebo in the right insular cortex (independent student t test applied to the subtraction result between conditions, voxel - level uncorrected p < 0.001) (a). in the dm group, the bvmt t score after insulin administration was related to vasodilatation in the mca territory (r = 0.58, radjusted = 0.44, p = 0.0098) (b). this relationship was not observed after placebo administration (r = 0.14, radjusted = 0.14, p = 0.34, ls regression models adjusted for age and sex) (c). in control subjects, after insulin administration the verbal fluency category t score was also related to vasodilatation in the right mca territory (r = 0.75, radjusted = 0.64, p = 0.0087, p = 0.024, ls regression models adjusted for age and sex) (d). (a high - quality color representation of this figure is available in the online issue.) in the whole cohort, cognitive performance on the bvmt and verbal fluency measures upon insulin administration was related to perfusion and vasodilatation within the mca territory and specifically to the insular cortex that regulates attention - related task performance. across all subjects, perfusion increases after insulin administration within the mca territory were associated with an improvement of bvmt t3, and for the bvmt delayed recall in the right mca territory (radjusted = 0.28, p = 0.04) and also with vasodilatation in the insular cortex (radjusted = 0.22, p = 0.04) (ls model adjusted for age, sex, and group). after insulin administration in the dm group, better visuospatial memory correlated with vasodilatation in the mca territory for immediate recall t2 (radjusted = 0.43, p = 0.01), bvmt t3 (radjusted = 0.39, p = 0.035), and total recall (radjusted = 0.44, p = 0.0098) (ls models adjusted for age, sex, and vasodilatation in leptomeningeal mca territory) (fig. these relationships were not observed after placebo administration, as shown in fig. 2c for total recall (radjusted = 0.14, p = 0.34) (ls models adjusted for age, sex, and vasodilatation in leptomeningeal mca territory). a similar trend was observed between bvmt immediate recall (t2 and t3) and total recall vasodilatation in the whole aca territory (p = 0.050.08). after insulin administration within the control group, better performance on bvmt immediate recall t3 was also related to mca vasodilatation (radjusted = 0.4, p = 0.035). this relationship between visuospatial memory and vasodilatation was not observed after placebo administration in either group. in control subjects on insulin, fas score (radjusted = 0.39, p = 0.04) and the composite verbal fluency measure (radjusted = 0.18, p = 0.045) were associated with greater vasodilatation in the right insular cortex (model adjusted for age). in control subjects on insulin, category performance was associated with greater vasodilatation in the right mca (p = 0.027) and decreased vasodilation in the left mca (p = 0.024) (r = 0.75, radjusted = 0.64, p = 0.0087, ls model adjusted for age and sex) (fig. 2d) and also greater left - right difference in vasodilatation in the insular cortex (r = 0.75, radjusted = 0.68, p = 0.0023). in the dm group on insulin, fas scores were also associated with more vasodilatation in the left (p = 0.02) and lesser vasodilatation in the right (radjusted = 0.26, p = 0.04, ls model adjusted for age and sex) insular cortex. baseline cognitive testing conducted on day 1 showed that the dm group performed worse than the control group on verbal learning measures (hopkins verbal learning test - revised learning was borderline, p = 0.052 ; delayed recall, radjusted = 0.31, p = 0.008 ; retention, radjusted = 0.21, p = 0.046, and radjusted = 0.1 recognition, p = 0.038), processing speed (trail making test a, radjusted = 0.2, p = 0.01) and executive function (trail making test b, radjusted = 24, p = 0.005) (ls models adjusted for education years) and had fewer years of education (p = 0.04) and lower global gray matter volume (p = 0.02). table 2 summarizes the time course of glucose (intravenous and finger stick) and cardiovascular vital signs between insulin versus placebo conditions, which were similar within each group. glucose levels and vital signs were stable and similar across insulin and placebo conditions in both groups. blood sample collection times and cognitive testing administration times did not differ between insulin and placebo. blood flow velocities (bfvs) in the aca and mca, measured by tcd, declined during administration in both insulin and placebo conditions for control and dm subjects by 9% (p = 0.050.001) but returned to baseline within 5 min after administration. bvmt performances after insulin administration tended to be higher than on - placebo performances, and control subjects performed better than dm subjects. overall, control subjects on insulin performed better than the dm group on insulin and on placebo on measures of immediate recall trials 2 and 3 (t2 and t3) and total learning (total recall) (fig. control subjects on insulin were the highest - scoring subgroup, while dm subjects on placebo scored the lowest. this relationship was observed for immediate recall t2 (ls model adjusted for age radjusted = 0.14, p = 0.029 ; control subjects on insulin compared with dm group on placebo p < 0.01), t3 (radjusted = 0.14, p = 0.026), and total recall (radjusted = 0.18, p = 0.02). brief visual memory scores for immediate recall trials 13 (t1t3) and total recall for the dm and control groups. overall, control subjects on insulin performed better than the dm group on insulin and on placebo ; p < 0.03 and p < 0.01 control subjects on insulin vs. dm group on placebo (ls models adjusted for age). for the whole cohort, performance on insulin p = 0.04, and was borderline for total recall, p = 0.052 (paired t test). these effects remained similar after adjustment for potential confounding effects of education on immediate recall t2 (radjusted = 0.12, p = 0.017) and t3 (radjusted = 0.1, p = 0.029) (ls model age, education adjusted). the effect of education was not significant in these models. for the whole cohort, the performance on insulin improved compared with placebo on t2 (p = 0.04) and was borderline for total recall (paired t test, p = 0.052). in both groups, subjects were also better able to correctly identify target figures on insulin than on placebo (paired t test, raw scores, p = 0.02) and registered fewer false alarms (paired t test, raw scores, p = 0.05), though normative data for these measures was highly skewed in the test population and no t scores were available. verbal fluency performances after insulin administration tended to be higher than on - placebo performances. control subjects on insulin performed better than dm subjects on insulin on fas (ls model adjusted for age radjusted = 0.26, p = 0.0045 ; ls model adjusted for age and education radjusted = 0.25, p = 0.018), switching (radjusted = 0.2, p = 0.006 ; radjusted = 0.17, p = 0.012), and composite verbal fluency (radjusted = 0.12, p = 0.02 ; radjusted = 0.11, p = 0.049). on placebo, control subjects were better only on fas not other verbal fluency measures (ls model adjusted for age and education, radjusted = 0.27, p = 0.019). regionally, changes in perfusion and vasoreactivity after insulin administration were observed in the mca territory, which contains the insular cortex and integrative areas for learning, memory, and language within the temporal and parietal lobes. baseline perfusion was lower in the dm group in the insular cortex (p = 0.039) as compared with control subjects (table 2). in the dm group, perfusion in the right insular cortex increased after insulin administration (p = 0.001) compared with placebo. voxel - based analyses have shown that increase of perfusion on insulin was greater in the dm group compared with the control group (p = 0.0003) (fig. voxel - based analysis demonstrates that within the dm group, intranasal administration of insulin induced more increased perfusion compared with placebo in the right insular cortex (independent student t test applied to the subtraction result between conditions, voxel - level uncorrected p < 0.001) (a). in the dm group, the bvmt t score after insulin administration was related to vasodilatation in the mca territory (r = 0.58, radjusted = 0.44, p = 0.0098) (b). this relationship was not observed after placebo administration (r = 0.14, radjusted = 0.14, p = 0.34, ls regression models adjusted for age and sex) (c). in control subjects, after insulin administration the verbal fluency category t score was also related to vasodilatation in the right mca territory (r = 0.75, radjusted = 0.64, p = 0.0087, p = 0.024, ls regression models adjusted for age and sex) (d). (a high - quality color representation of this figure is available in the online issue.) in the whole cohort, cognitive performance on the bvmt and verbal fluency measures upon insulin administration was related to perfusion and vasodilatation within the mca territory and specifically to the insular cortex that regulates attention - related task performance. across all subjects, perfusion increases after insulin administration within the mca territory were associated with an improvement of bvmt t3, and for the bvmt delayed recall in the right mca territory (radjusted = 0.28, p = 0.04) and also with vasodilatation in the insular cortex (radjusted = 0.22, p = 0.04) (ls model adjusted for age, sex, and group). after insulin administration in the dm group, better visuospatial memory correlated with vasodilatation in the mca territory for immediate recall t2 (radjusted = 0.43, p = 0.01), bvmt t3 (radjusted = 0.39, p = 0.035), and total recall (radjusted = 0.44, p = 0.0098) (ls models adjusted for age, sex, and vasodilatation in leptomeningeal mca territory) (fig. 2b). 2c for total recall (radjusted = 0.14, p = 0.34) (ls models adjusted for age, sex, and vasodilatation in leptomeningeal mca territory). a similar trend was observed between bvmt immediate recall (t2 and t3) and total recall vasodilatation in the whole aca territory (p = 0.050.08). after insulin administration within the control group, better performance on bvmt immediate recall t3 was also related to mca vasodilatation (radjusted = 0.4, p = 0.035). this relationship between visuospatial memory and vasodilatation was not observed after placebo administration in either group. in control subjects on insulin, fas score (radjusted = 0.39, p = 0.04) and the composite verbal fluency measure (radjusted = 0.18, p = 0.045) were associated with greater vasodilatation in the right insular cortex (model adjusted for age). in control subjects on insulin, category performance was associated with greater vasodilatation in the right mca (p = 0.027) and decreased vasodilation in the left mca (p = 0.024) (r = 0.75, radjusted = 0.64, p = 0.0087, ls model adjusted for age and sex) (fig. 2d) and also greater left - right difference in vasodilatation in the insular cortex (r = 0.75, radjusted = 0.68, p = 0.0023). in the dm group on insulin, fas scores were also associated with more vasodilatation in the left (p = 0.02) and lesser vasodilatation in the right (radjusted = 0.26, p = 0.04, ls model adjusted for age and sex) insular cortex. this proof - of - concept study evaluated the acute effects of a single dose of intranasal insulin compared with placebo on vasoreactivity and cognition in older dm and control adults using a randomized crossover design. the intranasal administration of insulin was safe, with no serious adverse events or hypoglycemic episodes, and the protocol was feasible for participants. the dm group presented with mild cognitive deficits in learning, retention, and executive function. insulin administration improved visuospatial memory and verbal fluency for the entire cohort, but within the control and dm group differences between insulin and placebo were not significant, likely due to a relatively small sample size. across both groups, these on - insulin improvements in cognitive performance were associated with greater vasodilatation in the mca territory and particularly within the right insular cortex. in dm subjects on insulin, visuospatial performance after insulin administration in the dm group and verbal fluency performance in the control group were related to greater vasodilation in the mca territory. the mca territory includes cortical areas representing learning and memory, as well as the insular cortex, which is an important relay region for autonomic functions, emotions, and memory. in particular, the right insular cortex provides a link across systems that are selectively responsive to attention - related problem solving during conditions that require attention and coordination during a task performance (25). our results suggest that improvement of cognitive performance on insulin may be related to regional perfusion and vasodilatation and may specifically activate anterior regions that regulate attention - related task performance. dm is associated with lower baseline perfusion, blunted vasodilatation to hypercapnia, and exaggerated vasoconstriction to hypocapnia, and the regions of altered vasoreactivity extend across aca and mca territories and anatomically across frontal, parietal, and occipital lobes (5,6). cerebral perfusion and vasoreactivity negatively correlate with the degree of insulin resistance, dm control, vascular inflammation, and other indicators of cerebromicrovascular disease (3,5,6). the exact mechanisms by which intranasal insulin may affect regional perfusion are not known but may include endothelium and nitric oxide (no)-dependent vasodilatation and reduction of vasoconstriction by regulating secretion of endothelin-1 (26). vasodilatation - associated increases in blood flow via insulin - stimulated production of no in vascular endothelium have not been well studied in the human brain. therefore, vasodilatation to hypercapnia, although not a specific measure of endothelial function, may serve as an effective proxy to neurovascular coupling within specific regions, as well as the ability to redistribute blood flow to those regions (6,21). therefore, we anticipate that intranasal insulin may have direct effects on neurovascular coupling, regional vascular tone, and neuronal activity (2629). cognitive performance correlates with blood flow and its redistribution to areas with increased neuronal activity (7). previous research has supported a link between vasoreactivity and cognitive performance (30). decreased vasodilatation and increased vasoconstriction reactivity associated with dm have been linked with regional gray matter atrophy and worse functionality in older dm adults (6). conversely, the relationship between improved vasodilatation on insulin with improved cognitive scores may suggest vasoreactivity as a potential diagnostic tool for determining responsiveness to intranasal insulin therapy. the relationship between vasodilatation in right insular cortex and performance of a visuospatial task is intriguing. the activation of the right insular cortex has been linked to better performance on cognitive tasks that are challenging or require longer processing, to simple tasks in older or impaired individuals (31), and to tasks that are associated with autonomic system arousal (32). we can not, however, refute the notion that intranasal insulin may interact with cerebral glucose metabolism and thus enhance the immediate recall and memory, as recently demonstrated in non - dm subjects with mild alzheimer disease (12). dm has been shown to accelerate brain aging by at least 5 years and to increase the risk of alzheimer disease such that even younger dm patients have greater learning and memory deficits than age - matched control subjects. therefore, targeting the population with dm and mild cognitive deficits may be useful for prevention of future cognitive decline and dementia later in life (33). studies evaluating effects of intranasal insulin on cognition suggested potential benefits but have been limited to small sample sizes and healthy young and older adults or non - dm adults with mild cognitive impairment or mild alzheimer disease (17,34,35). the on - insulin improvements of delayed verbal recall in non - dm adults with cognitive impairment associated with mild alzheimer disease were stronger in apoe4 4 allele - negative subjects compared with apoe - positive subjects (28). furthermore, preserved memory and functionality in these subjects was also associated with reduction of a 42 levels in cerebrospinal fluid (12). this pilot study evaluated the acute effects of a single dose of 40 iu intranasal insulin on two subsequent days and therefore had several limitations. we have observed group - treatment effects between insulin and placebo conditions, but within the groups differences were limited owing to the small sample size. potential confounders such as increased familiarity with the environment and potential learning effects despite randomized treatment and parallel versions of tests may have affected the results. both groups performed better on the verbal and numeric tasks on day 3 of testing, while the majority of participants in both groups received insulin on day 2. additionally, there were more women than men participants, which may have contributed to the presence of sex effects with verbal learning and memory. a possible reverse relationship between intranasal insulin dose and cognitive responses has been reported (17,36,37), but an optimal dose for dm subjects is not known. finally, we tested only a single dose of insulin, and therefore it is unclear whether lower or higher doses could be more effective and whether this dose may lead to long - term improvement of memory if administered over a longer period of time. this study provides preliminary evidence that intranasal insulin administration appears safe in older adults with type 2 dm, does not affect systemic glucose control, and may provide acute improvements in cognitive function in older nondemented dm and non - dm patients. the link between cognitive improvement and vasodilation in anterior brain circulation suggests that activation of anterior brain regions controlling visuospatial memory may be a potential mechanism of acute intranasal - insulin changes in cognitive performance. shared central insulin signaling in vascular and metabolic pathways may provide new therapeutic targets to couple perfusion regulation with homeostasis to prevent brain atrophy and consequently cognitive decline in older people with dm. however, larger and prospective studies are needed to determine the long - term safety and efficacy to prevent or slow down cognitive deterioration in older people with type 2 dm. | objectiveto determine acute effects of intranasal insulin on regional cerebral perfusion and cognition in older adults with type 2 diabetes mellitus (dm).research design and methodsthis was a proof - of - concept, randomized, double - blind, placebo - controlled intervention evaluating the effects of a single 40-iu dose of insulin or saline on vasoreactivity and cognition in 15 dm and 14 control subjects. measurements included regional perfusion, vasodilatation to hypercapnia with 3-tesla mri, and neuropsychological evaluation.resultsintranasal insulin administration was well tolerated and did not affect systemic glucose levels. no serious adverse events were reported. across all subjects, intranasal insulin improved visuospatial memory (p 0.05). in the dm group, an increase of perfusion after insulin administration was greater in the insular cortex compared with the control group (p = 0.0003). cognitive performance after insulin administration was related to regional vasoreactivity. improvements of visuospatial memory after insulin administration in the dm group (r2adjusted = 0.44, p = 0.0098) and in the verbal fluency test in the control group (r2adjusted = 0.64, p = 0.0087) were correlated with vasodilatation in the middle cerebral artery territory.conclusionsintranasal insulin administration appears safe, does not affect systemic glucose control, and may provide acute improvements of cognitive function in patients with type 2 dm, potentially through vasoreactivity mechanisms. intranasal insulin - induced changes in cognitive function may be related to vasodilatation in the anterior brain regions, such as insular cortex that regulates attention - related task performance. larger studies are warranted to identify long - term effects and predictors of positive cognitive response to intranasal insulin therapy. |
cardiorespiratory fitness (crf) maintenance is important for functional independence and physical capacity throughout aging [1, 2 ]. substantial declines in the ability to tolerate physical exertion generally predict mobility problems and cardiovascular morbidity and mortality, particularly in the sedentary elderly [3, 4 ]. despite the importance of crf assessment, very low functional capacity and frailty may hinder the use of exercise tests in this population [5, 6 ]. in this context, nonexercise prediction models become practical alternatives to estimate crf and may have important applications both in clinical and epidemiological settings. these models are developed by means of regression - based equations that usually include variables of simple and fast assessment, such as anthropometric measures, demographic characteristics, and daily habits. cross - validated an equation developed primarily in middle - aged adults by jurca. and suggested that nonexercise models could be used to estimate the crf of older adults. however, the prevalence of chronic diseases such as cardiovascular disease and diabetes increases dramatically with age, and is associated to lower physical capacity, inactivity, and limitations in the ability to exercise. it would be therefore important to take into account that elderly populations are not always healthy and free of cardiovascular and metabolic diseases, which on the contrary, are common in the later life. notwithstanding to date nonexercise models to assess the crf in elderly subjects with chronic diseases hence the present study aimed to develop a crf nonexercise prediction linear model of cardiorespiratory fitness and test its validity in elderly men presenting chronic cardiovascular and metabolic diseases. the sample consisted initially of 108 subjects admitted to the elderly care center of the open university of the third age (unati) of the rio de janeiro state university (uerj). these subjects went through clinical exams in order to detail their medical history and completed a brief questionnaire providing demographic information, which was used to determine whether they could perform maximal aerobic exercise testing. the inclusion criteria were asymptomatic patients, stable disease, and no abnormalities in rest electrocardiogram for at least six months. tests interrupted due to clinical reasons were not considered as maximal and therefore have been excluded from the study. twelve participants did not complete the exercise testing (five were not allowed by the physicians to perform the test, four asked to stop before achieving maximal effort, and three exhibited high blood pressure). from the initial sample, a total of 96 subjects remained in the study (6091 years), being randomly assigned into two groups : validation (70%) and cross - validation (30%). the command sample 70 from stata statistical package version 10.1 (stata, college station, tx) was used with this purpose. the validation group was used to develop the statistical model while the cross - validation group was used to confirm the generalization potential of the obtained model. the experimental protocol was approved by institutional ethical committee and participants provided a written informed consent for the use of their data for research prior to the commencement of the study, as stated in the declaration of helsinki. individual data on anthropometry, self - reported physical fitness, and physiological measurements were assessed. anthropometric measurements included weight and height from which the body mass index (bmi) was calculated. the physical activity history was assessed by the self - reported physical activity index (srpa). the subjects were asked to choose one of five activity categories that best described their usual pattern of daily physical activities. self - rated fitness was evaluated by means of the rating of perceived capacity scale (rpc), a 1 - 20-scale previously adapted and translated to the brazilian portuguese language, in which the subject chooses the most strenuous activity that can be sustained for at least 30 min. the rpc score is expressed in mets and the listed activities include walking, jogging, running, and cycling at different paces. instead of asking of the physical activity history, the blood pressure at rest and during exercise was measured by auscultation with a sphygmomanometer welchalln (tycos, arden, mn, usa). a 12-lead electrocardiogram was used to assess the resting heart rate (rhr) and maximal heart rate (micromed, brasilia, df, brazil). handgrip strength (hg) was measured with subjects keeping their shoulder adducted and neutrally rotated, with the arm fully extended and being encouraged to exert maximal grip force on a lafayette dynamometer 78010 (lafayette, in, usa). the highest value in kilograms (kg) was determined after four trials in the dominant and nondominant hands and the relative handgrip strength (i.e., handgrip strength normalized to body weight and represented as the ratio handgrip / weight) was then used for further analyses. all subjects performed a clinically supervised maximal exercise test in an electromagnetically braked cycloergometer (cateye ec-1600, osaka, japan) using an individualized ramp protocol. subjects were submitted to a familiarization trial to get used to the cycloergometer and mouthpiece on the day prior to the exercise test. prediction equation was used to estimate the test incremental workload in watts in order to achieve maximal exertion in approximately 10 minutes. paul, mn, usa) was used for gas exchange measures, using a medium flow pneumotachometer (10120 lmin). peak oxygen uptake (vo2 peak) was determined as the maximal oxygen uptake at the point of test termination due to volitional exhaustion. the borg cr-10 perceived exertion scale was used to estimate the degree of exertion and standard clinical criteria for terminating exercise testing have been applied. before each test, the equipment was calibrated as recommended by the manufacturer, using standard reference gases. the test was considered maximal when at least two of the following criteria were observed : (a) respiratory exchange ratio (rer) > 1.1, (b) vo2 plateau despite an increase in workload (increase 0.05). all subjects attended at least two of the criteria adopted to consider the cardiopulmonary exercise testing as maximal : respiratory exchange ratio (rer) > 1.1 (87% of the sample), vo2 plateau despite an increase in workload (75%), and (c) maximum volitional exhaustion (100%). three levels of physical activity were detected by the srpa index : inactivity or little activity other than usual daily activities (58% of the whole sample), participation in physical activities requiring low levels of exertion that result in slight increases in breathing and heart rate for at least 10 minutes at a time (38%), and participation in aerobic exercises such as brisk walking, jogging or running, cycling, swimming, or vigorous sports at a comfortable pace or other activities requiring similar levels of exertion for 20 to 60 minutes per week (4%). only three of four variables were significantly associated (p 5 mets. the auc further confirmed the high accuracy of the model in screening subjects presenting crf either below or above 5 mets. the negative effect of aging on crf as well as the use of self - reported fitness are both well documented in the literature and are frequently included in prediction models [8, 20 ]. although not previously used in other models, the relative handgrip strength was a very significant predictor of crf in the present study. one could argue that the inclusion of handgrip strength may limit the widespread applicability of this equation, given that it requires equipment that may not be routinely used in epidemiological studies. in fact, the handgrip dynamometry has been a very important tool to assess the functional status of elderly samples, and the inclusion of this measurement in epidemiological settings should be reconsidered. the handgrip strength test is very simple and inexpensive, and has been previously related to mortality, mobility, functional capacity, and correlated with walking speed and overall strength, which are variables strongly related to the functional independence and health in older persons [1, 21, 22 ]. despite of the fact that 79% (r) of variance in the prediction of crf was explained by the obtained statistical model, the see (table 2) suggests that more precise methods as maximal tests are recommended if the exactly crf value is needed. on the other hand, the auc values (table 3) suggested that the model has good accuracy to stratify elderly men with very low (i.e., crf 5 mets) and higher cardiorespiratory capacity (i.e., crf > 5 mets). in other words, the model was capable to identify elder subjects whose functional and exercise capacity are compromised. based on this finding, the proposed model can be useful for population - based investigations or epidemiological studies, especially those searching for associations between crf and other physical and mental health outcomes, such as cognitive function and wellbeing. in clinical studies our model mailey. tested the validity of the nonexercise model proposed by jurca. in old adults, and reported a multiple r of 0.72 (p < 0.001) for the regression in a sample composed mainly of old women, which were somewhat more physically active than our subjects. our model was developed in a sample of elderly men with cardiovascular and metabolic diseases and produced a multiple r of 0.89 (p < 0.001). it is also worthy to note that metabolic and cardiovascular diseases in the elderly are highly prevalent, reinforcing the relevance of the present model for this population. although comparisons between the two models are difficult because they were developed in different populations, we have included variables from mailey 's model in our regression model, and they were not significant. we have also compared the crf estimation accuracy of the models, and the results were favorable to the present equation (mailey 's model in the validation group : r = 0.47 ; epe = 1.7 mets, and in the cross - validation group : r = 0.33 ; epe = 1.6 mets). the adoption of a cycloergometer maximal exercise testing protocol to assess the metpeak must be justified. although treadmill tests are known to engage larger muscle mass and therefore may elicit higher peak vo2 [16, 23 ], some authors have proposed that cycloergometer tests would be more appropriate to assess the cardiorespiratory fitness in older subjects, mainly for safety reasons. for instance, it has been suggested that high - intensity treadmill exercise should be avoided in older subjects with balance restrictions or joint problems. moreover, the poor mechanical efficiency while running seems to reduce the performance of older compared to younger subjects during treadmill exercise, which would very likely limit the peak vo2 in maximal cardiopulmonary tests. such limitation has been considered by previous research that adopted cycloergometer protocols to assess the cardiorespiratory fitness in older populations [4, 26 ]. the main finding of this study was that crf of elderly men with cardiovascular and metabolic diseases and low physical capacity can be classified without aerobic tests using a combination of information on the subject 's daily activity levels, relative handgrip strength, and a self - report of physical fitness level. maximal aerobic tests have a higher cost, demand familiarization with ergometers, and are frequently difficult to perform in old adults due to poor balance and coordination, gait problems, and fear of exercising. it is also worthy to mention that the accuracy of some submaximal exercise testing models to estimate crf [28, 29 ] could be comparable to the accuracy of the present prediction model. it also represents an alternative to some walking tests because it does not require encouragement, which can be a source of disparity across trials, and does not require fatigue and dyspnea measurements. this study has some limitations, namely, the relatively small sample size, the prediction model restricted to unhealthy men. it is worthy to mention that since running tests may potentially produce higher metpeak values, the reproducibility of our findings in treadmill exercise testing should also be addressed, despite the fact that cycloergometer tests are frequently indicated due to safety and mechanical efficiency reasons. probably, changes in age, handgrip strength, and self - related fitness would influence the functional capacity and the mets levels, but only longitudinal studies could confirm this possibility. additionally, the main objective of the present study was similar to other studies that developed nonexercise models, which were not conceived to detect slight longitudinal variations. in brief, the model aims to classify and compare individuals within a given population, and does not intend to replace cardiopulmonary exercise testing to precisely assess the crf. therefore, it is possible that small changes in crf due to training can not be detected by our equation. however, the crf assessment is usually limited due the mobility issues and the risk of cardiovascular events especially in older people with chronic conditions. the present study presented an accurate fitness prediction model for elderly men with cardiovascular and metabolic diseases. the model provides a very fast and safe assessment of fitness, without any chance for cardiovascular events during assessment, which could be very feasible in many healthcare settings to estimate crf and stratify elderly subjects accordingly, and very attractive for epidemiological studies. | low cardiorespiratory (crf) is associated with health problems in elderly people, especially cardiovascular and metabolic disease. however, physical limitations in this population frequently preclude the application of aerobic tests. we developed a model to estimate crf without aerobic testing in older men with chronic cardiovascular and metabolic diseases. subjects aged from 60 to 91 years were randomly assigned into validation (n = 67) and cross - validation (n = 29) groups. a hierarchical linear regression model included age, self - reported fitness, and handgrip strength normalized to body weight (r2 = 0.79 ; see = 1.1 mets). the press (predicted residual sum of squares) statistics revealed minimal shrinkage in relation to the original model and that predicted by the model and actual crf correlated well in the cross - validation group (r = 0.85). the area under curve (auc) values suggested a good accuracy of the model to detect disability in the validation (0.876, 95% ci : 0.7930.959) and cross - validation groups (0.826, 95% ci : 0.6770.975). our findings suggest that crf can be reliably estimated without exercise test in unhealthy elderly men. |
since the discovery of helicobacter pylori (hp) in the 1980s, considerable attention has been given to this bacterium as a cause of gastritis and an established risk factor for gastric cancer [13 ]. helicobacter pylori is known to chronically infect more than half of the world 's population. infection is common in singapore, affecting 71% of adults above 65 years and 3% of children below 5 years. helicobacter pylori infection is associated with a complex interaction between genetic, socioeconomic, environmental, and bacterial factors. this results in multiple potential outcomes following infection, including chronic gastritis and gastric adenocarcinoma [10, 11 ]. due to the close association between hp, gastritis and gastric cancer, it is of interest to decrease the occurrence of hp infection and gastritis. to date, there is a scarcity of published literature on the impact of irregular meals on hp infection or gastritis. this study aims to determine whether a prolonged irregular meal pattern is associated with increased risk of gastritis and hp infection. the sample size was calculated based on a community survey of 113 people in singapore prior to the commencement of this study, which showed 16% had irregular meals. postulating that this prevalence would double in subjects with hp and gastritis, 120 subjects per group has a power of 80% and a 2-sided test of 5% to achieve a statistically significant result. all subjects were of chinese ethnic origin and aged 50 years and above, in order to minimize the confounding factors of age and race. a total of 323 subjects were divided into three groups according to hp and gastritis status. the hp and gastritis group (group a) consisted of patients diagnosed with hp and gastritis (n = 121). the gastritis group (group b) consisted of patients who had been diagnosed with gastritis but negative to hp (n = 100). all patients in group a and b had undergone endoscopic biopsy, with gastritis and hp diagnosed from mucosal biopsy in three locations (antrum, body, and cardia) and by consensus amongst three pathologists according to the updated sydney system for the classification and grading of gastritis. subjects in the control group (group c) had normal endoscopic biopsy results (n = 18) or no symptoms or history of gastritis or hp (n = 84 community - recruited subjects) (n = 102). we compared the diet patterns of the 18 participants with endoscopy results to the 84 without endoscopy and found the patterns were similar (p much greater than 0.05 in all parameters). details of the recruitment process are described in figure 1. this included questions regarding regularity of meals, the frequency and duration of any changes to usual meal timing, variation in the amount of food eaten, and the practice of skipping meals. subjects in groups a and b were surveyed regarding their eating patterns prior to the diagnosis of hp or gastritis. subjects in the control group (group c) were asked to respond regarding their eating pattern prior to endoscopy, or prior to interview for the community recruited subjects. we defined irregular meals as a deviation from regular meal timing for 1 hour or more at least once per week. questions regarding the practice of skipping meals were worded to detect subjects who omitted non - corresponding meals of the day (i.e., not the same meal every day). subjects who missed the same meal each day were considered to have a regular meal pattern consisting of one less meal per day. the questionnaire also surveyed probiotic consumption, the presence of stress or any major stressful event prior to diagnosis, to enable these to be addressed as confounders. all analyses were performed using spss 17.0 with statistical significance set at p < 0.05. multivariate logistic regression was performed to determine the risk predictors for the hp with gastritis and gastritis groups. unadjusted odds ratios were derived comparing group a versus control (group c) and group b versus control (group c) using chi - square or fisher 's exact test. adjusted odds ratios were derived controlling for gender, age, stress, and consumption of probiotics. table 1 describes the demographics of the study subjects. there were no significant differences in age and gender distribution across the 3 study groups. table 2 shows that the adjusted odds ratio (or) of developing hp with gastritis (group a) and gastritis (group b) increased as the time of meal deviation increased. a deviation in meal timing of equal to or more than 2 hours was associated with a significant risk of developing hp with gastritis or gastritis, with an adjusted or of 13.3 (95% ci 5.333.3, p < 0.001) and 6.1 (95% ci 2.515, p the adjusted or for developing hp with gastritis and gastritis also increased as the frequency of meal deviation increased (table 3). subjects in group a who deviated their meals equal to or more than twice per week had an adjusted or of 4.4 of developing hp infection with gastritis (95% ci 2.38.7, p < 0.001). those in group b had an adjusted or of 3.8 of developing gastritis (95% ci 1.97.6, p < 0.001). table 4 shows that subjects who deviated from their regular meals by two or more hours, twice or more per week, were associated with significantly higher incidence of hp infection with gastritis (adjusted or = 6.3, 95% ci 2.615.2, p < 0.001) and gastritis (adjusted or = 3.5, 95% ci 1.58.5, p < 0.005). there were significant differences in the mean period of meal deviation between the hp with gastritis, gastritis and control groups (7.9 years versus 8.1 years versus 4.5 years, p < 0.001) (table 5). although the proportion of subjects who skipped meals almost doubled in the hp with gastritis and gastritis groups in comparison to those in the control group (19% versus 9.8%), there was no significant difference between the groups (table 6). there was no significant difference between groups for subjects who had an inconsistent amount of food at each meal (table 7). this study is the first to examine an association between the degree of irregularity in meal timing and risk of hp and gastritis. after controlling for the potential confounders of gender, age, stress, and consumption of probiotics, we found that deviating from regular meal timing by two hours or more was associated with a thirteenfold increase in risk of developing hp with gastritis, and a sixfold increase in risk of developing gastritis. the association of dietary habits with the development of hp infection has been given relatively little attention. a number of studies have demonstrated evidence of an association between intake of specific food or nutrients and hp [1316 ]. however fewer studies exist examining the relationship between irregular meals and gastritis, and none have studied the degree of irregularity in meal timing [17, 18 ]. a retrospective questionnaire study involving 76 men and 19 women with peptic ulcers in japan found that eating irregular meals significantly increased the relative risk of peptic ulcer in men, but not in women. in this instance the small number of women subjects may not have provided enough power for statistical significance. one chinese study revealed a significant correlation between irregular meals and gastric cardia cancer with an odds ratio of 4.2. however in both studies, there was no mention how irregularity in meals was surveyed, and whether deviation in meal timing, omitted meals, and variations in food quantity were included. bulgarian researchers who found an increase in radiologically documented gastroduodenal ulcers during a period of economic crisis reported their impression that skipped meals and chain smoking were contributory factors. the role of traditional risk factors on the prevalence of duodenal ulcer disease was investigated at an endoscopy unit in jordan with high prevalence of hp amongst patients. skipping breakfast or more than one meal was found to be among important factors in the predisposition for ulcer disease in subjects with hp. in this study, meal regularity and habits the odds ratio increased as the deviation in meal timing increased in the case groups when compared to the control group. before a person can be infected with hp, the bacteria must penetrate the gastric mucosa. the gastric mucosa acts as a natural protective barrier, which limits the penetration of microorganisms. however we do not yet know whether irregularity in meal timing changes the mucosal membrane and increases susceptibility to bacterial penetration. it has recently been established that the barrier function of the mucosa can be disturbed under a variety of pathological insults. we hypothesize that people who have irregular meals are at higher risk of hp infection or gastritis because during the usual meal timing the stomach and intestines produce secretions, free radical scavengers or perhaps some other yet to be discovered chemical, in readiness to receive food. if food is not ingested during this time, the secretions or lack of secretions somehow cause the lining of the stomach to be susceptible to hp infection and gastritis. it has been established that hp survives in brief exposure to acidic ph of less than 4 and growth occurs only at the relatively narrow ph range of 5.5 to 8.0, with optimal growth at neutral ph [25, 26 ]. upon entry to the host, spiral morphology and flagellar motility facilitates penetration of the more ph neutral viscous mucosal layer for infection to occur [27, 28 ]. this could be indicative of a possible effect of meal timing deviation on the gastric ph that makes the mucosa susceptible to hp infection. the mean duration of meal timing deviation in this study was about 8 years for the case groups compared to 4.5 years in the control group. it has been suggested that to cause harm, hp must efficiently adapt to the gastric niche, a process that takes place over many years and involves regulation of bacterial genes in response to environmental factors. these environmental factors may include, but are not limited to, cigarette smoking, stress, irregularity in meal timing and other dietary factors. stress has been shown to increase gastric permeability to pathogens such as hp [30, 31 ]. although our study showed a significant association between irregular meal timing and gastritis as well as occurrence of hp infection, it did not determine whether irregularity in meal timing is the cause or effect of these. we postulate that frequent deviation from regular timing of meals is likely to cause gastritis or hp infection. glutathione level has been found to be elevated in hp infection and some forms of gastritis [33, 34 ]. in addition it may also cause low gastric acid secretion and studies have shown that clinical conditions with low gastric acid secretion are associated with increased risk of gastric cancer [35, 36 ]. meal timing may also impact physiological parameters such as endocrine variables. in our study, the significant differences in the regularity of meal timing of the hp with gastritis and gastritis groups in comparison to the control group supports the presence of the above mechanisms. the merits of this study are the fairly large sample size and the use of endoscopic biopsy as endpoint for diagnosis in three quarters of the study population. due to ethical issues, endoscopic biopsies were not carried out on the community - recruited subjects in the control arm. however, comparison of participants with and without endoscopy results showed similar diet patterns and baseline characteristics (analyses not shown in this paper). some subjects in the control group may have had hp without their knowledge, as individuals may remain asymptomatic despite having hp. if we had been able to definitively exclude hp or gastritis in these community - recruited subjects it may have further strengthened the results of this study, as 39% of subjects in the control group had irregular meals. a major limitation of this study was the retrospective design and its inability to provide causal link of hp infections and gastritis to irregular eating patterns. an individual with chronic gastritis or hp infection might likely be much more aware of their dietary habits than a healthy control. in addition, the dietitians administering the questionnaire were not blinded to the participants ' diagnosis. in conclusion, a variation in meal timing over a prolonged period appears to be associated with increased risk of symptomatic hp infection and gastritis. regular timing of meals may play an important role in the prevention of these two medical conditions. as there is a scarcity of published data studying an association between irregular meal timing and hp and gastritis, this pilot paper warrants future prospective studies to determine the effect of irregular meals on the development of gastritis and hp. | helicobacter pylori (hp) is associated with chronic gastritis and gastric cancer, and more than half of the world 's population is chronically infected. the aim of this retrospective study was to investigate whether an irregular meal pattern is associated with increased risk of gastritis and hp infection. the study involved 323 subjects, divided into three groups as follows : subjects with hp infection and gastritis, subjects with gastritis, and a control group. subjects were interviewed on eating habits and meal timing. multivariate logistic regression was used to compare groups. adjusted odds ratios (or) were derived controlling for gender, age, stress, and probiotic consumption. subjects who deviated from their regular meals by 2 hours or more had a significantly higher incidence of hp infection with gastritis (adjusted or = 13.3 ; 95% ci 5.333.3 ; p < 0.001) and gastritis (adjusted or = 6.1 ; 95% ci 2.515.0 ; p < 0.001). subjects who deviated their meals by 2 hours or more, twice or more per week, had an adjusted or of 6.3 and 3.5 of acquiring hp infection with gastritis (95% ci 2.615.2 ; p < 0.001) and gastritis (95% ci 1.58.5 ; p < 0.001), respectively. frequent deviation in meal timing over a prolonged period appears associated with increased risk of developing hp infection and gastritis. |
witkop defined taurodontism as teeth with large pulp chambers in which the bifurcation or trifurcation are displaced apically, so that the chamber has greater apico - occlusal height than in normal teeth and lacks the constriction at the level of cemento - enamel junction (cej). the distance from the trifurcation or bifurcation of the root to the cej is greater than the occluso - cervical distance. this localized diorder was labeled as taurodontism because of its appreance of or a similarity to a bull 's head with horns on x - ray, hence named after taurus the bull. it also has been found to mimic the molar of cud - chewing animals. in diverse populations, the objective of this study was to assess the frequency of taurodontism in the posterior teeth of the turkish population by radiographic analysis and to compare the results with published data in different population groups. panoramic radiographs from 6912 patients (3,860 women and 3052 man, mean age : 29.04 years range 15 to 50 years) attending krkkale university dental faculty hospital during the period from july 2009 to august 2011 were reviewed for the presence of taurodontism. exclusion criteria included patients who were less than 15 years of age at the time of radiographic examination, records with poor quality radiographs and records with radiographs of only primary teeth. a tooth with an apically displaced pulp chamber which did not show the usual constriction of the pulp at the cemento - enamel junction (cej) and had an apically displaced furcation area was considered as a taurodont. statistical analysis of the data was done using the statistical package for the social sciences (spss 15.0). chi - square test was also used to compare the prevalence of taurodontism between male and female subjects and upper and lower jaws. six thousand nine hundred and twelve patients, 3860 women and 3052 men between the ages of 15 and 50 years (average, 29.04 8.68 years) were considered in this study ; 97362 posterior teeth (including third molars) were evaluated. eightteen patients were found to have a taurodont teeth (10 women and 8 men [p = 0.98 ]). taurodontism was detected in only four premolars (two mandibular first premolars and two mandibular second premolars). the overall incidence of patients with taurodontism was 0.26% (0.26% for women and 0.26% for men). the distribution of taurodontism among different teeth in the upper and lower arches is shown in table 1. taurodonts were significantly more common in the mandibula compared with the maxilla (71.0% cf 29.0% respectively, p < 0.05). the prevalence of taurodont molars among all teeth examined was 0.047% (0.043% for molars, 0.004% for premolars). the mandibular second molar was the most common tooth involved followed by the mandibular first molars. radiographically, all these teeth showed no signs of a previous root canal treatment or apical periodontitis. distribution of taurodont teeth among 6912 patients, in the maxilla and mandible by tooth type taurodontism is a morphologic change generally occurring in multirooted teeth characterized by wide elongated pulp chambers and apical oor displacement. the prevalence of taurodontism was reported to be 8% in a jordanian, 46.4% in a young adult chinese, 5.6% in an israeli people, 9.9% in a dutch and 33 - 41% of certain african populations in our study, the prevalence of taurodont teeth was found to be 0.26%. this nding is in agreement with a previous study conducted in seven - year - old swedish children. the prevalence in the turkish population was reported to be from 4.5% to 7.4% in some studies which were markedly higher than from our findings. discrepancies in the same population may be explained by regional differences and number of study samples. in other studies assessing the prevalence of taurodontic teeth in different ethnic groups, much higher incidences were reported [table 2 ]. the wide variation in reported prevalence may be explained by the different cohorts studied, geographical differences and diffreerences in criteria used for interpretation of taurodontism and also the specifc teeth examined. survey of available studies on the prevalence of taurodont molars the present study should be considered with caution as it may not be representative for the overall turkish population. | the aim of this retrospective study was to evaluate the frequency of the occurrence of taurodontism in a turkish population with using panoromic radiographs. a retrospective study was performed using full - mouth periapical and panoramic radiographs of 6912 patients (3860 females and 3052 males) ranging in age from 15 to 50. a total of 97362 posterior (including third molars) were evaluated. a chi - square test was used to determine the difference in the prevalence of tauorodntism between genders. eighteen patients were found to have a taurodont molar (10 women and 8 men [p = 0.98 ]). the overall incidence of patients with taurodont molars was 0.26 % and the prevalence of taurodont molars from all teeth examined was 0.024%, and the prevalence taurodonts were significantly more common in the mandibula compared with the maxilla (71.0% cf 29.0% respectively, p < 0.05). it was almost equally distributed between males and females. taurodontism is not uncommon in turkish population but further larger scale studies are required to assess its prevalence in the general population to compare it with other ethnic groups. |
the serotonin system is involved in regulation of mood and is the predominant target for antidepressant treatment, primarily with selective serotonin reuptake inhibitors (ssris). the serotonin 4 (5-ht4) receptor is a gs - coupled receptor and is believed to act by modulating other neurotransmitter systems. the 5-ht4 receptor provides a new potential target for fast - acting antidepressant treatment (vidal., 2013). rodent experiments show that only 3 days of treatment with 5-ht4 agonists elicits actions similar to those induced by 23 weeks of treatment with classical antidepressants, including desensitization of 5-ht1a autoreceptors, increased tonus on hippocampal postsynaptic 5-ht1a receptors, enhanced phosphorylation of the creb protein, and neurogenesis in the hippocampus (lucas., 2007). recent rodent work confirms a fast - acting antidepressant and anxiolytic effect of 5-ht4 receptor stimulation and also, notably, implicates 5-ht4 receptor activation in the behavioral and neurogenic effects of ssris (mendez - david., 2013). in the flinder sensitive line rat model for depression, decreased levels of hippocampal 5-ht4 receptor binding were reported (licht., 2009), while regional changes in different directions were seen in two murine models of depression - related states, characterized by serotonin (5-ht) and hypothalamic - pituitary adrenal system changes of depression (licht., 2010). behaviorally, 5-ht4 agonists reverse effects of chronic mild stress on sucrose intake and reduce the effects of olfactory bulbectomy on mice locomotor activity, thereby displaying an antidepressant potential (lucas., 2007). accordingly, 5-ht4 receptor knock - out mice display a decreased reactivity to novelty seeking, which suggests a slight anxiety - like behavior (compan., 2004). the distribution of [c]sb207145 binding potentials in 3 young (2430 yrs) males scanned at the hrrt scanner. binding levels are high in the striatum, intermediate in the temporal and limbic areas, and low in the neocortex. (b) lower binding potentials in a subject who reported to have one first - degree relative with mdd, and (c) even lower binding potentials in a subject who reported to have 2 first - degree relatives with mdd. we have recently found evidence in humans that the cerebral 5-ht4 receptor is a biomarker for extracellular levels of serotonin (haahr, fisher, jensen,., 2013), and that carriers of the short variant of the promoter serotonin transporter (sert) gene have lower 5-ht4 receptor binding (fisher., 2012). thus, it seems that the 5-ht4 receptor is inversely regulated to the 5-ht tonus, albeit not responsive to acute changes in 5-ht (licht. no in vivo studies of the cerebral 5-ht4 receptor binding in depressed patients or at - risk populations have been published so far, but a postmortem study of 19 depressed suicide victims showed increased 5-ht4 receptor density in the frontal cortex and caudate nucleus (rosel., 2004). studying patients suffering from depression is intricate, and confounding effects of previous depressive episodes, co - morbidity with a current episode, and history of antidepressant treatment must be taken to account. an alternative approach is to study the serotonin system in relation to risk factors for depression. we have previously shown that personality risk factors associate positively with 5-ht2 receptor binding (frokjaer., 2008), and a familial risk for depression may enhance the effect (frokjaer., 2010). a relevant family history is the most potent risk factor for depression (kendler., 1999). the inheritance of depression is polygenetic (levinson, 2006), and twin studies have suggested that the heritability is around 40% (kendler., 2006b). people with a family history of depression are more prone to develop depressive symptoms (klaassen., 1999), and show compromised emotional processing after dietary depletion of the serotonin precursor protein tryptophan, which decreases synaptic serotonin transiently in cns (feder., 2010). whether healthy individuals with a family history of depression have different cerebral 5-ht4 receptor bindings compared to healthy individuals with no family history of depression may thus shed light on neurobiological mechanisms underlying risk for depression. the aim of this study was to investigate cerebral 5-ht4 receptor binding, measured with brain pet imaging, in healthy people with varying degrees of familial predisposition to depression. we hypothesized that a family history of major depressive disorder (mdd) is associated with lower cerebral 5-ht4 receptor binding based on the above - mentioned experimental studies. the study was approved by the copenhagen region ethics committee ([kf]01 - 274821, [kf]01 2006 - 20 and h - d-2007 - 0067 with amendments). exclusion criteria were significant medical history, drug or alcohol abuse, neurological or psychiatric disorders (also including depression and prior use of antipsychotics and antidepressants), pregnancy, or moderate - severe head trauma. all volunteers had a normal neurological examination, blood analyses within normal range, unremarkable brain magnetic resonance imaging (mri) scans, and were screened for depressive symptoms using the mdi 10 questionaire (bech., 2001 ; olsen., 2003) on the day of the pet scan. the participants also completed the danish version of the 240-item neo personal inventory revised self - report (neo - pi - r) personality questionnaire, which evaluates the broad personality dimensions of neuroticism, extraversion, openness, agreeableness, and conscientiousness (skovdahl - hansen., 2004). neuroticism is strongly related to lifetime prevalences of mdd, largely due to genetic factors (around 50%) that predispose to both neuroticism and mdd (kendler., 1993, 2006a) demographic data, risk factors for depression, and scanner data for individuals with and without familial risk for depression. data listed as numbers or means (range). fifty - one volunteers were recruited to the study by public advertisements or extracted from the civil registration system in denmark. six volunteers were included because they had a sibling or parent with mdd treated at an in- or out - patient hospital in denmark ; these volunteers also participated in a clinical trial study (knorr., all volunteers were scanned in the period from 2006 to 2011, and some datasets have been included in previously - published studies regarding validation of the tracer (marner., 2009 ; madsen, marner,., 2011) and studies of the 5-ht4 receptor in healthy volunteers to determine the association between the receptor binding and gender and age (madsen, haahr,., 2011), 5-httlpr genotype (fisher., 2012), body mass index (bmi) (haahr., 2012), memory (haahr, fisher, holst,., 2013), effects of ssri (marner., 2010 ; haahr, fisher, jensen,., 2013), and in relation to patients suffering from alzheimer s disease (madsen, neumann,. relevant effects found in these studies were also evaluated in the statistical analysis of this study. to identify participants with familial risk of depression, all participants were interviewed at the day of the pet scan using a danish version of the family history assessment module (fham) (rice., 1995). this module is designed to assess major psychiatric disorders in relatives of the participant. for ethical reasons it was not possible to contact the affected relatives themselves. instead, participants who reported to have one or more first - degree relatives with a major psychiatric disorder were subsequently interviewed by a trained physician with a structural interview regarding the symptoms and treatment of each affected relative. no participants had first - degree relatives diagnosed with schizophrenia or bipolar disorder. in this enriched cohort, 26 out of 57 healthy volunteers (46%) reported to have one or more first - degree relatives diagnosed with depression according to the dsm - iv criteria, which were used as the diagnostic criteria in this study. a total of 34 affected relatives were identified : 97% had been treated for depression by a general physician or psychiatrist, 77% had been treated with antidepressants, 44% had been hospitalized, and 15% had attempted suicide. consistent with the preponderance of women with depression, 68% of the affected relatives were women (14 mothers, 10 sisters, 8 fathers, 2 brothers, and 0 children). since short - allele carrier status of the 5-httlpr polymorphism in the promoter region of the sert gene, scl6a4, may modulate risk for developing depression (caspi., 2003) and affect the 5-ht4 receptor levels (fisher., 2012), blood samples were drawn to determine the 5-httlpr status as previously described (kalbitzer., 2010). high - resolution 3d t1-weighted (matrix 256 x 256 ; 1 x 1 x 1 mm voxels) images were segmented into grey matter, white matter, and cerebrospinal fluid using statistical parametric mapping (spm5 ; wellcome department of cognitive neurology). a set of 17 brain regions was automatically delineated with the pvelab software package (svarer., 2005) on each volunteer s mri in a user - independent fashion. all pet scans were based on a 120-minute dynamic acquisition starting with a bolus injection of [c]sb207145 given over 20 seconds. two different pet scanners were used over time, as the department added a scanner with a higher resolution. twenty - nine volunteers had the pet scans performed with an 18-ring ge - advance scanner (general electric, milwaukee, wi, usa) operating in 3d acquisition mode with an approximate in - plane resolution of 6 mm. after acquisition, attenuation- and decay - corrected recordings were reconstructed by filtered back projection using a 6 mm hann filter. the remaining 28 volunteers completed the pet scans performed with a high - resolution research tomography (hrrt) siemens pet scanner and the images were reconstructed with 3d - osem - psf (sureau., 2008) with a resolution of approximately 2 mm (olesen., 2009). the scan consisted of 38 time frames (6 x 5 seconds [s ], 10 x 15 s, 4 x 30 s, 5 x 120 s, 5 x 300 s, and 8 x 600 s). mean voxel movement between frames was assessed with air 5.2.5 (woods., 1992), and, only when exceeding 3 mm, movement correction was applied as the rigid transformation of each frame to a selected single frame with sufficient structural information (frame 26 : 1520min. post injection) using the scaled least squares cost - function in air. for automatic co - registration of the pet scan to the mri, the air algorithm was applied for ge - advance scans while spm5 was applied for hrrt scans and the quality of each co - registration was evaluated by visual inspection in three planes. the regional in vivo outcome measure for 5-ht4 receptor levels, the binding potential, bp nd, was modeled with the simplified reference tissue model as validated previously (marner., 2009). from the set of regions, volume - weighted means of bp nd were calculated for three brain regions considered important for mood disorders : the striatum (high 5-ht4 receptor binding, including caudate nucleus and putamen), the limbic regions (intermediate 5-ht4 receptor binding, including hippocampus, amygdala, thalamus, and anterior and posterior cingulate gyrus), and the neocortex (low 5-ht4 receptor binding, including parietal cortex, occipital cortex, lateral temporal cortex, insula, and orbito - frontal and lateral - frontal cortex) as previously described (madsen, haahr,., 2011). these regions were chosen since the striatum and the limbic regions previously have been shown to be involved in mdd (price and drevets, 2010), including findings of reduced grey - matter volumes, increased cerebral blood flow and metabolism, altered hemodynamic responses towards emotional stimuli, and reward - processing. the neocortex was included in our analysis as one large cortical region, as it has low 5-ht4 receptor binding, albeit frontal regions also may be involved in mdd. as the primary investigation, a multiple linear regression model was employed to study the association between having a family history of depression (binary) and the 5-ht4 receptor bindings for each of the three selected brain regions. as expected, age, gender, and scanner type were significant covariates and were included in the regression model. we also examined the effect of the number of affected relatives with a history of depression to investigate a possible risk - dose effect of family history of depression on 5-ht4 receptor binding. also, the interaction of being female and having a female relative with a history of depression was investigated to see if the heritable effect could be sex - specific (kendler. all statistical tests were two - sided, and p values were considered statistically significant when less than 0.05. on one hand, the assessment of familial risk for depression may be biased by the age of the volunteers : e.g., being older increases the likelihood of a higher number of mdd - diagnosed first - degree relatives. on the other hand, remaining mentally healthy in spite of a family history may also result from being protected against depression. also, the availability of efficient antidepressant treatment and more attention to the diagnosis in society over time may have an impact. therefore we post hoc estimated the model in the subset of the cohort < 40 years (n = 39). the study was approved by the copenhagen region ethics committee ([kf]01 - 274821, [kf]01 2006 - 20 and h - d-2007 - 0067 with amendments). exclusion criteria were significant medical history, drug or alcohol abuse, neurological or psychiatric disorders (also including depression and prior use of antipsychotics and antidepressants), pregnancy, or moderate - severe head trauma. all volunteers had a normal neurological examination, blood analyses within normal range, unremarkable brain magnetic resonance imaging (mri) scans, and were screened for depressive symptoms using the mdi 10 questionaire (bech., 2001 ; olsen., 2003) on the day of the pet scan. the participants also completed the danish version of the 240-item neo personal inventory revised self - report (neo - pi - r) personality questionnaire, which evaluates the broad personality dimensions of neuroticism, extraversion, openness, agreeableness, and conscientiousness (skovdahl - hansen., 2004). neuroticism is strongly related to lifetime prevalences of mdd, largely due to genetic factors (around 50%) that predispose to both neuroticism and mdd (kendler., 1993, 2006a) demographic data, risk factors for depression, and scanner data for individuals with and without familial risk for depression. data listed as numbers or means (range). fifty - one volunteers were recruited to the study by public advertisements or extracted from the civil registration system in denmark. six volunteers were included because they had a sibling or parent with mdd treated at an in- or out - patient hospital in denmark ; these volunteers also participated in a clinical trial study (knorr., all volunteers were scanned in the period from 2006 to 2011, and some datasets have been included in previously - published studies regarding validation of the tracer (marner., 2009 ; madsen, marner,., 2011) and studies of the 5-ht4 receptor in healthy volunteers to determine the association between the receptor binding and gender and age (madsen, haahr,., 2011), 5-httlpr genotype (fisher., 2012), body mass index (bmi) (haahr., 2012), memory (haahr, fisher, holst,., 2013), effects of ssri (marner., 2010 ; haahr, fisher, jensen,., 2013), and in relation to patients suffering from alzheimer s disease (madsen, neumann,. relevant effects found in these studies were also evaluated in the statistical analysis of this study. to identify participants with familial risk of depression, all participants were interviewed at the day of the pet scan using a danish version of the family history assessment module (fham) (rice., 1995). this module is designed to assess major psychiatric disorders in relatives of the participant. for ethical reasons it was not possible to contact the affected relatives themselves. instead, participants who reported to have one or more first - degree relatives with a major psychiatric disorder were subsequently interviewed by a trained physician with a structural interview regarding the symptoms and treatment of each affected relative. no participants had first - degree relatives diagnosed with schizophrenia or bipolar disorder. in this enriched cohort, 26 out of 57 healthy volunteers (46%) reported to have one or more first - degree relatives diagnosed with depression according to the dsm - iv criteria, which were used as the diagnostic criteria in this study. a total of 34 affected relatives were identified : 97% had been treated for depression by a general physician or psychiatrist, 77% had been treated with antidepressants, 44% had been hospitalized, and 15% had attempted suicide. consistent with the preponderance of women with depression, 68% of the affected relatives were women (14 mothers, 10 sisters, 8 fathers, 2 brothers, and 0 children). since short - allele carrier status of the 5-httlpr polymorphism in the promoter region of the sert gene, scl6a4, may modulate risk for developing depression (caspi., 2003) and affect the 5-ht4 receptor levels (fisher., 2012), blood samples were drawn to determine the 5-httlpr status as previously described (kalbitzer., 2010). high - resolution 3d t1-weighted (matrix 256 x 256 ; 1 x 1 x 1 mm voxels) images were segmented into grey matter, white matter, and cerebrospinal fluid using statistical parametric mapping (spm5 ; wellcome department of cognitive neurology). a set of 17 brain regions was automatically delineated with the pvelab software package (svarer., 2005) on each volunteer s mri in a user - independent fashion. all pet scans were based on a 120-minute dynamic acquisition starting with a bolus injection of [c]sb207145 given over 20 seconds. two different pet scanners were used over time, as the department added a scanner with a higher resolution. twenty - nine volunteers had the pet scans performed with an 18-ring ge - advance scanner (general electric, milwaukee, wi, usa) operating in 3d acquisition mode with an approximate in - plane resolution of 6 mm. after acquisition, attenuation- and decay - corrected recordings were reconstructed by filtered back projection using a 6 mm hann filter. the remaining 28 volunteers completed the pet scans performed with a high - resolution research tomography (hrrt) siemens pet scanner and the images were reconstructed with 3d - osem - psf (sureau., 2008) with a resolution of approximately 2 mm (olesen., 2009). the scan consisted of 38 time frames (6 x 5 seconds [s ], 10 x 15 s, 4 x 30 s, 5 x 120 s, 5 x 300 s, and 8 x 600 s). mean voxel movement between frames was assessed with air 5.2.5 (woods., 1992), and, only when exceeding 3 mm, movement correction was applied as the rigid transformation of each frame to a selected single frame with sufficient structural information (frame 26 : 1520min. post injection) using the scaled least squares cost - function in air. for automatic co - registration of the pet scan to the mri, the air algorithm was applied for ge - advance scans while spm5 was applied for hrrt scans and the quality of each co - registration was evaluated by visual inspection in three planes. the regional in vivo outcome measure for 5-ht4 receptor levels, the binding potential, bp nd, was modeled with the simplified reference tissue model as validated previously (marner., 2009). from the set of regions, volume - weighted means of bp nd were calculated for three brain regions considered important for mood disorders : the striatum (high 5-ht4 receptor binding, including caudate nucleus and putamen), the limbic regions (intermediate 5-ht4 receptor binding, including hippocampus, amygdala, thalamus, and anterior and posterior cingulate gyrus), and the neocortex (low 5-ht4 receptor binding, including parietal cortex, occipital cortex, lateral temporal cortex, insula, and orbito - frontal and lateral - frontal cortex) as previously described (madsen, haahr,., 2011). these regions were chosen since the striatum and the limbic regions previously have been shown to be involved in mdd (price and drevets, 2010), including findings of reduced grey - matter volumes, increased cerebral blood flow and metabolism, altered hemodynamic responses towards emotional stimuli, and reward - processing. the neocortex was included in our analysis as one large cortical region, as it has low 5-ht4 receptor binding, albeit frontal regions also may be involved in mdd. as the primary investigation, a multiple linear regression model was employed to study the association between having a family history of depression (binary) and the 5-ht4 receptor bindings for each of the three selected brain regions. as expected, age, gender, and scanner type were significant covariates and were included in the regression model. we also examined the effect of the number of affected relatives with a history of depression to investigate a possible risk - dose effect of family history of depression on 5-ht4 receptor binding. also, the interaction of being female and having a female relative with a history of depression was investigated to see if the heritable effect could be sex - specific (kendler., 2006b). all statistical tests were two - sided, and p values were considered statistically significant when less than 0.05. on one hand, the assessment of familial risk for depression may be biased by the age of the volunteers : e.g., being older increases the likelihood of a higher number of mdd - diagnosed first - degree relatives. on the other hand, remaining mentally healthy in spite of a family history may also result from being protected against depression. also, the availability of efficient antidepressant treatment and more attention to the diagnosis in society over time may have an impact. therefore we post hoc estimated the model in the subset of the cohort < 40 years (n = 39). a family history of depression was associated with a significant decrease in striatal 5-ht4 receptor binding (p = 0.038, -0.20 bp nd, 95% ci : [-0.39 ; -0.012 ] bp nd), whereas 5-ht4 receptor binding in the limbic regions (p = 0.20) and in the neocortex (p = 0.87) did not differ significantly between groups (table 2). the results were even more significant when only including individuals below 40 years in the model (n = 39 ; striatum p = 0.013 ; limbic regions p = 0.16 ; neocortex p = 0.99). multiple linear regression model analysis outcome of multiple linear regression model analysis used to determine the effect of a family history of depression on 5-ht4 receptor binding. age, gender, and scanner type were significant co - variates in all regions and are included in the model. when considering the risk - dose of first - degree relatives with a history of depression, a significant negative correlation was observed with 5-ht4 receptor binding in both the striatum (p = 0.001, -0.22 bp nd / relative, 95% ci : [-0.352 ; -0.097 ] bp nd, figure 2) and limbic regions (p = 0.012, -0.043 bp nd / relative, 95% ci : [-0.076 ; -0.010 ] bp nd), but no correlation was observed in the neocortex (p = 0.20, 0.017 bp nd / relative, 95% ci : [-0.044 ; 0.009 ] bp nd). the results are illustrated with examples of the distribution in different subjects in figure 1. a leave - one - out sensitivity analysis showed that the estimated association was not strongly driven by any single observation in the data. the estimated linear association between 5-ht4 receptor binding in the striatum (corrected for age, gender, and scanner type) and the number of first - degree relatives treated for major depression (p = 0.001), with pointwise 95% confidence limits and partial residuals (reference : male, mean age 36 years ; ge advance). a leave - one - out analysis showed that the estimated association was not strongly driven by any single observation in the data (p values in the range 0.00030.005). consistent with a previous study in a subset of this cohort (madsen, haahr,., 2011), a decline is found with aging in all regions (table 2). female gender status was associated with reduced limbic 5-ht4 receptor binding in the previous study ; however, in this larger study the association was significant in all regions (table 2). as expected (nilsson. 2010), the hrrt pet scanner generated higher 5-ht4 receptor bpnd than the ge scanner (table 2). inclusion of 5-httlpr genotype status, neuroticism score, and bmi in the model (all predictors were statistically insignificant in all three regions) resulted in very similar p values and parameter estimates of the associations between our primary predictor familial history of mdd and the regional 5-ht4 receptor binding. consistent with our hypothesis, we found that familial risk of mdd was associated with lower striatal 5-ht4 receptor binding (p = 0.038), but no significant effect was found in the neocortex and limbic regions. the effect was even more significant in participants below 40 years (striatum p = 0.013) in spite of the reduced sample. analysis using the same statistical model showed that the association to striatal 5-ht4 receptor binding was even more pronounced when considering the number of affected relatives (p = 0.001) and an association in the same direction was observed in the limbic region (p = 0.01), indicating that there may be a the association towards the striatum and the limbic regions is in concordance with previous findings of involvement of these regions in mdd (price and drevets, 2010). anhedonia is a common symptom in mdd and reward responsiveness (hedonic capacity) may be heritable (bogdan and pizzagalli, 2009). the striatum, particularly its ventral part, and the limbic regions are key structures in the reward system, and patients suffering from mdd have a reduced striatal activation response to rewards (pizzagalli., 2009 ; stoy., reversal learning is also associated with striatal responses and has a negative bias in mdd (robinson., 2011), and the 5-ht4 receptor has been suggested to be involved in cognitive function (king., 2008 ; the involvement of the limbic and striatal regions in mdd has also been demonstrated by induction of mood changes from deep brain stimulation (including symptoms of hypomania, dysphoria, and anhedonia), and experimental investigations are currently being conducted in treatment - resistant mdd (cusin and dougherty, 2012). our finding of lower striatal and limbic 5-ht4 receptor binding in relation to the heritability of depression may reflect that decreased receptor availability is a trait marker of mdd. this is consistent with observations in a genetic rat model of depression (licht., 2009) and a slight hyperanxiety - like behavior of the 5-ht4 receptor knock - out mice in studies of activity in the open field (compan., 2004) our finding of lower 5-ht4 receptor binding in mentally - healthy individuals at familial risk for developing depression could be interpreted as reflecting higher chronic endogenous serotonin levels, since the 5-ht4 receptor is inversely regulated to the 5-ht tonus (haahr, fisher, jensen,., 2013). in vivo studies have reported associations between depression and altered sert and elevated 5-ht1a binding (meyer, 2007 ; miller., 2009), and between behavioral phenotypes related to risk for depression and increased 5-ht2a binding (frokjaer., 2008). we can not determine whether the lower striatal 5-ht4 receptor binding found in our study represents a protective or compensatory mechanism for the included participants to remain mentally healthy, as part of being mdd resilient. it could be that those subjects at highest risk additionally down - regulate limbic 5-ht4 receptors to remain mentally healthy, maybe by modulation of the 5-ht tonus. the answer to this question can only be obtained through longitudinal follow - up studies, which would also reveal whether the low 5-ht4 receptor binding is predictive of development of depression later in life, or by examining unmedicated patients remitted from a depressed state. the heritability of mdd is higher in women than in men and some genetic risk factors for mdd are sex - specific in their effect (kendler., 2006b). a post hoc test showed no interaction between being a female and having a female relative with a history of depression (p = 0.51 in striatum, p = 0.84 in limbic regions, p = 0.74 in neocortex), even though females have lower limbic 5-ht4 receptor binding (madsen, haahr,. thus, based on our data it seems that the effect of familial risk for depression on 5-ht4 receptor binding is not sex - specific. some potential limitations of our study should be considered. the number of first - degree relatives could bias our investigation, since some have more siblings and children than others. as being older increases the likelihood of a higher number of mdd - diagnosed first - degree relatives however, the participants were quite young, and none reported to have children who had suffered from depression. on the other hand, being elderly and having stayed healthy despite a family history of mdd may index protective factors. yet, when we considered only individuals younger than 40 years old we continued to see a significant association between familial risk and striatal 5-ht4 receptor binding. one could speculate whether participants with an affected relative had an overrepresentation of other neuropsychiatric disorders, which could potentially contribute to decreases in 5-ht4 receptor binding., participants were screened for depressive symptoms on the day of the pet scan. despite our inability to interview the affected relatives of the participants themselves, participants were able to give detailed information regarding their relatives. for example, they reported that 97% of the affected relatives had been treated for depression by a general physician or psychiatrist and 77% with antidepressant drugs, which we find underpins the validity of the mdd diagnosis. we experienced that the characteristics of the affected relatives were more difficult to clarify for elderly participants who reported a parent suffering from depression. this might explain why the association between familial risk and striatal 5-ht4 binding appeared weaker when including participants above 40 years of age. however, mdd is a heterogeneous disorder and 5-ht4 receptor binding could be more strongly related to a more homogenous phenotype of mdd (bogdan., 2013), as, for example, in patients with predominant symptoms of anhedonia, anxiety, or suicidal behavior. however, we were not able to reliably characterize the affected relatives in such detail based on the interviews of the participants. the finding of lower 5-ht4 receptor binding in healthy individuals with familial risk for mdd suggests that the 5-ht4 receptor is involved in the neurobiological mechanism underlying familial risk for depression. our current finding is intriguing considering that the 5-ht4 receptor may be an effective target for antidepressant treatment (lucas. future studies are needed to elucidate whether 5-ht4 receptor binding is changed in the depressed state of mdd, and clinical trials are needed to determine the effects of 5-ht4 agonists on depressive symptoms and cognitive performances in mdd. dr knorr has been a consultant for astrazeneca. otherwise, the authors declare no conflicts of interest and no non - financial form of support has been given to the study. | background : the 5-ht4 receptor provides a novel potential target for antidepressant treatment. no studies exist to elucidate the 5-ht4 receptor s in vivo distribution in the depressed state or in populations that may display trait markers for major depression disorder (mdd). the aim of this study was to determine whether familial risk for mdd is associated with cerebral 5-ht4 receptor binding as measured with [11c]sb207145 brain pet imaging. familial risk is the most potent risk factor of mdd.methods:we studied 57 healthy individuals (mean age 36 yrs, range 2086 ; 21 women), 26 of which had first - degree relatives treated for mdd.results:we found that having a family history of mdd was associated with lower striatal 5-ht4 receptor binding (p = 0.038 ; in individuals below 40 years, p = 0.013). further, we found evidence for a risk - dose effect on 5-ht4 receptor binding, since the number of first - degree relatives with a history of mdd binding correlated negatively with 5-ht4 receptor binding in both the striatum (p = 0.001) and limbic regions (p = 0.012).conclusions : our data suggest that the 5-ht4 receptor is involved in the neurobiological mechanism underlying familial risk for depression, and that lower striatal 5-ht4 receptor binding is associated with increased risk for developing mdd. the finding is intriguing considering that the 5-ht4 receptor has been suggested to be an effective target for antidepressant treatment. |
the study protocol was approved by the ethics committee of potsdam university, potsdam, germany. participants were selected from the ongoing german metabolic syndrome berlin potsdam (mesybepo) study that currently includes 2,500 individuals with different states of glucose tolerance. details of baseline phenotyping have previously been described (17), and all individuals with at least 3 years of follow - up time were recruited to repeat phenotyping (21). the baseline examination of participants included anthropometric measurements, blood sampling, a 75-g oral glucose tolerance test (ogtt) for 120 min, which was performed after overnight fast of 10 hours, and a personal interview on lifestyle habits and medical history. for the current study, we examined a consecutive series of 800 subjects (subjects with metabolic syndrome, n = 325 ; subjects without metabolic syndrome, n = 475) at baseline and 189 subjects with follow - up data (incident metabolic syndrome, n = 47 ; incident impaired glucose metabolism [igm ], n = 33). all subjects had no history of diabetes, cardiovascular diseases, malignant disease, liver or chronic kidney failure, or inflammatory diseases at baseline visit. the metabolic syndrome was diagnosed according to harmonizing criteria of the metabolic syndrome (22). samples for insulin and c - peptide measurements were drawn at 0, 30, 60, 90, and 120 min of the ogtt. capillary blood glucose concentrations were measured using the glucose oxidase method on a super gl (dr. hba1c was measured using a hi - auto a1c ha-8140 system (menarini diagnostics, berlin, germany). serum triglycerides, total cholesterol, and hdl cholesterol were measured by standard enzymatic assays, and ldl cholesterol was calculated from these data (certified laboratory for clinical chemistry). serum insulin and c - peptide were measured using commercial elisas (insulin elisa and c - peptide elisa ; mercodia, uppsala, sweden). based on the ogtt data, we calculated the insulin secretion rate (isr) using the two - compartment model of c - peptide kinetics (23). the hicc - peptide was determined as a ratio of the incremental areas under the curve (auc) of ogtt (aucc - peptide 0120 min / aucinsulin 0120 min) (12,14,24). in addition, we calculated hicisr as a ratio of the incremental area under the isr curve (aucisr 0120 min) to the incremental area under the peripheral insulin concentration curve (aucinsulin 0120 min) (12). the ogtt - derived hicc - peptide was strong correlated with metabolic insulin clearance determined in hyperinsulinemic - euglycemic clamp experiments in our previous study (17). insulin sensitivity was quantified from the ogtt by gutt index (gutt isi0,120) (25). insulin response to glucose in the ogtt was estimated by calculation of 1st - phase insulin secretion index : 1,283 + (1.829 ins30 min [pmol ]) (138.7 blood glucose30 min [mmol ]) + (3.772 ins0 min [pmol ]), where ins is insulin (26). the difference between groups was calculated by one - way anova. the linear relationships between hic and anthropometric as well as metabolic markers were calculated using pearson correlation. to investigate the shape of the associations between both indices of hic and 1st - phase insulin secretion, we used restricted cubic spline regressions (27) with knots at the 5th, 50th, and 95th percentiles. the effect and the term for nonlinearity of the restricted cubic spline regression were statistically tested. the power (the probability of avoiding a type ii error) for the estimation of the ors of hic on metabolic syndrome was calculated with sas power and sample size 3.12. the statistical analyses were performed with spss 18 (spss, chicago, il) and sas 9.3 (sas institute, cary, nc). capillary blood glucose concentrations were measured using the glucose oxidase method on a super gl (dr. mller, freital, germany). hba1c was measured using a hi - auto a1c ha-8140 system (menarini diagnostics, berlin, germany). serum triglycerides, total cholesterol, and hdl cholesterol were measured by standard enzymatic assays, and ldl cholesterol was calculated from these data (certified laboratory for clinical chemistry). serum insulin and c - peptide were measured using commercial elisas (insulin elisa and c - peptide elisa ; mercodia, uppsala, sweden). based on the ogtt data, we calculated the insulin secretion rate (isr) using the two - compartment model of c - peptide kinetics (23). the hicc - peptide was determined as a ratio of the incremental areas under the curve (auc) of ogtt (aucc - peptide 0120 min / aucinsulin 0120 min) (12,14,24). in addition, we calculated hicisr as a ratio of the incremental area under the isr curve (aucisr 0120 min) to the incremental area under the peripheral insulin concentration curve (aucinsulin 0120 min) (12). the ogtt - derived hicc - peptide was strong correlated with metabolic insulin clearance determined in hyperinsulinemic - euglycemic clamp experiments in our previous study (17). insulin sensitivity was quantified from the ogtt by gutt index (gutt isi0,120) (25). insulin response to glucose in the ogtt was estimated by calculation of 1st - phase insulin secretion index : 1,283 + (1.829 ins30 min [pmol ]) (138.7 blood glucose30 min [mmol ]) + (3.772 ins0 min [pmol ]), where ins is insulin (26). the linear relationships between hic and anthropometric as well as metabolic markers were calculated using pearson correlation. to investigate the shape of the associations between both indices of hic and 1st - phase insulin secretion, we used restricted cubic spline regressions (27) with knots at the 5th, 50th, and 95th percentiles. the effect and the term for nonlinearity of the restricted cubic spline regression were statistically tested. binary logistic regression was used for the calculation of odds ratios (ors). the power (the probability of avoiding a type ii error) for the estimation of the ors of hic on metabolic syndrome was calculated with sas power and sample size 3.12. the statistical analyses were performed with spss 18 (spss, chicago, il) and sas 9.3 (sas institute, cary, nc). at baseline, 325 of the participants fulfilled the criteria for metabolic syndrome (table 1). these subjects had higher bmi, waist circumference, triglycerides, fasting glucose, and systolic and diastolic blood pressure, as well as surrogate markers of 1st - phase insulin secretion, and were more insulin resistant compared with subjects without metabolic syndrome. ogtt - derived indices of hic were markedly lower in subjects with metabolic syndrome (hicc - peptide 6.7 2.6 vs. 5.5 2.3 arbitrary units [au ], p < 0.001, and hicisr 2.2 0.8 vs. 1.9 0.8 pmol / min, respectively) and remained significant after adjustment for bmi and age. moreover, subjects with normal glucose tolerance (ngt) and metabolic syndrome had decreased hic compared with subjects without metabolic syndrome (hicc - peptide 5.90 2.54 vs. 6.73 2.60 au, p < 0.05, and hicisr 2.09 0.86 vs. 2.21 0.84 pmol / min, p < 0.05, respectively) (table 1). clinical characteristics of the study population both hic indices correlated significantly with each other (r = 0.94, p < 0.001). positive linear correlation of hic indices with gutt isi0,120 was observed in subjects with metabolic syndrome (r = 0.44, p < 0.001, for hicc - peptide and r = 0.50, p < 0.001, for hicisr) and in subjects without metabolic syndrome (r = 0.34, p < 0.001, for hicc - peptide and r = 0.39, p < 0.001, for hicisr). moreover, an inverse relationship was found between hic and 1st - phase insulin secretion index (r = 0.23, p < 0.001, for hicc - peptide and r = 0.15, p < 0.001, for hicisr) in the analysis of the general cohort. to investigate the mechanism by which insulin secretion interacts with hic, we tested the relationship between these variables separately in subjects with and without metabolic syndrome (fig. 1). we found close nonlinear relationships between hic and 1st - phase insulin secretion only in the subjects with metabolic syndrome (r = 0.26, pnonlin (p value for nonlinear model) < 0.007, for hicc - peptide) (fig. 1b)not in subjects without metabolic syndrome (r = 0.16, peffect < 0.001, and r = 0.16, pnonlin = 0.24, for hicc - peptide and r = 0.09, peffect < 0.001, and r = 0.09, pnonlin = 0.63, for hicisr) (fig. again, the results remained significant after adjustment for waist circumference, age, and sex. relationship between hic estimated as hicisr (c and d) and hicc - peptide (a and b) and ogtt - derived indices of insulin secretion (1st - phase insulin secretion index [is ]) in the entire cohort (n = 800 ; subjects with metabolic syndrome, n = 325 ; subjects without metabolic syndrome, n = 475). an inverse correlation between different parameters of metabolic syndrome such as waist circumference, diastolic blood pressure, fasting glucose, and triglycerides was observed in the entire cohort (table 2). in contrast, plasma hdl cholesterol correlated positively with hic (r = 0.11, p = 0.003, for hicc - peptide and r = 0.11, p = 0.002, for hicisr). relationship between indexes of hic and markers of metabolic syndrome in the next step, we analyzed the relation between hic and incident metabolic syndrome. we observed no statistically significant difference in either index of hic between subjects with incident metabolic syndrome and subjects without metabolic syndrome (hicc - peptide 7.01 3.05 vs. 6.23 2.13 au, p = 0.11, and hicisr 2.32 0.98 vs. 2.09 0.73 pmol / min, p = 0.15, respectively). logistic regression analysis indicates a trend that hic independently predicted the risk of developing the metabolic syndrome (hicc - peptide or 1.13 [95% ci 0.971.31 ], p = 0.12, and hicisr 1.38 [0.882.17 ], p = 0.16) (crude model). additional adjustment for age, sex, waist circumference, index of 1st phase of insulin secretion, and time of follow - up (hicc - peptide or 1.13 [95% ci 0.961.32 ], p = 0.14, and hicisr1.41 [0.872.29 ], p = 0.16), which slightly attenuated results, but hic still remained an independent predictor of future metabolic syndrome (fig. risk of subsequent metabolic syndrome according to the respective median of ascending tertiles of two hic indices. adjustment for age, sex, body weight, waist circumference, 1st - phase insulin secretion, and follow - up time. in addition, hicisr showed a trend for association with increased risk of igm incidents after adjustment for age, sex, waist circumference, time of follow - up, and 1st - phase of insulin secretion (hicc - peptide or 1.12 [95% ci 0.921.36 ], p = 0.26, and hicisr 1.31 [0.742.33 ], p = 0.36), although point estimates reached no statistical significance. additional adjustment for waist - to - hip ratio, diastolic blood pressure, hdl cholesterol, triglycerides, and baseline fasting and 2-h glucose again modified results, but point estimates remained comparable with the crude analysis for incident metabolic syndrome and incident igm (data not shown). the power (the probability of avoiding a type ii error) for the estimation of the ors of hic on metabolic syndrome was 0.30 and 0.32 for hicc - peptide and hicisr, respectively. decreased hic is an early phenotypical marker of disturbances in insulin metabolism and was observed in various disorders associated with metabolic syndrome and t2 dm (6,1316,28). however, most studies were not designed to test the hypothesis that insulin clearance is strongly associated with existing metabolic syndrome and may predict this condition. in this large - cohort prospective study, we found an association between two ogtt - derived indices of hic and different components of metabolic syndrome and a trend indicating their possible association with an increased risk of incident metabolic syndrome and igm. moreover, we observed an inverse nonlinear correlation between hic and 1st - phase insulin secretion index in subjects with metabolic syndrome and a positive linear correlation between hic and ogtt - derived index of insulin sensitivity for the general mesybepo cohort. in our study, we identified highly significant correlations between ogtt - derived hic indices and different components of metabolic syndrome, in agreement with previous results from other studies (4,6,13). the imbalance of hepatic insulin metabolism appears to be a first change in the development of weight gain related insulin resistance (29). conversely, weight loss increases hic in both humans (29) and animals (30). in accordance with this, our study subjects with ngt and metabolic syndrome had lower hic compared with subjects without metabolic syndrome, suggesting that impairment of insulin clearance may occur before the development of disturbances in glucose metabolism. moreover, we found a trend for the association between two ogtt - derived indices of hic and increased risk of incident metabolic syndrome and igm, although point estimates reached no statistical significance. the ogtt - derived hic was strongly correlated with metabolic insulin clearance, as determined in hyperinsulinemic - euglycemic clamp experiments in our previous study (17), and may be helpful for the identification of subjects with high risk of metabolic syndrome, even in the absence of other signs of igm. reduced hepatic insulin elimination may intensify insulin resistance via chronic elevations of circulating fasting and postprandial insulin concentrations (20,31,32). the gutt insulin sensitivity index, calculated as a ratio of postloading glucose disposal to the mean of fasting and 2-h postinsulin concentrations, has been suggested as the best predictor of t2 dm after 58 years of follow - up (33). our data showed a significant and positive correlation between the gutt insulin sensitivity index and hic, supporting complete capture of other important domains of t2 dm in this index (32). in accordance with previously published data (11,3436), we observed an inverse relationship between insulin secretion and hic, potentially representing a physiological mechanism by which insulin secretion may regulate hic. thus, decreased hic in subjects with metabolic syndrome may not compensate for lower insulin sensitivity but, rather, represent an additional element of insulin disturbance, possibly directly dependent on changes in insulin secretion. we can speculate that the decrease in the hic may also be an important mechanism in the case of insulin secretion stimulating diets like diets with a high glycemic index and the phenomenon of soft drink induced metabolic syndrome being associated with nonalcoholic steatohepatitis (37). on the other hand, based on the epidemiological character of our study, we can not entirely rule out the fact that hic may simply cluster with metabolic syndrome without necessarily belonging to the syndrome as one of the defining components. however, mechanisms leading to the alteration of insulin degradation in humans are complex and not understood in detail (31). the insulin - degrading enzyme (ide) is thought to be a major enzyme responsible for insulin degradation (31). however, the liver is the main site of insulin clearance, removing ~75% during the first portal passage (31,36). hyperglycemia downregulates the insulin - induced ide activity in the liver cell model (19) and in this way may provoke the known decrease of ide activity in t2 dm (31). on the other hand, insulin clearance is a highly heritable trait (8), and polymorphisms in the ide gene are associated with increased t2 dm risk and decreased ogtt - derived hic in nondiabetic subjects (17). we observed a close correlation between hic and hdl cholesterol, a marker of liver fat metabolism. hic correlated inversely with liver fat content and hepatic glucose production in diabetic and nondiabetic subjects (16). taken together, decreased hic is possibly the earliest marker of hepatic insulin resistance and is directly linked to insulin action in the liver with consequent effects on the hepatic lipid metabolism and liver inflammation. we measured hic indirectly in two ways, based on previously reported techniques of insulin clearance calculation (12,17). although direct assessment of portal concentration of hormones in human subjects has been established (36), this is not a practicable method for the use in large cohorts. in addition, the power to detect more moderate changes of hic in our prospective study population is likely to be insufficient, and doing so would require the investigation of considerably larger prospective cohorts. in conclusion, we found decreased hic in middle - aged subjects with metabolic syndrome. the decrease of hic showed a trend for association with a risk of incident metabolic syndrome and incident impaired glucose homeostasis independent of obesity and age. thus, ogtt - derived indices of hic may be helpful for the identification of people with high risk of metabolic syndrome. | objectiveinsulin clearance is decreased in type 2 diabetes mellitus (t2 dm) for unknown reasons. subjects with metabolic syndrome are hyperinsulinemic and have an increased risk of t2 dm. we aimed to investigate the relationship between hepatic insulin clearance (hic) and different components of metabolic syndrome and tested the hypothesis that hic may predict the risk of metabolic syndrome.research design and methodsindividuals without diabetes from the metabolic syndrome berlin brandenburg (mesybepo) study (800 subjects with the baseline examination and 189 subjects from the mesybepo recall study) underwent an oral glucose tolerance test (ogtt) with assessment of insulin secretion (insulin secretion rate [isr ]) and insulin sensitivity. two indices of hic were calculated.resultsboth hic indices showed lower values in subjects with metabolic syndrome (p < 0.001) at baseline. hic indices correlate inversely with waist circumference, diastolic blood pressure, fasting glucose, triglycerides, and ogtt - derived insulin secretion index. during a mean follow - up of 5.1 0.9 years, 47 individuals developed metabolic syndrome and 33 subjects progressed to impaired glucose metabolism. both indices of hic showed a trend of an association with increased risk of metabolic syndrome (hicc - peptide odds ratio 1.13 [95% ci 0.971.31 ], p = 0.12, and hicisr 1.38 [0.882.17 ], p = 0.16) and impaired glucose metabolism (hicc - peptide 1.12 [0.921.36 ], p = 0.26, and hicisr 1.31 [0.742.33 ] p = 0.36), although point estimates reached no statistical significance.conclusionshic was associated with different components of metabolic syndrome and markers of insulin secretion and insulin sensitivity. decreased hic may represent a novel pathophysiological mechanism of the metabolic syndrome, which may be used additionally for early identification of high - risk subjects. |
a 15-year - old female patient was referred to seoul national university hospital with palpitation and chest pain. three years previously, she had been diagnosed with graves disease and her transthoracic echocardiogram (tte) and electrocardiogram had revealed mild mitral regurgitation (mr), mild tricuspid regurgitation (tr), and atrial fibrillation (af) ; however, the patient was lost at follow - up. she had not been compliant with medication and had had no medication for at least the 3 previous months. one week before she was referred, she had visited a hospital complaining of general weakness, palpitation, chest discomfort, cold sweat, and loss of 6 kg of body weight in 3 weeks ; she was then diagnosed with thyroid storm. after 1 week of medical treatment for thyroid storm and improvement of symptoms, she was referred to seoul national university hospital for further management. at the point of time that she was referred, the initial laboratory test showed high serum - free t4 (2.12 ng / dl ; normal range, 0.70 to 1.80 ng / dl), low serum thyroid stimulating hormone (tsh) (< 0.05 iu / ml ; normal range, 0.4 to 4.1 iu / ml), and high titer of autoantibodies (thyroglobulin antibody, 110 iu / ml ; normal range, 0 to 100 iu / ml ; tsh receptor antibody, 13.8 iu / l ; normal range, 0 to 1 iu / l) implying autoimmune hyperthyroidism such as graves disease. grade iv systolic murmur with irregular heart beat was auscultated, and the electrocardiogram revealed af (fig. the tte revealed 58.5% of left ventricular ejection fraction (lvef), moderate to severe mr with anterior leaflet prolapse, moderate tr with mild leaflet prolapse, and 68.8 mm of left ventricular end diastolic diameter, which implied left ventricular dysfunction and enlargement (fig. after 11 days of medical treatment, the thyroid function test was normalized (free t4, 1.24 ng / dl ; tsh, 0.01 iu / ml), but the heart failure and valvular dysfunction did not improve. we concluded that the heart including the valves and chordae had already been changed structurally and therefore, performed surgery. as intraoperative findings, the mitral valve showed diffuse chordae thinning and elongation with diffuse leaflet prolapse, and the tricuspid valve showed diffuse chordae thinning and elongation with severe anterior leaflet prolapse. therefore, we performed mitral valvuloplasty making two artificial chordae at a2 and a3, respectively, mitral annuloplasty with a 32-mm cosgrove ring, tricuspid valvuloplasty making 4 artificial chordae at the anterior leaflet, de vega - type tricuspid annuloplasty, and cox maze procedure. after the surgery, the tte revealed improved cardiac function with 66.1% of lvef, mild mr, and trivial tr, and decreased the left ventricular end diastolic diameter to 49.5 mm (fig. the patient s cardiac rhythm was af when she was discharged on postoperative day 16 but was converted to normal sinus rhythm from the first out - patient follow - up (postoperative day) to the last follow - up (2 years after operation) (fig. her thyroid function has also been controlled well (free t4, 1.09 ng / dl ; tsh, 1.26 iu / ml at last follow - up) with medication including oral methimazole and levothyroxine. thyroid hormone has effects on cardiovascular hemodynamics through its indirect effect on the peripheral vasculature and body metabolism, and through its direct effect on the heart. t3 decreases the systemic vascular resistance (svr) by promoting vasodilation with its genomic and nongenomic actions altering the vascular smooth muscle and the endothelial cell function. the decrease in svr causes the mean arterial pressure to decrease, which, when sensed in the kidneys, activates the renin - angiotensin - aldosterone system and increases renal sodium absorption. t3 also increases erythropoietin synthesis, which leads to an increase in the red cell mass. these changes combine to promote increases in the blood volume and the stroke volume. at the myocyte level, t3 enhances contractility and relaxation of the myocardial cells through nontranscriptional effects on various ion channels and through transcriptional effects that regulate the release and uptake of sarcoplasmic reticular calcium and phosphorylation of phospholamban [13 ]. the most common cardiovascular symptoms and signs of hypothyroidism include bradycardia, mild hypertension (diastolic), narrowed pulse pressure, cold intolerance, and fatigue. decreased thyroid hormone action causes increased svr, decreased cardiac contractility, and decreased cardiac output. further, it is well known that hypothyroidism is associated with accelerated atherosclerosis and coronary artery disease with elevated serum lipid levels. a variety of case reports have demonstrated that hypothyroidism may cause a prolongation of the qt interval that predisposes the patient to ventricular irritability, and torsade de pointes may result in rare cases. most of the changes in the cardiac structure and function are potentially reversible with thyroid hormone replacement. in hyperthyroidism patients, the cardiac output increases as a result of the combined effect of the increases in the resting heart rate, contractility, ejection fraction, and blood volume with a decrease in svr. the condition that results from excess thyroid hormone may induce cardiac rhythm disturbance and heart failure. almost every hyperthyroidism patient has sinus tachycardia, and the incidence of af among the hyperthyroidism patients is approximately 13.8%, which is much higher than that of the normal population. approximately 6% of the hyperthyroidism patients show heart failure as an initial clinical presentation, and half of them have left ventricular systolic dysfunction. although the symptoms of heart failure and lvef improve after the medical treatment of hyperthyroidism, persistent dilated cardiomyopathy develops in about one - third of these patients. heart failure in the hyperthyroidism patients is mainly induced by sinus tachycardia or af, as well as hypertension. in particular, a higher incidence of mitral valve prolapse has been reported in autoimmune hyperthyroidism such as graves disease and hashimoto s disease. xenopoulos. reported a case of right heart failure associated with graves disease, which required tricuspid valve repair for successful treatment of right heart failure. in our case, medical correction of hyperthyroidism was too late to reverse the development of persistent dilated cardiomyopathy and the structural change of the heart including the valves ; therefore, surgical treatment was necessary. to the best of our knowledge, this is the first case of a cardiac complication of graves disease requiring surgical treatment reported in the korean journal of thoracic and cardiovascular surgery. as an effort to prevent such severe complications, the cardiac function and rhythm should be monitored periodically in hyperthyroidism patients, and proper treatment of hyperthyroidism is essential. thyroid dysfunction should also be checked with a detailed history, physical examination, and thyroid function test in every patient with newly onset congestive heart failure. | cardiac complications such as arrhythmia and heart failure are common in graves disease. early detection and proper treatment of hyperthyroidism are important because cardiac complications are reported to be reversible if the thyroid function is normalized by medical treatment. we report here a case of cardiac complication of graves disease that was too late to reverse with medical treatment and required surgical treatment. |
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